RaeumnonormWWW.) ' ' mcuuuaanmsmcecw Dissertation far the. Degree iof‘PhQD. ' i‘ a_ - Mtcmemsmeumveasmv; ‘ _ ~ ZEVAREWENY_;§A (~1975 f_d“~" Date 0-7639 This is to certify that the thesis entitled REGULATION OF ATP SULFURYLASE IN CULTURED TOBACCO CELLS presented by ZIVA REUVENY has been accepted towards fulfillment of the requirements for Ph.D. Biochemistry degree in jot professor /4 .‘ J‘s, July 8, 1975 w ABSTRACT REGULATION OF ATP SULFURYLASE IN CULTURED TOBACCO CELLS BY Ziva Reuveny The influence of changes in sulfur and nitrogen nutrition on the level of ATP sulfurylase (E.C. 2.7.7.4, ATP:sulfate adenyltransferase) in cultured tobacco cells was investigated. A new assay for the enzyme was devised, the key feature of which is the quantitative pre- cipitation of NaZSO4 in ethanol:water (5:1), leaving the product, adenosine 5'-phosphosulfate (APS), in solution with an 80% yield. Using this separation procedure and [3581-804-2, the rate of synthesis of picomole amounts of [3ssl-APS can be assayed, which makes possible kinetic and regulatory studies of ATP sulfurylase based on APS formation. Extracts from tobacco cells and several higher plants synthe- sized [3SS]-APS but not [358]-3'-phosphoadenosine-5'-phosphosulfate (PAPS). Therefore, the [3SSJ-APS remaining in solution after the separation procedure is a valid assay of the rate of the ATP sul- furylase reaction. The tobacco cell ATP sulfurylase is not induced by sulfate. It is regulated through a negative feedback mechanism by the end product(s) of the pathway. ATP sulfurylase is repressed during Ziva Reuveny growth on readily assimilated sulfur sources such as sulfate, L- cysteine, or L-methionine, but is derepressed during sulfur-limited growth on slowly assimilated sulfur sources such as djenkolate or glutathione or during sulfur starvation. The ATP sulfurylase specific activity begins to rise within 12 hours after the derepression condi- tions are initiated, and continues to increase up to 25-fold within 3 to 4 days. The sulfur compounds which affect the development of ATP sul- furylase in vivo have no effect on the enzyme activity in vitro. Derepression is inhibited by cycloheximide at a concentration which strongly inhibits incorporation of amino acids into protein, indicating that the mechanism of derepression depends, in some way, upon protein synthesis. After addition of a repressing sulfur source to derepressed cells, a decline in total ATP sulfurylase activity can be detected, indicating a role for enzyme inactivation or degradation in the regulation of the enzyme in the tobacco cells. Derepression does not occur in tobacco cells starved for nitro— gen, a circumstance in which turnover synthesis of protein is known to continue. Upon addition of a nitrogen source derepression occurs, along with a resumption of net protein synthesis, indicating a positive role for nitrogen assimilation in the regulation of ATP sulfurylase. Therefore, it is hypothesized that ATP sulfurylase of the tobacco cells is derepressed by the absence of an end product of the sulfate pathway, SX, provided that there also is a positive effector signal, NY, from the nitrogen assimilation pathway. Ziva Reuveny Molybdate and selenate are structural analogs of sulfate for the reaction of APS synthesis by tobacco cell ATP sulfurylase. Either of these anions, vfimni included in the culture media with sulfate, derepressed the ATP sulfurylase. Molybdate can cause derepression only when added at 10-fold the concentration of sulfate, a condi- tion which inhibits growth,and net accumulation of protein is inhi- bited, suggesting that the derepression resulted from sulfur starvation. Selenate, which is a competitive inhibitor of APS synthesis in vitro, causes a derepression of ATP sulfurylase in vivo when added to the culture media at concentrations at which neither growth nor protein accumulation in the cells is affected. At higher molar ratios of selenate to sulfate, selenate is toxic and derepression does not occur. Selenate is extremely toxic to derepressed cells growing on djenkolate but is much less toxic to repressed cells growing on cysteine. It is suggested that the selenate-dependent derepression of ATP sulfurylase may be via an antagonism between the hypothetical corepressor of the sulfate pathway, SX, and a hypo- thetical anti-corepressor, SeX. REGULATION OF ATP SULFURYLASE IN CULTURED TOBACCO CELLS BY Ziva Reuveny A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1975 W? or I m " . . 12in «w wan-52‘?! ma ma mm n3“: was firm. new ms was? 13-12,3: fl: 31m "Man puts an end to darkness, Every recess he searches. . . But wisdom, where can it be found? Where is the place of understanding? Man knows not the path to it, It is not found in the land of the living." Job XXVIII: 3, 12-13 ii ACKNOWLEDGEMENTS Being a graduate student at the Plant Research Laboratory (also known as "Lang's Gang") has been a unique and highly rewarding experience. The prerogative of knowing and interacting with col— leagues and friends here over the years has been of great benefit to my education. It has been a particular privilege to be a student of Dr. Philip Filner, whose perceptions of learning, thinking, and science have given me much more than just guidance, encouragement and understand- ing during the course of this work. The many ideas, thoughts and concepts which have been originated and built via our interactions shall always be of fundamental value for me. I would like to express a few special thoughts for my friend and colleague Judy Cherniack, whose invaluable spiritual support and editorial assistance during the preparation of this disserta- tion have been an extension of our trust and friendship over the years, with particular meaning for me. I would also like to thank Dr. Deborah P. Delmer for her aid and invariably sound advice in preparing this manuscript. The critical comments provided by Drs. C. P. Wolk, L. L. Bieber, R. L. Bandurski, and A. Ehmann are gratefully appreciated. iii This work would not have been possible without the continuous morale contributions from my family in Israel and my very special friends here and at home. Love and patience from my parents, trust and understanding from Ora and Amir Reuveny, and true beautiful friends such as Glenda and Steven Scheer, Harriet Cooper, and Bill Rosenberg have been an endless source of strength and light during my stay here. This research was supported by the U.S. AEC/ERDA Contract AT-(ll—1)-l338. iv TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . The Sulfate Assimilation Pathway . . . . . . . . . . Sulfate Activation . . . . . . . . . . . . . . . . . Assimilatory Sulfate Reduction . . . . . . . . . . . Biosynthesis of Cysteine and Methionine. . . . . . . Sulfate Esters and Sulfonic Acids. . . . . . . . . . Properties of ATP Sulfurylase. . . . . . . . . . . . Assay for ATP Sulfurylase Activity: Difficulties in Perspective . . . . . . . . . . . . . . . . . . Control of the Sulfate Assimilation Pathway. . . . . Group VI Anions and the Sulfate Transport System . . Interactions of Group VI Anions with ATP Sulfurylase Selenium: Micronutrient Role in Bacteria and Animals. Selenium Metabolism in Plants: Selenium Accumulators. The Relationship Between Selenium Metabolism and the Sulfate Assimilation Pathway . . . . . . . . . Selenium: Resistance and Toxicity . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . Separation of Sulfate and APS by Differential Solubility of the Two Compounds in Ethanol- Water Mixture. . . . . . . . . . . . . . . . . . . Determination of Radioactivity . . . . . . . . . . . Paper Electrophoresis. . . . . . . . . . . . . . . . Thin Layer Chromatography. . . . . . . . . . . . . . The Cultured Tobacco Cell System . . . . . . . . . . Special Modified Media . . . . . . . . . . . .J. . . Maintenance of Tobacco Cells on the Modified Media . Growth of Seedlings. . . . . . . . . . . . . . . . . Growth of Lemna Plants . . . . . . . . . . . . . . . Preparation of ATP Sulfurylase . . . . . . . . . . . Assay of ATP Sulfurylase Activity. . . . . . . . . . V Page . viii xii 10 15 15 16 21 26 33 34 37 38 39 42 45 45 46 46 47 47 48 49 50 50 51 RESULTS ATP Determination. . . . . . . . . . . . . . . . . . . Column Chromatographic Separation of the Reaction Mixture on DEAE A-25 Sephadex. . . . . . . . . . . . Chemical Characterization of the [3581-Product Eluted from the DEAE Sephadex Column . . . . . . . . Column Chromatographic Separation of the Sulfur Compounds from the Reaction Mixture on Dowex-l-Nitrate. . . . . . . . . . . . . . . . . . . Desalting on Charcoal. . . . . . . . . . . . . . . . . Preparation of [3581-APS of a High Specific Radioactivity. . . . . . . . . . . . . . . . . . . . Determination of Protein Content . . . . . . . . . . . Determination of Total Uptake and Incorporation of Radioactive Amino Acid into Protein. . . . . . . . . Chemicals. PART A: DEVELOPMENT OF ATP SULFURYLASE ASSAY BASED ON THE DIFFERENTIAL SOLUBILITY OF SULFATE AND APS IN ETHANOL. I. II. III. Differential Solubility of Sulfate and Sulfate-Containing Nucleotides in EthanOI-Water Mixture o o o o o o o o o o 0 Formation of an Ethanol-Soluble Product by Tobacco Cell Extract . . . . . . . . . . Characterization and Identification of the Ethanol-Soluble [3SS]—Product Formed by Tobacco Cell Extract as APS . . . . . . . . (a) Electrophoretic Analysis . . . . . . . (b) Thin Layer Chromatography. . . . . . . (c) Charcoal Adsorption. . . . . . . . . . (d) Acid Lability. . . . . . . . . . . . . (e) Attempted Identification of th Ethanol—Soluble [3SS]-Product as APS by the Reverse Reaction of ATP Sulfurylase. . . . . . . . . . . . . . (f) Characterization of the Chemical Composition of the [3581-Product . . . (9) Determination of APS as the Only Sulfur-Containing Product Synthe- sized by Tobacco Cell Extract. . . . . (h) Summary of Conclusions Regarding the Measurement of Incorporation of [358]- Sulfate by Tobacco Cell Extract Into Product . . . . . . . . . . . . . vi Page 52 52 53 53 54 55 55 56 56 58 58 58 65 69 69 7O 7O 7O 75 81 82 84 Page IV. ATP Sulfurylase Assay Based on the Dif— ferential Solubility of Sulfate and APS . . . 86 PART B: THE REGULATION OF ATP SULFURYLASE IN CULTURED TOBACCO CELLS. . . . . . . . . . . . . . . . . . . . . 91 B.l Regulation of ATP Sulfurylase by Sulfur Compounds. . . . . . . . . . . . . . . . . . . 91 I. The Effects of Sulfur Compounds on Growth and ATP Sulfurylase Activity in Tobacco Cells . . . . . . . . . . . . . . . . . . . . 92 II. Dependence of the Regulation of ATP Sul- furylase on Growth Rate, as Determined by Nitrogen Source. . . . . . . . . . . . . . 97 III. Kinetics of the Development of ATP Sul- furylase as a Function of the Sulfur Source . 98 IV. Specificity of the ATP Sulfurylase Repres— sion-Derepression Mechanisms for Sulfur Amino Acids . . . . . . . . . . . . . . . . . 109 V. The Effect of Nitrogen Starvation on the Derepression of ATP Sulfurylase . . . . . . . 112 VI. The Effect of Inhibition of Protein Syn- thesis on the Derepression of ATP Sulfurylase . . . . . . . . . . . . . . . . . 116 B.2 Regulation of ATP Sulfurylase by Group VI Anions. . . . . . . . . . . . . . . . . . . 118 I. Group VI Anions as Inhibitors of APS Fomation in Vi tr0. o o o o o o o o o o o o o 120 II. The Effects of Group VI Anions on Growth and ATP Sulfurylase Level of Tobacco Cells. . 122 III. Derepression of ATP Sulfurylase by Selenate: Dependence on the Sulfur Source . . . . . . . 133 IV. Selenate Toxicity as a Function of the Sulfur Source Utilized for Growth . . . . . . 136 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . 142 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . . . . . 159 vii Table 10 11 12 13 14 LIST OF TABLES . . . . . 35 Quantitative preCipitation of [ S]-su1fate by the separation method. . . . . . . . . . . . . . . . . . 35 . Recovery of added [ Sl-APS from complete reaction mixtures by the separation technique . . . . . . . . . . . . 35 PreCipitation of [ Sl-PAPS by ethanol . . . . . . . . . 35 Time dependent formation of ethanol-soluble [ 8]- product by tobacco cell extract. . . . . . . . . . . Requirements for the enzymatic synthesis of the ethanol-soluble [35S]-product by tobacco cell extract. . 35 Formation of sulfate from the ethanol—soluble [ S]- product catalyzed by ATP sulfurylase purified from yeast. . . . . . . . . . . . . . . . . . . . . . . . APS formation during various conditions. . . . . . . . APS formation catalyzed by extracts from higher plants Growth of tobacco cells on various sulfur sources. ATP sulfurylase activity of tobacco cells grown on various sulfur sources . . . . . . . . . . . . . . . . Lack of effects of the sulfur amino acids on APS formation in vitro . . . . . . . . . . . . . . . . . Growth and ATP sulfurylase activity: dependency on both sulfur and nitrogen sources . . . . . . . . . . . Relationship of ATP sulfurylase development to fresh weight and soluble protein during growth on various sulfur sources . . . . . . . . . . . . . . . . . . Growth and ATP sufurylase activity during the derepres- sion by djenkolate and the repression by sulfate or cysteine . . . . . . . . . . . . . . . . . . . . . viii Page 60 62 66 68 69 80 89 9O 93 95 96 99 103 105 Table 15 16 17 18 19 20 21 22-A Resumption of net protein synthesis concomitantly with the decline of ATP sulfurylase level upon addition of sulfate to sulfur-starved cells. . . . . Decay of ATP sulfurylase upon addition of sulfate to derepressed cells . . . . . . . . . . . . . . . . Absence of net protein synthesis in nitrogen-starved cells and its resumption upon addition of nitrogen . Inhibition of APS formation in vitro by Group VI anions O O I O O O O C O O O O O O O O O O C O O O 0 Effects of Group VI anions on growth and ATP sul- furylase in vivo . . . . . . . . . . . . . . . . . . The effect of molybdate on the development of ATP sulfurylase in cultured tobacco cells. . . . . . . . The effects of selenate on soluble protein and ATP sulfurylase in cells utilizing sulfate for growth. . Inhibition by selenate of growth of tobacco cells on various sulfur sources. . . . . . . . . . . . . . The effect of L—arginine on the protection by L- cysteine of tobacco cells from inhibition of growth by selenate. . . . . . . . . . . . . . . . . . . . . The effect of L-cysteine on the djenkolate- dependent susceptibility to growth inhibition by selenate. . . . . . .-. . . . . . . . . . . . . . ix Page 110 111 115 121 128 130 132 137 139 140 Figure 10 11 12 LIST OF FIGURES Page Procedure for separation of sulfate and APS by differential solubility in ethanol:water (5:1) . . . . . 59 35 Recovery of the [ S]—APS added in the final ethanol fraction: verification by electrophoresis . . . . . . . 64 Radioelectrophoretogram of complete reaction mixture showing the [35$]-product synthesized by tobacco cell extract. . . . . . . . . . . . . . . . . . . . . . . . . 67 Radioelectrophoretograms showing the ethanol-soluble [35$]-product derived from assay mixture with tobacco cell extract after precipitation with ethanol. . 72 Radiochromatograms showing absence of PAPS in the ethanol-soluble [3SSl-product formed by tobacco cell extract. . C C . C O O C C O O C C O I C . . C C O O O O 73 Radiochromatograms showing absence of sulfate in the ethanol-soluble [3581-product synthesized by extract from tobacco cells . . . . . . . . . . . . . . . . . . . 74 Radioelectrophoretograms of the acid hydrolysis product of the ethanol-soluble [3581-product . . . . . . 77 Radioelectrophoretograms showing enzymatic conversion of the [35$]-product to [3581-su1fate by tobacco cell extract. . . . . . . . . . . . . . . . . . . . . . . . . 79 Chemical composition of the [BSSJ-product eluted from DEAE sephadex column, showing the molar ratio of sulfur:adenine:phosphate. . . . . . . . . . . . . . 83 3 Analysis of the [ SSl-derivatives formed by tobacco cell extract: chromatography on Dowex-l—nitrate column . . . . . . . . . . . . . . . . . . . . . . . . . 85 ATP sulfurylase assay based on APS formation: pro- portionality with time and enzyme concentration. . . . . 87 Kinetics of the development of ATP sulfurylase as a function of the sulfur source. . . . . . . . . . . . . . 101 X Figure Page 13 Kinetics of the development of ATP sulfurylase: derepression by djenkolate and repression by sulfate or cysteine. . . . . . . . . . . . . . . . . . . . . . . 102 14 Derepression of ATP sulfurylase during growth on glutathione or during sulfur starvation on nitrate . . . 106 15 Kinetics of the repression—derepression development of ATP sulfurylase during growth on urea . . . . . . . . 107 16 Kinetics of the decline of ATP sulfurylase activity upon addition of sulfate to sulfur-starved cells . . . . 108 17 Lack of effect of non-sulfur amino acids on the repression-derepression regulation of ATP sulfurylase. . 113 18 Dependency of the development of ATP sulfurylase activity on nitrogen assimilation: lack of derepres- sion by djenkolate in cells starved for nitrogen . . . . 114 19 Inhibition by cycloheximide of the uptake and incor- poration into protein of [14C1-L-arginine in the tobacco cells. . . . . . . . . . . . . . . . . . . . . . 117 20 Inhibition by cycloheximide of the development of ATP sulfurylase during derepression on djenkolate. . . . 119 21-A Dependence of the rate of APS formation on sulfate concentration. . . . . . . . . . . . . . . . . . . . . . 124 21-B Double reciprocal plot (Lineweaver-Burk) of the rate of APS formation versus sulfate concentration. . . . . . 124 22 Double reciprocal plot of the concentration- dependent inhibition of APS formation by selenate. . . . 125 23 Dixon plot of the concentration-dependent inhibition of APS formation by selenate . . . . . . . . . . . . . . 126 24 Derepression by selenate of ATP sulfurylase in cells grown on sulfate . . . . . . . . . . . . . . . . . . . . 131 25 The effects of various sulfur sources on the dere- pression of ATP sulfurylase by selenate. . . . . . . . . 135 xi APMO APS APSe DPH g.f.wt. (N,S)- MID OAS PAP PAPS PAPSe PP PPT SAM LIST OF ABBREVIATIONS adenosine 5'-phosphomyolybdate adenosine 5'-phosphosu1fate adenosine 5'—phosphose1enate 3',5' diphosphonucleoside 3' phosphohydrolase gram fresh weight MID medium lacking both nitrogen and sulfur sources O-acetylserine adenosine 3',5'—diphosphate 3'-phosphoadenosine-5'-phosphosu1fate 3'-phosphoadenosine-5'-phosphose1enate inorganic phosphate inorganic pyrophosphate precipitate S-adenosylmethionine xii INTRODUCTION Among the few metabolic pathways of higher plants which begin with an exogenous compound is the sulfate assimilation pathway. Higher plants can utilize either sulfate or more reduced sulfur compounds to satisfy their requirements for growth (123), but sulfite and sulfide are toxic to plants (cf. 154). Higher plants incorporate sulfur predominantly into cysteine and methionine in cell protein (147). Therefore, in order to minimize accumulation of toxic intermediates, and to optimize production of end products, it can be predicted that the sulfate assimilation pathway of higher plants should be regulated by the quantities and chemical forms of sulfur available and by the overall potential for protein synthesis. Regulation of a biosynthetic pathway usually involves the regulation of its first enzymatic step which, in the case of the sulfate assimilation pathway, after the uptake of sulfate, would be the activation of sulfate to adenosine 5'-phosphosulfate (APS), a reaction catalyzed by ATP sulfurylase. There has been only one previous attempt to investigate in higher plants the regulation of ATP sulfurylase by sulfur compounds, but negative results were obtained (104). However, regulation of this enzyme has been found repeatedly in microorganisms (24-27,126,129,185). A reexamination of the question of regulation of ATP sulfurylase in higher plants, therefore, was warranted. 2 The cultured tobacco cell system has been found to be useful in studies of the regulation of the nitrate assimilation pathway, because nutritional conditions are strictly controlled and can be readily manipulated (179-181). Furthermore, in earlier studies with the tobacco cells, evidence was obtained of a role for sulfur amino acids in the regulation of the uptake of sulfate (123-124). From all of these considerations, it seemed highly probable that if ATP sul- furylase were regulated in higher plants, the phenomenon could be detected and studied in the cultured tobacco cells. Studies on the regulation of enzymes of biosynthetic pathways can involve either a genetic or physiological approach. In the absence of methods for genetic manipulation of higher plant cells, the physiological approach has to be adopted. Accordingly, the general experimental procedure was to seek changes in levels of ATP sulfurylase activity in response to specific perturbations in sulfur nutrition. Before such changes could be detected, however, an assay for ATP sulfurylase was required which would give a reliable quantitative estimate of the physiologically important reaction, APS synthesis. The research presented in this dissertation consequently can be divided into two parts: first, development of the enzyme assay and, second, application of the assay in regulatory studies. The regulatory studies with the tobacco cell system were guided by the following general questions: (a) Is ATP sulfurylase induced by its substrate, sulfate? (b) Is the enzyme subject to regulation by end product(s) of the pathway? 3 (c) Is there any connection between regulation of sulfate assimilation and regulation of nitrate assimilation, since the end products from both pathways flow into protein? LITERATURE REVIEW The Sulfate Assimilation Pathway Sulfur is essential for all living organisms because of its occurrence in the sulfur amino acids of protein, in sulfur-containing bases of tRNA, and in a number of vital cofactors, e.g., thiamine pyrophosphate, biotin, lipoic acid and coenzyme A. Sulfur also occurs in sulfate esters and sulfolipids of various organisms (108,147). While the ability to synthesize sulfate esters from sulfate, which does not involve a change in oxidation level of the sulfur, is found in all biological kingdoms, the ability to carry out the 8—e1ectron reduction of inorganic sulfate (oxidation state +6) to the sulfide level (oxidation state -2) is restricted to plants and microorganisms (108). Plants and most bacteria reduce sulfate to satisfy their nutri- tional requirement for sulfur. This is termed assimilatory sulfate reduction (148). In a small group of anaerobic bacteria, however, sulfate is reduced during anaerobic respiration and thereby serves as a terminal electron acceptor. This process is called dissimila- tory or respiratory sulfate reduction. Inorganic sulfate is the principal form of sulfur used by plants for growth. The sulfate assimilation pathway in plants is believed to have two branches: reduction of sulfate followed by incorporation into amino acids containing sulfur on the one hand, and incorporation of sulfate into sulfate esters (and possibly sulfonates) on the other (cf. 48-49,108): ATP -2 -2 SULFURYLASE 50"” m ’ SULHJR FEW —. 3—. —.§ PIUIEINS m ADA. PPS \Em ri—~ :3... Nevertheless, cysteine and methionine, in protein, are responsible for the bulk (>90%) of the organic sulfur in plants (147). Conse- quently, the demand for these protein amino acids largely determines the sulfur requirements of plants. Sulfate Activation Sulfate uptake and its control have been studied in bacteria, fungi, algae and higher plants (cf. 48). The reduction of sulfate to sulfide is highly endergonic, requiring an input of some 180 Kcal/mole (cf. 48). Assuming that the reaction proceeds by 2-electron steps, sulfite would be the first reduced intermediate. However, reduction of sulfate to sulfite requires ca. 60 Kcal/mole (168), which is unlikely to be carried out biochemically in a single step (168). Therefore, a stepwise activation of the sulfate, prior to reduction, can be anticipated. Biochemical and genetic evidence obtained to date has 6 verified thatactivationlof sulfate is a prerequisite to its utili- zation in either biosynthetic or energy—yielding reactions (108,148). Therefore, the ability to activate sulfate is widely distributed among living organisms. The enzymatic mechanism for the activation of sulfate, and the intermediates involved, were elucidated by Lipmann and Robbins (cf. 149) and Wilson and Bandurski (cf. 150). The first step involves the formation of the unique mixed anhydride bond between sulfate and adenosine 5'-monophosphate in adenosine 5'—phosphosulfate (APS), catalyzed by ATP sulfurylase (eq. 1). NH. + N -2 M9 2 9' ' j ATP + 80 —+ o=s—o—P—O-CH. N N + PP. (eq. 1) 4 +— 3 g o I cum: APS APS can be further phosphorylated on the 3'OH group of the ribose moiety, to yield 3'-phosphoadenosine—5'-phosphosu1fate (PAPS). This reaction is catalyzed by APS kinase (eq. 2). Mm Mg+2 i:[%\N 0‘ 0' APS + ATP —-+ o=g_o_:,_o_c,,, PAPS + ADP AF , - 6,000 cal (eq. 5) +— The finding by Robbins and Lipmann (140) that APS kinase has a high affinity toward APS is consistent with the idea that the function of this enzyme is the removal of a product of a thermodynamically unfavorable reaction. It is noteworthy that in the presence of inorganic pyrophospha- tase with the ATP sulfurylase, the equilibrium concentration of APS 8 was found to increase by only ca. one order of magnitude (41,154), which is lower than expected from calculations based on the reported free energy changes for the reactions of ATP sulfurylase and inor- ganic pyrophosphatase (see eqs. 3-4). Thus, an extremely low Km (APS) for the APS kinase reaction may be required even in the presence of inorganic pyrophosphatase. PAPS was assumed for many years to be the chemical form of sulfate which is actually reduced in organisms which assimilate sulfate. However, APS rather than PAPS is known to be the activated substrate for the dissimilatory sulfate reduction as shown by Peck and co-workers (cf. 148), although the equilibrium constant of ATP sulfurylase isolated from these dissimilatory bacteria was very similar to the yeast enzyme (91). Furthermore, recent studies strongly suggest that APS is also the activated substrate in sulfate reduction in Chlorella and spinach (cf. 48). The apparent lack of participation of PAPS as the active sulfate in sulfate reduction in certain organisms indicates that there must be alternative solutions in Vivo to the problem of the unfavorable equilibrium governing APS formation. In contrast to the extensive documentation of the occurrence and properties of ATP sulfurylase, reports of the occurrence of APS kinase are relatively few in number. Partially purified enzyme was obtained only from yeast (140) and Nitrobacter (141), while its occurrence in other bacteria, fungi and algae was deduced from nutri— tional studies with mutants and from the synthesis of PAPS from sulfate and ATP by crude extracts (cf. 108). 9 The occurrence of APS kinase in higher plants was until recently a matter of dispute. Although some workers have reported the synthe- sis of [BSSl-PAPS in extracts of a few higher plant species (105,142), other workers could detect [3SSI-APS but not [3SS]-PAPS under seemingly identical conditions (104,143-144,156). The failure to detect PAPS formation in extracts of higher plants led Ellis to propose that APS rather than PAPS might be the substrate for the first reduction step (104). It now appears, however, that APS kinase does occur in higher plants but, nevertheless, APS, not PAPS, is indeed the actual sub- strate for reduction (cf. 48). Two possible sources of difficulty in the detection of PAPS formation in plant extracts are: (a) a lack of adequate sulfhydryl protection; and (b) rapid reconversion of PAPS to APS by enzymes that hydrolyze the 3' phosphate of PAPS. The formation of PAPS by both spinach and Chlorella extracts was observed upon the addition of sulfhydryl reagents (145-146). Since addition of -SH containing compounds is required to obtain APS forma- tion with Chlorella extracts but not with higher plant extracts, the possible requirement for sulfhydryl protection for PAPS forma- tion may have been overlooked with higher plants. There are two hydrolytic enzymes known which will convert PAPS to APS. The first enzyme is the 3' nucleotidase which is widely found among higher plants and has been purified from rye grass (cf. 48,155). The failure to detect PAPS formation in a strain of Salmonella (151) appeared to be due to the presence of a potent 3' nucleotidase, since PAPS was readily detected upon inclusion of 3' AMP in the incubation mixture. The second enzyme, which was discovered by Schiff et al. in Chlorella 10 (cf. 48,153) and which appears to exist in spinach as well (146), is a 3',5' diphosphonucleoside 3' phosphohydrolase (DPH). This enzyme, although it hydrolyzes the 3' phosphate group on PAPS, will not use 3' AMP efficiently as a substrate (cf. 48). Recently, Burnell and Anderson (155) demonstrated an APS kinase activity, i.e., PAPS formation, in crude spinach extracts in the presence of 3' AMP. In the absence of the 3' nucleotide, APS was the only species detected. The absence of the 3' nucleotidase activity in the fraction which was capable of 3' AMP-dependent PAPS formation was interpreted to mean that 3' AMP was an activator of the APS kinase. It is clear that further studies of APS kinase in the plant kingdom have to be carried out before unequivocal conclusions regard- ing the role of PAPS formation and APS kinase in sulfate reduction can be reached. Assimilatory4Sulfate Reduction The exact pathway by which sulfate is reduced to sulfite and incorporated into cysteine in organisms which assimilate and reduce sulfate is still unclear. The evidence obtained from enzymological, nutritional and genetic studies with bacteria and fungi (cf. 108) has indicated that APS, PAPS, sulfite and sulfide are all intermediates in the incor- poration of sulfate into cysteine in these organisms. In addition to ATP sulfurylase and APS kinase, the enzymes catalyzing the subse- quent two steps, i.e., PAPS reductase and sulfite reductase, have been studied in these organisms (108). 11 Most of our knowledge on the PAPS reduction comes from the studies of Wilson, Bandurski and co-workers (summarized in 150 and 154) with yeast. The PAPS reducing activity in yeast extracts has been analyzed into three protein fractions: enzyme A, enzyme B, and a low molecular weight protein, fraction C. Enzyme A catalyzed the NADPH-dependent reduction of a disulfide group in fraction C, which then participated in the reduction of the active sulfate in PAPS to sulfite, catalyzed by enzyme B. The subsequent reduction of sulfite to the sulfide level can be catalyzed by sulfite reductase. This activity was found in many organisms (cf. 108) and has been highly purified from yeast, bac- teria and higher plant sources (cf. 49 and 108). These pure sulfite reductases, which are all complex enzymes, are capable of catalyzing the 6—electron reduction in the presence of various electron donors. The enzymes purified from bacteria and yeast also possess a nitrite reductase activity which appears to be due to the same enzyme, while the sulfite reductase from higher plants has been reported to lack the nitrite reductase activity (169). This dual activity has raised some question as to the role of the sulfite reductases from yeast and bacteria in sulfate reduction (cf. 48). Nevertheless, extensive biochemical-genetic studies with mutants of Salmonella (170), the wide distribution of this enzyme in species capable of reducing sulfate, and its coincident repression by cysteine, along with PAPS reductase (148), indicate that it is involved in the assimilatory pathway of sulfate reduction. No such evidence exists, however, for the sulfite reductase of photosynthetic organisms. 12 The enzymological studies with the reductase systems of yeast by Bandurski and co-workers (cf. 108 and 154) indicated that a protein- bound sulfite, rather than a free sulfite, was the product of the PAPS reductase and the substrate for further reduction. Thus, the concept of "bound intermediates" during sulfate reduction from PAPS to cysteine emerged. Bound sulfite and bound sulfide species were predicted also because of the toxicity of the free species (49,154). Evidence in support of the concept of a bound sulfite as a product of PAPS reduction has become available with the recent isolation by Wilson (171) of a low moecular weight protein, Pr-SSOB-, formed enzymatically in reaction mixtures containing PAPS and enzyme B. These preliminary studies have suggested that the protein is reduced by fraction C prior to the transfer of the sulfonyl group from PAPS and, therefore, appeared to be the bound sulfite in yeast. Recent studies with extracts of Chlorella and spinach, carried out by the groups of Schmidt and Schiff (cf. 48,172-174), focus on the pathway of sulfate reduction in these photosynthetic organisms. This pathway appeared to utilize APS, rather than PAPS, as the physiologically active species for reduction. This APS reducing system involves the transfer of the sulfonyl group of APS to a protein carrier to form a bound sulfite as carrier—S-SOB-, catalyzed by the enzyme APS sulfotransferase. This bound sulfite, which closely resembles that of yeast, is then acted upon by a second enzyme, the thiosulfonate reductase, which in the presence of ferrodoxin catalyzes the 6-e1ectron reduction of the bound sulfite to a bound thiol group. The proposed carrier-S-SH is thought likely to participate directly 13 as the substrate for the cysteine forming enzymes. Thus, under experimental conditions extracts of Chlorella and spinach incorporated sulfate directly into cysteine (48,172-174). Because no free inter- mediates were found, this reductive pathway was termed the "bound intermediates pathway" (cf. 48,174). The enzyme B and Pr-S-SO3 recently found in yeast (171) appear to be analogous to the enzyme APS sulfotransferase and Carrier-S-SOB- found in Chlorella (172). How- ever, in the photosynthetic organisms, APS appears to be the sulfonyl donor in a sulfotransferase reaction, as opposed to PAPS in yeast and bacteria. The significance of utilizing APS vs. PAPS for sulfate reduction in photosynthetic organisms is by no means clear. It is unlikely to be due to the presence of the DPH activity, nor does it appear to reflect a property of the sulfotransferase, which uses APS as a substrate, as both of these activities have been shown now to exist also in E. coli (152), although PAPS is the actual sulfonyl donor in this organism. The suggestion by Schiff and Hodson (48) that the use of PAPS for esterifications and APS for reduction may reflect a regulatory advantage is intriguing. Yet the proposed role for the DPH enzyme as regulating the flow between PAPS and APS is still obscure. The latter enzyme, partially purified from Chlorella, also has been reported to catalyze the synthesis of cyclic AMP from APS (153). Convincing evidence that the product is actually cyclic AMP has not been published, however. Additional complications come from the study of APS kinase in spinach extracts (155). The stimula- tion by 3' AMP of both PAPS formation and the overall rate of sulfate incorporation into cysteine led to the suggestion that PAPS is the 14 actual sulfonyl donor for reduction in spinach as well (155). The characterization of the enzyme thiosulfonate reductase in Chlorella and spinach, which coexists with the sulfite reductase (48,174) needs some elaboration. Studies with the thiosulfonate reducatse purified from Chlorella (173-174) indicated that it could not reduce free sulfite, but carried out the reduction of the sul- fonyl group of S-sulfoglutathione (GS-SO3H) to the sulfide level in the presence of NADPH and ferredoxin. S-Sulfoglutathione could not serve as a substrate for the sulfite reductase isolated from Chlorella. Thus, it was proposed {172-174) that in Chlorella and spinach there are two pathways for sulfate reduction: both begin with the APS sulfotransferase reaction, but one pathway utilizes the thiosulfonate reductase and bound intermediates, while the other employs the sulfite reductase and free intermediates. Because the product of the APS sulfotransferase appears to be a bound sulfite, the reduction via the sulfite reductase requires prior release of the sulfonyl group as free sulfite. The hypothesis that sulfate reduction via the thiosulfonate reductase in Chlorella and spinach is the main pathway in vivo is supported by a mutant which cannot grow on sulfate, nor reduce it in vivo. Extracts of this mutant lack the thiosulfonate reductase activity but possess the sulfite reductase activity (174). Thus, it appears likely that the reduc- tion of sulfate via the thiosulfonate reductase is the preferred pathway in vivo in Chlorella, while the reduction via the sulfite reductase could be functional when free sulfite is available in vivo. The existence of these two pathways for sulfate reduction was recently 15 reported for E. coli as well (152). Thus, studies with genetically defined mutants lacking either thiosulfonate reductase or sulfite reductase or both should now be feasible. Such studies should clarify the roles of the two enzymes in sulfate reduction in vivo. Biosynthesis ofggysteine and Methionine After its reduction, sulfide is incorporated into cysteine, and cysteine is the source of sulfur in methionine synthesis. The bio- synthetic routes for cysteine and methionine in plants are generally similar to those found in bacteria (cf. 49). The formation of cysteine involves two enzymatic steps: (a) the activation of the carbon acceptor, serine, to O-acetyl-L-serine, catalyzed by serine transacetylase; and (b) the synthesis of cysteine from the activated serine and a thiol group, catalyzed by O—acetyl serine sulfhydrylase. The formation of methionine from cysteine is carried out via the transsulfuration pathway (cf. 49) and the intermediate cysta- thionine, to form homocysteine which is then transmethylated to methionine. Sulfate Esters and Sulfonic Acids Sulfate activation is required prior to the incorporation of sulfate into sulfate esters and sulfonates as well. Sulfate esters are found among animals and plants (cf. 108), but reports on sulfate esters in bacteria are rather rare (cf. 48,133). Animal tissues contain sulfate esters such as steroids, phenols and polysaccharides (cf. 108). Their formation involves the transfer of the sulfonyl group of PAPS to suitable acceptors, in reactions catalyzed by specific sulfotransferases. 16 The most abundant sulfate ester in plants is choline-O—sulfate. It is found in fungi, algae, and higher plants (132). Additional sulfate esters are the flavonoid sulfates in higher plants (cf. 108), the polysaccharide sulfates in certain algae and Chlorella (cf. 48), and the mustard oil glycosides in certain plant species (131). There are no reports on sulfated polysaccharides in higher plants (cf. 48). Little, however, is known regarding the synthesis of the sulfate esters in plants. Incorporation of [3SSI-su1fate in vivo into poly- saccharide fractions in a red alga (111-111a), Fucus (130) and Chlorella (cf. 48), or into choline-O-sulfate and flavonoid sulfates in fungi, algae and plants (58) have been documented. Involvement of sulfotransferases and PAPS in these syntheses in the plant kingdom has not been proven. However, it seems likely on the basis of comparative biochemistry (cf. 48). Among the sulfonates found in plants, the most important one is the ubiquitous plant sulfolipid (sulfoquinovosyl diglyceride), dis- covered by Benson et a1. (cf. 135). In spite of its relatively high concentrations in green plants, particularly in the chloroplast membrane, the pathway of its biosynthesis is not known. It has been suggested to proceed from PAPS as the sulfonyl donor and phospho- enolpyruvate as the acceptor (136). Properties of ATP Sulfurylase ATP sulfurylase (E.C. 2.7.7.4, ATP:sulfate adenyltransferase) has been detected in bacteria (45,91-92,126,189), animals (157,162, 165), fungi (41,95-96,98—99,140-140a), algae (Cf. 48) and in a wide 17 range of higher plants (45-47,101-105,143-144,156), where it was found in extracts of roots, leaves, shoots and chloroplasts. Highly purified ATP sulfurylase has now been obtained from yeast (99,140-140a), Penicillium (95-96), spinach (45), Nitrobacter (107), and rat liver (162). Enzymes from several additional higher plants (46-47,156) and yeast (98) have been partially purified. These enzymes are all soluble and appear to have a broad pH optimum of 7.5-9.0, specificity toward ATP, and a requirement for a divalent cation, usually satisfied by Mg+2. However, there have been reports that purified ATP sulfurylases will catalyze ATP formation (140a, 166-167), and a pyrophosphate-ATP exchange reaction (45) in the absence of added Mg+2. These activities were attributed to the pre- sumed presence of traces of divalent cations in reagent and enzyme solutions. The enzymes from plant sources are relatively independent of sulfhydryl protection compared with enzymes from Nitrobacter and liver. Kinetic studies with ATP sulfurylase are rather limited and are confined mostly to the reverse reaction and the molybdolysis assay (see pp. 24, 35). Based on the formation of ATP from APS and pyro— phosphate in plants (65,144,156), fungi (96) and bacteria (107), the reported Km's range from 0.3-7.0 x 10.6 M for APS and 0.1-7.0 x 10"5 M for pyrophosphate. Studies based on the sulfate-dependent pyro- phosphate-ATP exchange reaction with enzyme from several higher plant sources (45-47) yielded Km's of 1-5 x 10.3 M for sulfate and 0.25 x 10.3 M for ATP. Km's for the molybdolysis reaction have been studied in yeast (66,98) and Penicillium (96) and are ca. 6 x 10“5 M 18 for MgATP -2 and 2 x 10-4 M for molybdate. Studies based on measurement of accurate initial rates of APS formation have not appeared. 0n the contrary, many investigators commented that APS was detected only in the presence of inorganic pyrophosphatase (e.g., 45,47,156) and that, even in the presence of a large excess of inorganic pyrophosphatase, the rate of APS formation was not proportional to enzyme concentration (e.g., 47,96). In spite of the fact that the rate of APS synthesis by the ATP sulfurylase from spinach continuously declined during the 1 hour incubation period (45,47), Shaw and Anderson (45) used such data to calculate the Km of 8 x 10.3 M for sulfate and 5 x 10.3 M for ATP. A Km for sulfate of ca. 5 x 10-3 M was also presumed for ATP sulfurylase of yeast (cf. 166), based on the overall production of PAPS from sulfate. Reported molecular weights for the highly purified enzymes are: 100,000 for yeast (140a), 900,000 for rat liver (162), 700,000 for Nitrobacter (107), and 440,000 for the Penicillium enzyme (95-96). The latter ATP sulfurylase, which has been extensively characterized (95-96), appears to be an octamer composed of 8 subunits of 56,000 molecular weight each. Additional data suggested that there are l cysteine and 4 cystine residues per subunit. Partially purified ATP sulfurylase from corn (156) and beetroot (46) appear to be of 42,000 and 230,000 molecular weight, respectively. Indications for the existence of isozymes of ATP sulfurylase were found in Furth mouse mastocytoma (165) and beetroot hypocotyl (46), as 2 distinct peaks of activity appeared during ion exchange chromatography. 19 Specific activity values obtained with the highly purified ATP sulfurylases from different sources are comparable for a given assay. The activities obtained with the molybdolysis assay for ATP sulfurylase from yeast (140a) and Penicillium (95) were 42 and 19 umoles pyro- phosphate x min"1 x mg protein-l, respectively. The yeast enzyme studied by Robbins and Lipmann had activity of ca. 1 umole APS x min- x mg protein-1. ATP sulfurylase from yeast (98) and rat liver (162) catalyzes the reverse reaction at ca. 0.65 umoles ATP x min.-1 x mg protein-1. Reports on inhibition by APS of ATP sulfurylase reactions have been published (66,96,98). However, kinetic evidence suggests that this is a simple product inhibition effect due to the unfavorable equilibrium constant of the forward reaction, as opposed to an allo- steric effect (96). The reaction of APS formation by ATP sulfurylase has generally been regarded as a nucleophilic displacement by sulfate ions on the inner phosphorus atom of ATP, resulting in elimination of pyrophosphate (154,163). However, the low nucleophilicity of the SO4 , with the repulsion to be expected from the negatively charged ATP, even in the + presence of Mg 2, led to the suggestion of a more probably alternative mechanism in which enzyme-AMP complex is formed prior to the attack by SO4 (cf. 108). A mechanism, in which formation of an enzyme-AMP complex and pyrophosphate release occur prior to the interaction by sulfate, was suggested for highly purified ATP sulfurylase from rat liver (162). This was based largely on the finding that the enzyme catalyzed the 20 pyrophosphate-ATP exchange reaction in the absence of sulfate ions. This mechanism, consistent with a ping-pong type (Cleland, cf. 65), also led to the prediction of exchange between the sulfur atoms of APS and sulfate. However, this exchange could not be demonstrated in the absence or presence of other substrates (162). In contrast, the few thorough kinetic studies of the highly purified ATP sulfurylases from Penicillium (96), yeast (98) and spinach (65) are all consistent with a sequential type mechanism (Cleland, cf. 65) for the reaction, in which both substrates bind to the enzyme before either product is released. The following data support a sequential mechanism: (a) initial velocity patterns obtained by the molybdolysis assay (96,98) or by the formation of ATP from pyrophosphate and APS (65) showed that the Km for each substrate varies with the concentration of the alternate substrate, and that there is no irreversible step between the addition of the two substrates; (b) initial velocity and dead-end inhibitor studies during the equilibrium conditions of the sulfate-dependent pyrophosphate- ATP exchange reaction catalyzed by the spinach ATP sulfurylase (65) likewise yielded patterns which were consistent with a sequential mechanism: (c) isotope exchange studies demonstrating that the 32? exchange between Ppi and ATP was catalyzed by ATP sulfurylase of spinach (16,45) and Penicillium (96), but only when sulfate was . . 35 present. LikeWise, the S exchange between SO4 and APS catalyzed by the yeast enzyme (98) was completely dependent on the presence of . -2 either pyrophosphate or MgATP . Furthermore, product inhibition studies of both the reverse reaction and exchange reaction, catalyzed by the spinach ATP 21 sulfurylase (65) indicated an ordered sequential mechanism, in which MgATP".2 is the first substrate to react with the enzyme, and MgP207— is the first product released. The forms of sulfate and APS in this proposed sequence of the reaction is not known, but according to this proposal, the PPi—ATP exchange could proceed in the absence of free APS (65). Although this ordered sequential mechanism requires an exchange of 358 between 804-2 and APS as well, this could not be detected under a variety of conditions, for some unknown reason, for the spinach enzyme (65). Such 353 exchange was only reported for the yeast enzyme (98). The conflict in evidence concerning the mechanism of the ATP sulfurylase reaction, obtained with the plant enzymes and the rat liver ATP sulfurylase, is difficult to account for on the basis of organism differences, because sequential addition of substrates to ATP sulfurylase of Furth mouse mastocytoma was indicated (164). Assay for ATP Sulfurylase Activity: Difficulties in Perspective In the past it has been difficult to conduct studies designed to elucidate the regulation of ATP sulfurylase because of the limita- tions of available assay procedures (cf. 108). The most direct method to assay ATP sulfurylase activity is the determination of the rate of APS formation upon incubation of the enzyme with sulfate and ATP. However, there are two major obstacles to this assay method: (a) the very unfavorable equilibrium constant of the forward reaction, of ca. 10-8, which precludes the accumula- tion of more than one or two nmole/ml of APS; this has been found to 22 be true even in the presence of inorganic pyrophosphatase; and (b) the lack of a specific chemical method for direct quantitation of APS (cf. 108,167). As a result, many of the studies of ATP sulfurylase were carried out with assays of the reverse reaction (eq. 6), which the equilibrium constant favors, the products being ATP and pyro— phosphate, for which there are highly specific assays. .1. Mg 2 APS + PPi -———+ ATP + so ’2 (eq. 6) +—— 4 Thus, the extensive characterization of the ATP sulfurylase of yeast, carried out by Robbins and Lipmann (140—140a), employed the reverse reaction in which ATP was measured spectrophotometrically in a coupled system with glucose—6-phosphate dehydrogenase, hexokinase, and NADP, while pyrophosphate was estimated as phosphate released after hydrolysis with inorganic pyrophosphatase. The highly puri— fied enzyme from yeast (140a) exhibited 1:1 stoichiometry between ATP formed and pyrophosphate consumed. In crude extracts of yeast, however, the presence of inorganic pyrophosphatase activity inter- fered with the assay (140). The disappearance of pyrophosphate in the back reaction has been used to assay ATP sulfurylase partially purified from animal sources (157,165). The high level of inorganic pyrophosphatase found in plants (e.g., 45—47,102—104) precludes the use of the pyrophosphate disappearance assay. The hexokinase and glucose-6-phosphate dehydrogenase coupled assay of ATP production was used in studies of ATP sulfurylase purified from Penicillium (95,96). Assays based on the reverse reaction in which ATP forma— tion was followed by the bioluminescence generated by the 23 ATP-dependent luciferin-luciferase system (of the firefly) were carried out in studies of ATP sulfurylase of Nitrobacter (144), spinach (107,158), corn (156) and enzyme from animal sources (162, 165). Alternatively, the formation of [32P]-ATP from [32F]— pyrophosphate was used for the spinach enzyme (65). Assays based on ATP formation are not, however, suitable for crude extracts from plants, because of their high ATPase activity (e.g., 43,45-47, 95,102-103,158). In their early studies with the yeast ATP sulfurylase, Robbins and Lipmann (l40-l40a) also measured the disappearance of APS by the reverse reaction (eq. 6). Their assay method was based on the enzymatic conversion of APS to PAPS which is then measured by a second enzymatic assay. The latter involved the enzymatic transfer of the activated sulfate group from a carrier to a phenol acceptor, catalyzed by a phenyl sulfotransferase. In both the direct transfer (159) and the catalytic PAPS-PAP assay (160) described in eq. 7 and eq. 8, respectively, PAPS is measured by the colorimetric disappear- ance (eq. 7) or appearance (eq. 8) of p—nitrophenol. p-Nitrophenol + PAPS -—-—+ PAP + p—Nitrophenyl—sulfate (eq. 7) p-Nitrophenyl-sulfate + Phenol ) p-Nitrophenol + Phenyl—sulfate _P___., (or PAP (eq. 8) This indirect APS determination is, however, not suitable for assay- ing APS formed or consumed by crude preparations of ATP sulfurylase from which APS kinase and the transferase activities have not been removed. Also, the conversion of APS to PAPS upon addition of APS kinase would be difficult to accomplish in the presence of enzymes (ll [.1 ll. 1 III. 24 which degrade PAPS (see above), as was noticed in extracts from liver (140a). Therefore, this method for APS determination was not used in subsequent studies of ATP sulfurylase. The equilibrium barrier of the forward ATP sulfurylase reac- tion can alternatively be overcome by assay methods based on an exchange reaction. An ATP sulfurylase assay based on the sulfate- dependent exchange of [32P]-pyrophosphate into [32P]-ATP was used by Bandurski and Wilson (41,43) for the yeast enzyme, and recently was adapted by Anderson's group (161) for studies of ATP sulfurylases from higher plants (44-47,65). In addition to the interference by ATPase and pyrophosphatase, which excluded the use of this assay in unpurified extracts (45-47), this assay requires the separation of ATP from pyrophosphate. Under the conditions used by Anderson et a1., they could not detect exchange of 35S between $04-2 and APS (65), nor was the exchange of [32P]-pyrophosphate into [32Pl-ATP always associated with formation of an isolatable adenylate-anion mixed anhydride (47). Consequently, there is some question as to whether or not the enzymes of Anderson et a1. actually catalyzed APS synthesis under their exchange assay conditions. Formation of the mixed anhydride need not occur if the ping-pong mechanism is correct. The molybdolysis assay developed by Wilson and Bandurski (41, 43), in which molybdate is used as an analog of sulfate, is perhaps the simplest assay of ATP sulfurylase. Unlike the assays described above, ATP sulfurylase activity is measured in the forward reaction, as molybdate-dependent pyrophosphatase (or phosphate) released, 25 presumably via [APMo], a hypothetical unstable analog of APS. This assay has been widely used in studies of ATP sulfurylase (e.g., 91, 93,96-98,102). A high ATPase activity in plants (see above) has been a major obstacle to the use of this assay. Additional diffi- culties releated to non—enzymatic phosphate release and other molybdate effects have been reported (e.g., 75,98,190). None of the assays mentioned above is suitable for measuring the physiologically significant reaction, APS formation, in enzyme pre- parations. With an equilibrium constant of ca. 10-8, an assay method is required to detect and quantitate pmole amounts of APS. The enzyme coupled assay for APS developed by Robbins and Lipmann (140- 140a) was, unfortunately, not sensitive to concentrations of APS below 5 nmoles/ml (140) and, therefore, was not suited for assaying APS formed from sulfate, even with the highly purified ATP sulfurylase of yeast (140a), since the equilibrium concentration of APS did not exceed 2 nmoles/ml. The required sensitivity for assaying such low concentrations of product can be achieved by assaying the incorporation of [35S]- $04”2 into [3SS]-APS. This radioisotope assay unfortunately has not been employed in detailed kinetic or regulatory studies of ATP sul- furylase, because of the lack of a method for rapid separation of the excess [355]»504-2 from [BSSl-APS in large numbers of samples. The techniques which have been used in the past for such a separation were preparative chromatography and electrophoresis (e.g., 41,104, 156). These methods are, nevertheless, laborious for use in puri- fication, kinetic or regulation studies in which rapid performance 26 of multiple assays is needed. Separation of the [3ssl—APS from [35$]-sulfate by adsorption to charcoal was also used in conjunction with the radioisotope assay (e.g., 45,96). This, however, was not sensitive enough to detect the small amounts of APS formed (45,96). Also, many investigators indicated that the charcoal treatment caused a marked degradation of APS (cf. 69). As a result, the formation of [3SS]-APS has usually been used only for the rigorous identification of APS as the product of a putative ATP sulfurylase reaction, and one of the more convenient assays (such as molybdolysis, the reverse reaction, or pyrophosphate-ATP exchange reaction) has then been used in subsequent studies (e.g., 45,95-96,98,107,l44). A radioisotope assay has been used most frequently for studies with crude extracts of bacteria (e.g., 126,151,193) where the [355]- . 3 . 3 . $04 is incorporated into [ 5Sl-PAPS Via [ 5Sl-APS (154). This, however, measures the combination of ATP sulfurylase and APS kinase activities. Control of the Sulfate Assimilation Pathway Evidence based on biochemical and genetic studies indicated that in bacteria and fungi the sulfate assimilation pathway is regulated in response to specific changes in the availability of environmental sulfur. Studies carried out by Dreyfuss, Monty, and Kredich and Tomkins in Salmonella typhimurium (cf. 126), and by Pasternak, Ellis, Wheldrake, and Jones-Mortimer in E. coli (cf. 185), showed that the enzymes involved in sulfate assimilation are subject to both negative and positive control. Cysteine, the end product of the pathway, is an allosteric inhibitor of both the sulfate permease and serine 27 transacetylase. In addition, all the enzymes which catalyze the assimilation of sulfate to cysteine are subject to repressiona control by cysteine. The syntheses of these enzymes which are dependent upon a decline in the intracellular concentration of cysteine, also require O-acetylserine (OAS) which functions as an internal inducer, and an intact cys B region (126,185), which appears to code for a protein (188). The nature of the interactions between OAS and the cys B gene product is not known, nor is the molecular mechanism of the cysteine-mediated repression. The activity of ATP sulfurylase in E. coli (189-190) and Salmonella (126) is repressed by growth on cysteine and derepressed by slow growth on djenkolate or glutathione sulfur compared with growth on sulfate. If sulfate or cysteine is added with glutathione or djenkolate, the ATP sulfurylase level remains repressed (189). The sulfur sources do not appear to act by inhibition or activation of the enzyme. This is evident from assays of mixed extracts from repressed and derepressed cells (126), as well as by the lack of effect of sulfur compounds when added to ATP sulfurylase in vitro (189). Thus, the changes in the level of ATP sulfurylase activity in response to the sulfur source are likely to involve regulation of the synthesis of the enzyme (126,189). There is evidence that in aThe terms, "induction", "repression", and "derepression" are used in this dissertation to describe changes in rates of development of total extractable ATP sulfurylase activity. It should be under— stood that the molecular bases of these changes are not yet known, either in the tobacco cell system or in any other system so far described in the literature. Thus, throughout the dissertation, changes in ATP sulfurylase activity should be considered cases of apparent induction, repression or derepression. 28 these bacteria cysteine (or a close derivative) is the corepressor and the activity of ATP sulfurylase is inversely related to the intracellular concentration of cysteine (193). The slower growth of these bacteria on glutathione or djenkolate compared with sulfate or cysteine (126,189,191-192) indicated that the ability to derepress enzymatic activity by these sulfur sources was probably due to their slower conversion into cysteine. This is in accord with the concept developed by Moyed and Umbarger (cf. 191) that slow growth of an organism on a certain nutrient may be the result of the low rate of its conversion into a repressing metabolite, and consequently may be associated with a derepression of the enzymatic pathway leading to this metabolite. Djenkolate was selected originally by Dreyfuss and Monty (191-192) because of the characteristic slow growth of Salmonella on it as a sulfur source. L-Djenkolic acid (L-cysteine thioacetal of formaldehyde) is, however, of plant origin and was first isolated from the Djenkol bean (cf. 194). It is among the many cysteine derivatives which occur naturally in plants as non-protein amino acids (195). Neither its function nor its metabolism in plants is known. The assimila- tion of the djenkolate sulfur in Salmonella, though not known, was not via inorganic sulfur intermediates because it supported growth of mutants defective in the sulfate assimilation pathway (192). In both E. coli and Salmonella, methionine, which is synthe— sized via cysteine, does not appear to be involved directly in the regulation of the enzymes of sulfate assimilation (196). Changes in the level of ATP sulfurylase specific activity, in response to sulfur nutrition, have been also noticed in several fungi, 29 such as yeast (24-27,66-67), Aspergillus (129), Penicillium (96), and Neurospora (75). Among the sulfur compounds tested, neither cysteine, methionine, glutathione nor choline-O-sulfate produced any effect on the activity of these enzymes in vitro (66,96,98). Sulfide was the only sulfur compound found to inhibit ATP sulfurylase activity in vitro in yeast (66,98) and Penicillium (96) in a way which suggested its being an end-product inhibitor of the enzyme. In Aspergillus, a decrease in ATP sulfurylase activity of 30-60% was observed in the presence of L-cysteine or L-methionine in the growth media (129). Studies with mutants, however, indicated that cysteine rather than methionine is mediating the repressive response (129). On the other hand, in yeast (66) and Penicillium (96), methionine was found to be the only repressive sulfur source, since growth of these fungi on cysteine resulted in an actual increase in ATP sul- furylase specific activity compared with growth on sulfate. Examina— tion of the studies in yeast (66) may account for these surprising results, because cysteine was a rather poor sulfur source to support growth of yeast compared with sulfate and methionine. Thus, the derepression of ATP sulfurylase by cysteine in yeast may have come abount by mere sulfur starvation. No growth data for the various sulfur sources tested in Penicillium were reported (96). But neither growth on djenkolate, nor sulfur starvation, had a strong derepres- sive effect on the ATP sulfurylase level (96), although both are very effective in derepressing the sulfate uptake system of Penicillium (40,71). It is therefore likely that the growth of this fungus in 30 this study (96) was not completely dependent on the sulfur source added. Nevertheless, the conjectured conclusion that ATP sulfurylase activity in yeast is repressed by methionine and derepressed by cysteine (66) was interpreted by De—Robichon-Szulmajster and co— workers (cf. 23) to mean that in yeast, methionine rather than cysteine is the regulator of the sulfate assimilation pathway. That laboratory also developed the concept that in yeast, methionine synthesis is independent of the cysteine formation and linked directly to the sulfate reduction pathway by the direct sulfhydra- tion pathway (23). This conclusion was recently refuted by Flavin et al. (cf. 49), who have shown that methionine is formed via the transsulfuration pathway, i.e., via cysteine, in yeast as well. Nevertheless, the regulation by methionine and its derivatives of the sulfate assimilation pathway in yeast has been reported in many studies carried out by De-Robichon-Szulmajster's group. They presented an elaborate scheme for a 2-level control of 4 different enzymes (methionine Group I enzymes) including ATP sulfurylase, sulfite reductase and 2 enzymes of the direct pathway of methionine biosynthesis (24-28,67). The activities of methionine Group I enzymes were reported to be coordinately repressed by the addition of either methionine or S-adenosylmethionine (SAM) to the media, and coordi— nately derepressed by methionine-limited growth (24-28,67). According to their scheme, the mechanism of the methionine-mediated repression of these enzymes is distinct from the SAM-mediated response (26,67). The former involves binding of a "regulatory methionyl-tRNAmet" with aporepressor protein to form aporepressor- 31 corepressor complex which is hypothesized to act as a repressor at the transcriptional level (25,27). On the other hand, SAM is pro- posed to cause repression by acting upon the translational level (26,28,67). It is my opinion, however, that these interpretations of their data should be taken with caution, because none of the regulatory elements has been rigorously identified. It is never— theless apparent from their results that ATP sulfurylase in yeast is indeed subject to regulation by sulfur nutrition as was shown earlier by De-Vito and Dreyfuss (66). However, conclusions regard- ing the mechanism of this control must await further studies. Additional complications are evident from studies with Neurospora, by Metzenberg, Marzluf and co—workers (cf. 197). Although the process of sulfur entry into the fungal mycelia is regulated by a complex system of both positive and negative signals, in response to sulfur availability, ATP sulfurylase activity responded differently (75,197). Conditions of sulfur sufficiency (high methionine),which strongly repress the activity of the sulfate permeases of Neurospora, increased the activity of ATP sulfurylase (75), while sulfur-starvation, which increases the sulfate transport rate, inhibited development of the ATP sulfurylase activity. Limited studies with higher plants are likewise confusing. There is evidence indicating that the sulfate transport system of higher plants is subject to regulation by sulfur nutrition. Hart and Filner (123) showed that both cysteine and methionine act as end-product inhibitors of the sulfate uptake in tobacco cells. Smith (124) extended this study to demonstrate an increase in the rate of 32 uptake upon sulfur starvation. Higher rates of sulfate transport upon sulfur deficiency were reported earlier for clover plants (128). Nevertheless, studies carried out by Ellis (104) with several higher plants, including Lemna, grown on either sulfate, cysteine, methionine or glutathione, failed to yield expected changes in ATP sulfurylase activities in response to the various conditions of sulfur nutrition. The activities observed after growth in the presence of these organic sulfur sources were some- what higher than in plants utilizing sulfate. These results are difficult to account for because, in the Lemna cultures, unlike the seedlings, growth was dependent upon the sulfur source added. But the activity which developed upon sulfur starvation, or slower growth on cysteic acid, were not tested. Therefore, Ellis' conclu- sion (104) that ATP sulfurylase activity in higher plants is not repressed by cysteine or methionine, does not exclude the possi- bility of its regulation in response to sulfur-limited growth. Indi- cations that sulfur nutrition influenced the ATP sulfurylase activity came from preliminary studies in developing soybean seedlings (103), where a 2- to 2.5-fold increase in activity was found in leaves of plants that grew in the absence of sulfate in the nutrient solution. These changes were detected after many days, suggesting that an endogenous supply of sulfur existed. Therefore, studies of changes in enzyme levels by nutritional factors need to be carried out only under strictly controlled nutritional conditions. 33 Group VI Anions and the Sulfate Transport System Oxygen, sulfur, selenium and tellurium constitute Group VIA of the periodic table of elements. Chromium, molybdenum, and tungsten make up Group VIB. There is abundant evidence in the literature that sulfate, selenate, molybdate, tungstate and chromate are all actively taken up by a common permease, i.e., the sulfate transport system in bacteria, fungi, algae, and higher plants. Sulfate transport is inhibited specifically by one or more of the Group VI anions in, e.g., E. coli (89), Salmonella (77), Neurospora (74,84), Chlorella (39,81), Euglena (88), Scenedesmus (109) and higher plants (38). The inhibition of sulfate uptake by one or more of these anions was demonstrated to be competitive in Salmonella (78), Aspergillus and Penicillium (40), Neurospora (74) , Chlorella (39,81) , the red alga Porphyridium (111), and barley roots (36-37,85). The transport of the Group VI anions in general has the same dependencies, sensitivities, etc., as sulfate uptake in the above described systems, and Km's and transport rates similar to those for sulfate have been observed (40,74,81). Furthermore, the transport system for selenate, molybdate, or chromate of Salmonella (78), Aspergillus and Penicillium (40), Néurospora (74) or Chlorella (83) is repressed by the same sulfur sources that repress the active transport system for sulfate. 0n the other hand, under conditions which derepress the sulfate transport system, a coincident derepres- sion of transport of the Group VI analogs occurs. In addition, 34 mutants of Penicillium (40), Neurospora (74), Aspergillus (76) and Salmonella (77-78), defective in sulfate transport, were equally defective in transporting the Group VI analogs. Other studies indi- cated that Group VI anions are also analogs of sulfate in the processes of efflux of internal sulfate in Neurospora (79) and the transinhi- bition of the sulfate permease in Penicillium (72), both of which are believed to be mediated by the sulfate transport system. The toxicological effects of Group VI anions often observed in both microorganisms (39,73-74,76-77,89,121) and higher plants (cf. 33) have been attributed to their entry via the sulfate transport system, since resistance to the toxic anion(s) was seen: (a) in mutants lacking the sulfate transport system (74,76,78); (b) during growth in the presence of sulfur sources which repressed the sulfate permease (74,78,89,121); and (c) by increasing the [sulfateJ/[anion] molar ratio in the growth medium (39,60,78, cf. 33). Under all these con- ditions resistance to an anion involved its exclusion from the cells. The particularly strong toxic effect of chromate was utilized by Pardee and co-workers (77) in studies with Salmonella, and later by Marzluf's group (73-74) with Neurospora, for selecting mutants lack- ing the sulfate transport activity, which are resistant to chromate. Interactions of Group VI Anions with ATP Sulfurylase The concept that Group VI anions can function as structural analogs of sulfate in reactions catalyzed by ATP sulfurylase developed out of the studies by Wilson and Bandurski (41-43) with ATP sulfurylase partially purified from yeast. The enzyme acted upon either sulfate, selenate, molybdate, chromate or tungstate as substrates. The nature 35 of the reaction, however, varied with the added anion. In the .presence of molybdate, chromate, or tungstate, no stable mixed anhydride of adenylate and anion was formed, but the enzyme cata- lyzed an anion-dependent cleavage of ATP to AMP and pyrophosphate. In contrast, with sulfate or selenate as substrates, only small amounts of pyrophosphate were formed. Both, however, supported an anion-dependent exchange reaction of [32P]-pyrophosphate with ATP, which was not observed with the Group VIB anions. Indications that the seleno-analog of APS was formed by the yeast enzyme were pro- vided (41), but the synthesis of APSe has still not been established rigorously. The anion dependent release and accumulation of pyrophosphate from ATP was documented also for ATP sulfurylase of spinach (101) and bacteria (91). Using ATP sulfurylase prepared from several higher plants, Anderson and co-workers (44-47) have verified that only selenate will substitute for sulfate in the [32Pl-PPi/ATP exchange reaction. Based on their finding, Wilson and Bandurski (41) developed the so-called molybdolysis assay for ATP sulfurylase: measurement of molybdate-dependent release of phosphate from ATP, in the presence of ATP sulfurylase and inorganic pyrophosphatase. This assay has played a major role in many studies of ATP sulfurylase. Also, out of these studies came the recognition that Group VI anions are specific inhibitors of reactions catalyzed by ATP sulfurylase. The molybdolysis assay of ATP sulfurylase has been employed in studies of the enzyme isolated from bacteria (91-92,97), animals 36 (93-94), fungi (41,66-67,95—96,98—100), algae (cf. 48) and higher plants (97,101-103). The molybdate-dependent release of pyro- phosphate was inhibited upon addition of sulfate (41,66,96,98,102). Group VI anions also inhibit other reactions catalyzed by ATP sulfurylase. Thus, molybdate, selenate, tungstate and chromate inhibited the incorporation of [35$]-sulfate into APS (47,104,107) and PAPS (105-106) by enzymes isolated from Nitrobacter (107), higher plants (47,104-105) and the alga Ochromonas (106). Molybdate and selenate inhibited the sulfate-dependent pyrophosphate exchange reaction (45) catalyzed by ATP sulfurylase purified from spinach leaves. Anions of Group VI also inhibited sulfate reduction by extracts of bacteria and yeast (cf. 108) and the incorporation of sulfate in vivo into an organic cell constituent in algae (111), where sulfate uptake is not affected. These effects were attributed to the inhibition by the anion of ATP sulfurylase activity. Among Group VI anions, molybdenum is the only one, in addition to sulfur, for which a nutritional requirement of plants has been established conclusively (116). Molybdenum is a constituent of molybdo-enzymes such as nitrogenase (120), nitrate reductase (117) and others (cf. 117). However, there is no evidence which suggests that molybdate needs to be acted upon by ATP sulfurylase in order to perform its function in these enzymes. Chromium, tungsten and tellurium are not regarded as nutrients in plants, and they do not appear to be further incorporated into analogs of sulfur-compounds, via ATP sulfurylase (113,118-119). In contrast, the incorporation of selenate into organo-seleno 37 compounds is widely known in various organisms, including plants. Since 1880, when the concept of a biological analogy between sulfur and selenium was expressed by Cameron (cf. 33), it became increasingly evident that the sulfate assimilation pathway has a key role in selenium metabolism. Selenium: Micronutrient Role in Bacteria and Animals The discovery of selenium deficiency syndromes in both animals (7) and bacteria (2) indicated a functional role for selenium during normal growth and development of these organisms. The essentiality of selenium in these systems became evident as several distinct selenium-containing proteins have been identified. This includes the formate dehydrogenase (1,3-4,13) and Protein A of the glycine reductase (l) of bacterial origin, glutathione peroxidase of animal origin (5-6,14-15) and several others (cf. 1,16-17). As indicated by the deficiency symptoms, selenium has an indispensable role in dtermining the activity of these proteins. The selenium of the functional selenoproteins is thought to represent a unique organo- selenium compound (l,14,l7), and a carrier role in electron transfer reactions has been postulated (cf. 1,4,6,l4). Nevertheless, the exact chemical nature and the function of the seleno groups in these proteins remain to be elucidated. The occurrence of specific proteins with a high content of selenium (1,6,13,15) reflects a very selective incorporation process which functions with selenium in the micromolar range of concentra- tions (1,3-4,6,15). This unique distribution pattern necessitates 38 the existence of at least one selenium-specific step (possibly branching off of the sulfate pathway). At high concentrations, however, selenium becomes very toxic and leads to inhibition of growth and metabolic disorders in animals (12) and bacteria (11). The biochemical events underlying the utilization of selenium as a required nutrient, though unknown, are likely to be, at least partially, different than those involved in selenium toxicity. The latter is thought to be a result of usurpation of the sulfur path- way and nonselective replacement of sulfur by selenium that causes lethality. Selenium Metabolism in Plants: Selenium Accumulators The pioneering work of Beath et al. (cf. 31), after the impli- cation of selenium as the toxic agent of certain range plants that caused livestock disorders, led to the concept of selenium indicator plants. This small group of plants, representing only several species of a few genera, are restricted in their distribution to seleniferous soils from which they extract extremely high levels of selenium and accumulate it in organic compounds. Selenium accumulator plants characteristically contain up to 10,000 times more than the few ppm of selenium found in most plants. Moreover, whereas selenium is toxic to most plants at very low concentrations (see below), no harmful effects were detected in the selenium accumulators (31,33). Nevertheless, a micronutrient role for selenium in the accumulator species is still a matter of conjecture (cf. 32-33). 39 Biochemical studies indicated that the accumulator plants, in contrast to bacteria and animals, contain selenium exclusively as low molecular weight soluble organo-seleno compounds, with negligible traces in the protein fraction (34-35). The soluble compounds were identified as seleno-amino acids, of the non-protein type, and the low molecular weight peptide derivatives (32-33). In general, as was pointed out by Shrift (cf. 32-33), the dominant species was Se-methylselenocysteine accompanied by selenohomocysteine, seleno- cystathionine, y-L-glutamyl-Se-methylselenocysteine and several other unidentified peptides. In non-accumulator plants, however, exposure to selenium resulted in a predominant incorporation of the element into the protein fraction, presumably as selenocysteine and selenomethionine (34-35). Se-methylselenomethionine was the most abundant soluble compound in non-accumulator plants with little or no trace of the soluble seleno-compounds characteristic of the accumulator plants (32-33). These studies indicated two important features of selenium metabolism in plants: (a) that there are physiological and bio- chemical differences between accumulator and non-accumulator plants with respect to selenium; and (b) in both classes, selenium is incorporated exclusively into analogs of sulfur compounds, known to occur in plants. The Relationship Between Selenium Metabolism and the Sulfate Assimilation Pathway The recovery of selenium in plants as reduced seleno-amino acid derivatives, all of which are sulfur analogs, suggested that selenate 40 and sulfate are likely to be coassimilated via a common pathway (of. 33). Although it is still a matter of contention, both enzymatic studies in vitro and feeding experiments in vivo support this hypothesis. Evidence that selenate is transported by the sulfate permease in many systems, including accumulator and non-accumulator species (38), was presented in the previous section. It is also conceivable that selenate is activated by ATP sulfurylase (see above). Anderson and co-workers (44-47) compared the activities of ATP sulfurylase purified from several accumulator and non-accumulator plants, with either sulfate or selenate as a substrate in the pyrophosphate-ATP exchange reaction. Their results can be summarized as follows: (a) selenate replaced sulfate as an alternative substrate in all the 8 enzymes tested; (b) the kinetics of sulfate-selenate competition were consistent with 2 substrates competing for a single enzyme; (c) both the selenate and sulfate activities were inseparable, and maintained a constant ratio during purification; (d) the Km for selenate in all the enzymes examined was consistently lower than the Km for sulfate: (e) the Km's of ATP sulfurylase for selenate and sulfate in accumulator plants were almost identical to those found in the non-accumulator species. The same was true for the Vmax values of the two anions; (f) the ATP sulfurylases of accumulators and non-accumulators were very similar in all other aspects studied. These results, however, are based on the ability of selenate to participate in the pyrophosphate exchange reaction only. Attempts to demonstrate aux APSe formation from selenate, with enzyme from 41 either accumulators or non-accumulators, so far have been unsuccessful (47). This was not due to the formation of an unstable, rapidly hydrolyzed mixed anhydride of AMP and anion, as AMP was not detected either. An enzyme-bound APSe was hypothesized by the authors as compatible with the proposed mechanism for ATP sulfurylase (65). Based on these findings, it appears likely that selenate can enter the sulfate assimilation pathway via ATP sulfurylase. Unfor- tunately, nothing is known about the subsequent reactions which pre- sumably involve the reduction of selenate to the selenide level. The predominant incorporation of labeled selenate, in a variety of plant species, into the seleno-analogs of cysteine (34-35,52), methionine (34-35,50,52-53,62), S—methylcysteine (51,62), S-methyl- methionine (50-51), and the identification of the seleno-analogs of cystathionine (35,55,57,61) and homocysteine (SS), in plants that actively metabolize selenium, suggested that the seleno-amino acids are synthesized in parallel with cysteine and methionine (cf. 49), possibly catalyzed by the same enzymes. Studies with labeled precursors, in which the in vivo formation of Se-methylselenocysteine (the dominant seleno-product among selenium accumulators) was compared with the synthesis of the cor- responding S-methylcysteine, also pointed to the likelihood of a common enzymatic path. Thus, the in vivo incorporation of radioactivity from carbon precursors (56), selenium precursors (55,57) and methyl donors (55-57) into Se-methylselenocysteine were all consistent with the synthesis of S-methylcysteine by subsequent methylation of the preformed 42 cysteine (55-57). The in vivo studies further indicated that seleno- methionine participated as methionine in both, as methyl donor and as methyl acceptor for the transmethylation enzyme (55,57). The latter was also shown in vitro, where selenomethionine was activated, as a substrate that is competitive with methionine, by the methionyl adenosyl transferase of yeast (68, cf. 1). Plants, however, have a wider range of sulfur-containing com- pounds (48,108), some of which are believed to be synthesized by a pathway separate or branched from the sulfate reduction pathway (48). Attempts to show the incorporation of selenate into seleno-analogs of either choline sulfate, flavonoid sulfates, sulfolipids (58), cnrglutathione (50-51) in several lower and higher plants, including selenium accumulators, were unsuccessful. The synthesis of PAPSe by the yeast enzyme (58) could not be demonstrated either. But recently, the seleno-analog of the mustard oil glucoside, sinigrin, was identified in non-accumulator plants (63). Selenium: Resistance and Toxicity The ubiquity of selenium toxicity throughout the plant kingdom (31,33) is believed to result also from the lethal incorporation of the seleno-analogs into cell protein (32-33). Various sulfur com- pounds are, therefore, able to counteract the toxic effect of selenium (33). The selenium accumulator plants, however, exhibit a unique resistance to selenium. The exact relation of the seleno-compounds found in accumulator species to the resistance phenomenon is not known. However, Peterson and Butler suggested (35) that the accumu- lation of seleno products in these plants represents a detoxification 43 mechanism, whereby selenium is shunted into certain innocuous, non- protein seleno-amino acids, as a step in a mechanism for exclusion of selenomethionine and selenocysteine from protein. This mechanism, which probably involves more than one step (35), is thought to have evolved in the accumulator plants and could be a factor in the tolerance of these species to high levels of selenium. In the absence of such a mechanism in non-accumulator plants, the incor- poration of selenium into protein amino acids (34-35) would result in lethal synthesis of non-functional protein and consequent death (35). This hypothesis is compatible with numerous physiological and biochemical observations, as indicated above, but the crucial comparative studies of the specific enzyme systems which were postu- lated to play a role in the detoxification mechanism of accumulator plants (35) have not yet been done. For example, the suggestion of Peterson and Butler (35) that the specificity of the amino-acid activating enzymes of accumulator species might participate in the exclusion mechanism of the seleno-analogs of cysteine and methionine from protein has not been investigated. This proposal is especially attractive because: (a) an example of differentiation between a natural amino acid, proline, and its toxic analog, azetidine-Z- carboxylic acid, is afforded by the prolyl-tRNA synthetases of certain species which differentiate between proline and the analog, whereas the synthetases of other plants are unable to differentiate between the two; the proline analog is toxic only to those species in which no discrimination against the proline analog occurs (Peterson and Fowden, cf. 44); and (b) the activation of selenomethionine was 44 shown to occur by the methionyl-tRNA synthetase, with the met-tRNA as an acceptor, purified from E. coli (20), rat liver (21), and more recently from yeast (68), all organisms which incorporate seleno- methionine into protein, and are subject to selenomethionine toxicity. MATERIALS AND METHODS Separation of Sulfate and APS by Differen- tial Solubility of the Two Compounds in Ethanol-Water Mixture The method for the separation of APS from sulfate (Figure l on p. 59) is based on the relative insolubility of Na2804 in ethanol: water (5:1) compared to NaZAPS. Ice cold absolute ethanol, 2.5 m1, is added to 0.5 ml reaction mixture containing 20 mM [3SSI-NaZSO4 (ca. 108 cpm), and [BSSI-APS in the range of 10 pmoles-lO nmoles (up to 9 x 104 cpm). The precipitate (PPT) of [3SSJ-Na 804, which 2 forms immediately upon the addition of ethanol, is then removed by centrifugation at 45,000 xg for 10 minutes (Sorval SS-34 rotor), and then discarded. The resultant supernatant fraction is then subjected to three additional precipitations, each consisting of the addition of 200 nmoles of carrier NaZSO4, in 0.2 ml water, followed by centrifugation at 45,000 xg for 10 min and discarding of the precipitate. [3 Sl-APS is recovered in the final supernatant solution (Figure 1). All steps were carried out at 4 C using 15 ml Corex tubes. In experiments designed to follow the efficiency by which sul- fate was precipitated during the separation technique (Figure 1), the four successive precipitates were dissolved in water and aliquots 35 . . . . . were taken for the [ S] determination by seintillation counting. 45 46 When the recovery of [BSSl-APS in the final supernatant frac- tion after the 4 precipitation steps (Figure l) was investigated, radiochemically pure [3SS]-APS of high specific activity (see below) was used. This [35S]-APS was added in 0.05 ml to the complete reaction mixtures free of [3SS]-NaZSO4 at the end of the incubation period, just prior to the addition of ethanol. Upon completion of the 4 steps of separation (Figure l), the [358] derived from the added [3SS]-APS was determined in the 4 precipitates and the final supernatant solution by scintillation counting. The identity of the ethanol-soluble [358] as [3SSJ-APS was verified by various methods (see below). Determination of Radioactivity Aliquots of 0.05-0.3 ml were pipetted into scintillation vials. Radioactivity was determined by liquid scintillation counting (Beckman LS-133), with 10 ml per vial of scintillation fluid pre— pared according to Formula II of Research Products International Corporation: 100 g of naphthalene, 5 g of PPO, 0.3 g of dimethyl POPOP, 730 ml of dioxane, 135 ml of toluene and 35 ml of absolute methanol. Formula II scintillation fluid has a high water compati- bility, up to 15%, and gave a 96% counting efficiency for [358] and [Me]. Paper Electrophoresis Samples of 10-100 ul were run on 45 x 6 cm strips of Whatman 3MM paper in a 0.1 M solution of sodium acetate-NaOH (pH 4.5) at 10 v per cm for 5 hr at 5 C, using a flat bed electrophoresis 47 apparatus (E-C Apparatus Corporation). Nucleotides were visualized by their fluorescence quenching at 254 nm. Radioactivity was located with a Radiochromatogram Scanner (Packard Model 7200), and quanti- tated by cutting the paper into 0.5 cm segments which were placed in scintillation vials and immersed in 10 ml Formula II scintillation fluid, for scintillation counting. Thin Layer Chromatography Aliquots of 5-10 ul were applied to 20 x 2.5 cm thin layer plates precoated with either cellulose or polyamide 6 (Baker-flex, J. T. Baker Chemical Company). The plates were developed in n- propanol:ammonia:water (8:2:3) at 25 C according to Schmidt (146). Nucleotides and radioactivity were determined as described above for paper electrophoresis. Silica gel coated plates, both commercial and laboratory made, were unsatisfactory because a radioactive breakdown product always formed during the run. The Cultured Tobacco Cell System The XD line of tobacco cells used throughout these studies was derived from stem pith of Nicotiana tabacum L.cv.Xanthi (178). Sterile cultures were maintained continuously in a chemically defined liquid medium, MID, containing 2.5 mM nitrate as the sole source of nitrogen and 3.0 mM sulfate as the sole source of sulfur (178). Cells were grown in 500 ml shake cultures at 28 C. Subcultures were started by diluting an aliquot of a 12- to 16-day-old culture 20-fold into fresh media. 48 For the in vitro studies, i.e., the development of an assay for ATP sulfurylase, and characterization of the enzyme activity, extracts were prepared from tobacco cells which had been grown on MID for 3-5 days. In experiments in which the regulation of the development of ATP sulfurylase in vivo was investigated, the cells were grown on various modified MID media. Special Modified Media Media lacking both nitrogen and sulfur [(N,S)- MID] were pre- pared by replacing the nitrate and the sulfate of the MID media with the corresponding chloride salts, as described earlier (123,179). In addition, the anionic sulfur which contaminates the reagent grade sucrose was removed by passage through an anion exchange resin, Dowex AG 2X8 prior to use in the culture media (123). The following compounds were used, or tested, as nitrogen sources: 2.5 mM potassium nitrate, 3 mM urea (180), 0.05-0.1% casein hydrolysate (vitamin free) (179), and a mixture of 15 amino acids (L—alanine, L-arginine, L— aspartate, L-glutamate, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-proline, Lpserine, L-threonine, L- tryptophan and Levaline) each at 0.1 mM. To meet the sulfur requirement for growth, the following sulfur containing compounds were used, each at 0.1 mM: sodium sulfate, L-cysteine, L-methionine, glutathione (reduced), and L-djenkolic acid. The addition of L—methionine to cells growing on nitrate was always accompanied by equimolar L-arginine in order to prevent the 49 inhibition of growth and development of nitrate reductase activity caused by methionine (179). Nitrogen and sulfur compounds were added to the media as aliquots of sterile stock solutions by a proper dilution. Stock solutions of 0.5 M KNOB, 0.01 M Na $04, 10% casein hydrolysate and 5 mM L-djenkolic 2 acid were sterilized by autoclaving. Stock solutions of 0.01 M of amino acids and 0.3 M urea were sterilized by filtration through Millipore GS filters. In studies of the effects of Group VI anions in vivo, aliquots of the following autoclaved solutions were added to media supplemented with nitrogen and sulfur: 0.1 M Na2W04-2H20, 0.1 M NaZCrO4-4H20, 0.1 M NazMoO4-2H20, 0.025 M NazTeO4-2H20 and 0.1-0.0005 M Na25e04. Solutions of 5 mM seleno-DL-methionine and 1 mM seleno-DL-cystine were sterilized by filtration prior to their addition to the media. Equimolar L-arginine was added with the methionine analog. Media and supplemented solutions were adjusted to pH 6.0-6.3 prior to sterilization. Maintenance of Tobacco Cells on the Modified Media Cells for studies of ATP sulfurylase activity in vivo were routinely maintained on (N,S)- MID supplemented with the following mixtures of compounds: 2.5 mM KNO3 plus 0.1 mM Na2504, 3.0 mM urea plus 0.1 mM Na $04, or 0.05—0.1% casein hydrolysate. Experiments 2 were started by either subculturing or by sterile harvesting, wash- ing and resuspending (181) stationary phase cells (in which both the nitrogen and the sulfur sources had been completely exhausted) into 50 fresh (N,S)- MID in which the nitrogen supplement was unchanged, and the sulfur source was varied. Growth of Seedlings Seeds of pea, tomato or cucumber were surface sterilized with 1% sodium hyphochlorite (commercial bleach diluted 5-fold with dis- tilled water), rinsed with sterile distilled water, and germinated under sterile conditions on a Whatman no. 1 filter disc in 400 ml beakers covered with petri dishes. The seeds were supplied every few days with autoclaved nutrient solution. The nutrient solution con- 0, 590; KH2PO4, 3 MgSO4-7H20, 125; ZnSO4-7H20, 0.112; MnSO4°H20, 0.782; CuSO4-5H20, 0.042; H3303, 1.45; M00 Fe [13% Fe (w/w)], 76.88. tained, in mg/liter: Ca(NO3)2-4H 68.5; KNO , 253.3; 2 3, 0.008; sequestrene Na2 The seedlings were grown at 23 C with a 16 hr photoperiod and harvested after 14 days of growth. Growth of Lemna Plants Aseptic cultures of Lemma gibba were grown in 2.5 L low form Fernbach flasks, containing 750 m1 of autoclaved nutrient solution (104) in which 0.2 mM NaZSO4 is the only sulfur source. The cultures were grown at 28 C with 16 hr photoperiod and harvested after 17 days of growth. ngparation of ATP Sulfugylase Tobacco cells were harvested by vacuum filtration on Whatman no. 1 filter paper. After determining the fresh weight, the cells were suspended in 0.1 M ice—cold glycine-NaOH buffer (pH 9.0) containing 5 mM ascorbic acid. Five or ten milliliters of the buffer were used 51 per gram fresh weight. The cells were then homogenized by 30 strokes of a motor driven Thomas teflon-glass homogenizer at 3 C. The homogenate was centrifuged at 27,000 xg for 10 min at 4 C. The resulting supernatant fraction was used as crude ATP sulfurylase after appropriate dilution with the homogenizing buffer. As little as 0.1 g of cells was sufficient for the enzymatic assay. Crude ATP sulfurylase extracts of seedlings and Lemna plants were prepared as described above for the tobacco cells except that homogenization of plant material was carried out in a pre-chilled micro—blender (Eberbach Corporation) for 15 seconds. Assay of ATP Sulfurylase Activity ATP sulfurylase activity was measured by the enzymatic incor- poration of [358] from sulfate into APS. Radioactivity in [358]- APS was determined after its separation from [3SSI-su1fate. The complete reaction mixture contained: 20 mM glycine—NaOH (pH 9.0); 2; 20 mM [35$]_Na2504 (5 uCi/umole); 10 mM Na4ATP; 7-8 units of yeast inorganic pyrophosphatase; 1-400 1 mM ascorbic acid; 10 mM MgCl ug protein of enzyme extract. For each assay, 0.5 m1 of the reaction mixture was prepared at ice temperature, the last addition being enzyme. The reaction was started by placing the tubes in a water bath at 30 C. Incubation times were 0 to 30 min. The reaction was stopped by adding 2.5 m1 of ice-cold absolute ethanol and mixed with a vortex stirrer. The enzymatically formed [3SSI-APS was then sepa- rated from [3SSJ-sulfate (see Figure l) and quantitated by determining the radioactivity in the final ethanol supernatant solution, using zero time and boiled enzyme assays as controls. Boiled enzyme was 52 prepared by incubation of reaction mixture less ATP, [3SSI—sulfate and inorganic pyrophosphatase in boiling water for 5 min prior to the assay. ATP Determination ATP was determined by means of the luciferin-luciferase enzyme system with a Lab-Line ATP-Photometer (Lab-Line Instruments, Inc.). A commercial luciferin-luciferase extract, partially purified from the firefly, was dissolved in cold 0.04 M glycylglycine buffer (pH 7.4) containing 3 mM MgC12, as described earlier (182), at l ml/S g. The solution was then sedimented for 2 min at 1,000 xg to pellet insoluble material. The resultant supernatant was used as the enzyme source. ATP assays were conducted at room temperature in scintillation vials which contained 0.1-1.0 ml aliquots of unknowns in 2.0 m1 of the glycylglycine—MgCl2 buffer. To initiate the light producing reaction, 100 pl of cold luciferin-luciferase mixture was rapidly injected into the vial with an Eppendorf pipette. Standard ATP in the range of 5-100 pmoles resulted in a reproducible response which was proportional to the ATP concentration. Column Chromatographic Separation of the Reaction Mixture on DEAE A—25 Sephadex The method of Wilson (171) was adapted. DEAE A-25 sephadex was packed in a 0.7 x 7.0 cm column and then equilibrated at 4 C with 0.05 M of ammonium formate (pH 7.0-7.5). The complete reaction mix- ture, which had been incubated for 60 min, was placed in boiling water for 90 seconds to precipitate the protein. Protein was sedimented by 53 a subsequent centrifugation at 1,000 xg for 5 min. The resultant supernatant of the assay mixture was then applied to the column. Compounds were eluted with a linear gradiant of 40 m1 NH4HCO3, ranging from 0.5 to 1.1 M at a flow rate of 2.5 ml per hr. Fractions of 1.0 ml were collected. An aliquot from each fraction was taken for the [358] determination by scintillation counting. Chemical Characterization of the [BSSJ—Product Eluted from the DEAE Sephadex Column Aliquots from each of the fractions which contained the [358]- product eluted from the column at the APS region were also analyzed for their adenine and phosphate content. Adenine was determined by the absorbance at 260 nm after it had been corrected for the base- line absorption between 245 and 275 nm. Total phosphate was measured by a micro adaptation of the assay described by Ames (184), using 5' AMP as a standard. These determinations were used to compute the molar ratio of adenine:sulfur:phosphate in the product. Column Chromatographic Separation of the Sulfur Compounds from the Reaction Mixture on Dowex-l-Nitrate The following is essentially the method developed by Iguchi (176) and modified later by Levinthal and Schiff (175) and Schmidt and Schwenn (145). Dowex-l—chloride x 8 (200-400 mesh) was converted to the nitrate form, packed in a 20 x 0.9 cm column and equilibrated, as described previously (175). The sample, 0.5 m1 of a complete reaction mixture, was diluted 1:10 with water after a 30 min incubation period and then applied to the column under gravity pressure. Stepwise elution was 54 carried out at room temperature, under gravity flow rate of about 0.65 ml per min, with the following: (A) 105 m1 of 0.07 M NH4NO3 in 9% acetone (pH 9.7), which elutes sulfite and sulfide, in that order; (B) 200 ml of 0.1 M NaNOB, which elutes sulfate; (C) 90 m1 of 0.3 M NaNO3, which elutes thiosulfate and additional unknown com- pounds; (D) 54 ml of a gradient of 0.3-2.0 M NaNO3, which elutes PAPS and APS in that order (145); (E) 18 ml of 2.0 M NaNO3. Radio- activity in the 3.0 ml fractions collected was determined in 0.1 m1 aliquots by scintillation counting. The identities of the [358]- compounds which eluted from the Dowex-l-nitrate column were verified by electrophoretic analysis as described above. The peak eluted by high salt (D) required desalting prior to electrophoresis because of the interference of high salt with electrophoresis. The fractions in this peak area were combined, concentrated by flash evaporation and desalted by adsorption and elution from char- coal (see below). Desalting on Charcoal Samples of 1.0-2.0 ml containing [3SS]-APS and salts were mixed by vortexing with 100 mg per ml of activated charcoal. The charcoal was collected on a fiberglass filter (Millipore AP) under vacuum. Salts were washed with ca. 5-7 ml water. Nucleotides were eluted by a few milliliters of ammonia:ethanol:water (2:50:50), with a yield of 88%. The solution of eluted nucleotides was evaporated to dryness and the residue was redissolved in either water or 50% ethanol. 55 Preparation of [3SS]-APS of a High Specific Radioactivity [3SSJ-APS is not available from commercial sources. Consequently, the method which has been develOped to assay ATP sulfurylase activity in extracts from tobacco cells (see above) was employed to prepare radiochemically pure [3SSJ-APS of a high specific radioactivity. In order to prepare [3SS]-APS, the basic reaction mixture was modified to include 0.1 mM [3SSJ-Na2SO4 (l mCi/umole) and 320 ug protein of tobacco cell extract. Eight tubes, each of which contained 0.5 ml of the modified reaction mixture, were incubated for 60 min at 30 C. The [3SSJ-APS formed was recovered in the final ethanol supernatant after it had been separated from the [3551-sulfate as described above (see Figure 1). The ethanolic fractions containing the [3SS]-APS were combined, concentrated by flash evaporation and desalted by adsorption and elution from charcoal. The latter would also remove the residual contaminant of [3ssl-sulfate. The [358]- APS which eluted from the charcoal was found by electrophoresis and chromatography to have a radiochemical purity of 99%. The procedure described above yielded ca. 2.5-3 nmoles [3581- APS of l uCi/nmole. This [3SSI-APS of high specific radioactivity was used for the determination of the recovery of [3ssl-APS and in 3 some of the procedures used to characterize the [ sSJ-product of the reaction catalyzed by tobacco cell extracts. Determination of Protein Content Protein content of plant extracts was determined by the method of Lowry et al. (177), using bovine serum albumin dissolved in 1.0 M 56 NaOH as a standard. Protein in unknowns was precipitated with 10% (w/v) trichloroacetic acid, heated at 100 C for 5 min, sedimented, washed with 95% ethanol, dried and dissolved in 1.0 M NaOH. Determination of Total Uptake and Incorpora— tion of Radioactive Amino Acid into Protein Tobacco cells were harvested and homogenized as described earlier in this section. Protein content of the total homogenate was deter- mined (see above). Total radioactivity recovered in the trichloro- acetic acid supernatant, plus the ethanol wash solution, plus the 1.0 M NaOH redissolved protein fraction was used as the measure of total uptake of the labeled amino acid. The radioactivity recovered in the protein fraction was used as the measure of total incorpora— tion into protein. The counting efficiency (96%) was not affected by 0.1 m1 of either 10% trichloroacetic acid or 95% ethanol. The quenching caused by 0.1 ml of 1.0 M NaOH was corrected by the addition of 0.1 m1 of 2.0 M HCl to the scintillation vials prior to the addition of the scintillation fluid. Chemicals ATP, APS, L-cysteine, L—methionine, L-djenkolic acid, reduced glutathione, seleno-DL—cystine, seleno-DL—methionine, cycloheximide, firefly luciferin-luciferase extract, ATP sulfurylase purified from yeast (6 units/mg), and yeast inorganic pyrophosphatase (type III, 647 units/mg) were obtained from Sigma Chemical Company. Na TeO ~2820 and Na SeO were purchased from the Ventron Corporation 2 4 2 4 Alfa Products. [3SSJ-Na SO [3531-H SO 35 2 4, 2 4 and [ Sl-PAPS were from 57 New England Nuclear Corporation (NEN). [14C1—arginine (UL) was obtained from ICN. Activated charcoal ("Darco" G-60) was from Sargent—Welch Scientific Company. Sequestrene Na Fe (13%) was from Geigy Indus— 2 trial Chemicals. Casein hydrolysate was from Difco Laboratories, and the Dowex anion exchange resins were from Bio-Rad Laboratories. ‘11‘1‘11 RESULTS PART A: DEVELOPMENT OF ATP SULFURYLASE ASSAY BASED ON THE DIFFER- ENTIAL SOLUBILITY OF SULFATE AND APS IN ETHANOL The objective of this study was to develop a rapid quantitative assay for total extractable ATP sulfurylase activity, based on the physiologically significant reaction of the enzyme, APS formation. The new assay, which was developed as described below, is based upon the differential solubilities of the substrate, sulfate, and the product, APS. I. Differential Solubility of Sulfate and Sulfate- Containing Nucleotides in Ethanol-Water Mixture Sodium sulfate, 20 mM, is insoluble in ethanol:water (5:1). Twenty mM [3SSJ-Na SO 2 4 (5 uCi/umole) was completely removed from the ethanol supernatant fraction of complete reaction mixture by the 4 successive precipitation steps described in Figure 1 (Table 1). Although [BSSl-Na SO 2 4 from NEN could be completely precipitated by 3 . this procedure, [ 5Sl-HZSO from the same company contained a radio— 4 active impurity which could not be coprecipitated with excess NaZSO4. This resulted in up to a few thousand cpm in the final ethanol 35 . . . supernatant when [ Sl—H SO was used. Therefore, it is essential 2 4 58 59 REACTION MIXTURE ETHANOL O.5ml,l0,u moles N02504 2.5 ml 3530,12 l.0xl08 cpm AP35S LOxlO4 cpm ””304 . N0235$04 200 ,1 moles 45 K x 9: I0 mm __. 0.2 ml \ 1 45 K x 9; l0 min 1 S PPT I [9.45x I07 cpm ] PPT II [5.45 3: l06 cpm ] 1 45Kxg;l0min 1 l PPT m [495x 10" cpm ] 45 K x q; IOmin FINAL SUPERNATANT PPT IV [500): lochm ] Contains APS, but not 804'3or PAPS Figure 1. Procedure for separation of sulfate and APS by differential solubility in ethanol:water (5:1). 60 Table 1. Quantitative precipitation of [3SS]—su1fate by the sepa- ration method CRM x10"2 Complete Complete Complete zero time boiled minus enzyme Total added 1,000,000 1,000,000 1,000,000 Fraction ppT 1 946,920(95)a 931,000(93) 942,250(94) PPT 11 61,600(99) 66,110(99) 53,280(99) PPT III 445(99) 568(99) 422(99) PPT IV 5(100) 7(100) 6(100) SUPERNATANT 0 0 w 0 a . . . . . . Numbers in parentheses are effiCiency of preCipitation at each step, in percent. Reaction mixture contained: 30 mM glycine-NaOH (pH 9.0); 1.5 mM ascorbic acid; 10 mM MgC12; 20 mM [3SSl-Na2804 (5 uCi/umole); 10 mM ATP; 7.2 units of yeast inorganic pyrophosphatase: 0.011 mg protein of tobacco cell extract, in total volume of 0.5 ml. Tubes were incubated for 0 or 20 min, at 30 C. At the end of the incubation 2.5 m1 of absolute ethanol were added, followed by the 4 precipi- tation steps described in Figure 1. Precipitates were redissolved in water, and aliquots of all fractions were analyzed by scintilla- tion counting. 61 to use radiochemically pure [BSSI-NaZSO4 in order to achieve the maximum sensitivity possible with this procedure. The efficiency of sulfate precipitation is not influenced by the presence of either native or denatured extract in the complete reaction mixture. On the other hand, sodium APS, up to 50 uM, is soluble in ethanol:water (5:1). To examine the efficiency of the recovery of APS by the separation technique (see Figure 1), complete reaction mixtures containing unlabeled sulfate were incubated for various times. At the end of the incubation periods, radiochemi- cally pure [35$]-APS (l uCi/nmole) was added to the tubes, followed immediately by the addition of ethanol. The 4 precipitation steps resulted in the recovery of over 80% of the [358], added initially as [3SS]-APS, in the final supernatant fraction (Table 2). This ethanol-soluble [355] was verified by electrophoresis to be entirely in APS (Figure 2-A, 2—B). When [3SSI-APS was added to reaction mixtures which had been incubated with two different concentrations of extract to allow the synthesis of unlabeled APS in the range of 0-20 uM, the recovery of [35$]-APS added in the ethanol supernatant remained rather constant, at ca. 80% (Table 2). Thus, the recovery of [3SSI-APS by this separation method is not influenced by the concentrations of extract, nor by the concentrations of APS which are in the range expected for enzyme assays. 35 These results also indicate the stability of the added [ S]- APS under the conditions of the separation technique. 62 .H manna cfl oonauomoo mm coauomumm mm3 mcoeuomum 0:» ca Hmmma m0 mwm>amc< .H ousowm ca oonwnomoo mm uso oofiuumo ones mmmum Godumuwmfiooum v 058 .Hocmnuo HE m.~ mo cofluwoom man up aaoumwooesfi omzoaaom .mp5» some on oooom mm3 Moum3 cw Aoaoaa\flgu.n.mov mmdtnmmMH no a: om .coflumnsocw on» no new onu 94 .cd: om one OH .0 How O on um ooumnsoca mums moose .oEsHo> HE m.o Hmuou ca HE H.o.\cflououm on ma .uomuuxm Haoo ooomnou ocm “ommumnmmosmouhm casemuoow mo opens N.h «mad :8 0H “vommmz 28 on «NHOOZ SE OH aoflom ownuoomm SE m.m «Ao.m may momz|oowoham 28 cm “oocfimucoo mlvomummmmH usonufl3 mouquHE Somme oE>Ncm .Hmuou no unmouom mm .cowuooum some ca oouo>ooou momma mum mononucoumm ca muonfiozm 3863.3 8863.6... :88me 8868.8 8863.2. ezNc0 HE H.o meanco HE H.o Texaco HS H.o R ammm>oumm zau mmm osoecsoou nodumummom onu an mousuxwe cofluomou ouoamsoo Eoum mm¢|_mmmL oooom mo >uo>ooom .N manna 63 Figure 2. Recovery of the [3SSJ-APS added in the final ethanol fraction: verification by electrophoresis. Supernatant and PPT I fractions, described in Table 2, were evaporated to dryness, redissolved in water, and aliquots were taken for electrophoretic analysis as described in Materials and Methods. Authentic APS standard was detected by its fluorescence quenching at 254 nm. Arrows indicate [35$]- labeled standards. A. [3SSI-APS added. 35 . . B. [ S] recovered in the ethanol supernatant fraction. C. [35$]precipitated in PPT I fraction. [35$] RADIOACTIVITY (cpm xlO'Z) 40 30 N 0 6 <3 01 O N O 6 C) 64 ’ A £5 HIPS ' 3134-2 t « n- J P d )- -I APS B .—-C C 4O MIGRATION DISTANCE (cm) 6‘) Figure 2 65 The 10-13% [358] derived from the added [BSSl-APS, which was ethanol-insoluble and precipitated largely during the first step (Table 2), was mostly (70%) [3SS]-sulfate as shown by electrophoresis (Figure 2-C). The wide, asymmetrical peak (Figure 2-C) is probably due to the high level of salts and protein which are also found in this fraction. Unlike APS, sodium PAPS was completely insoluble in the ethanol: water mixture (5:1). The addition of ethanol to complete reaction mixture containing unlabeled sulfate and ca. 35 uM [3SS]-PAPS resulted in the precipitation of 99% of the [358] added as PAPS (Table 3). Therefore, sulfate and PAPS exhibit solubility properties, in ethanol:water mixture (5:1), which are markedly different from those of APS. These findings suggested that these characteristics could be further exploited as a basis for a rapid and simple method for the separation of APS from sulfate, and thus replace the conventional methods of chromatography and electrophoresis which are inherently slow. Such a separation procedure may then be used in conjunction with the radioisotope assay of ATP sulfurylase, to quantitate the amount of [3SS]-APS synthesized from [BSSl—sulfate if the enzymatically formed APS is stable and is not further converted to PAPS or other sulfur-containing derivatives. Investigation of these ideas in the cultured tobacco cells was consequently initiated. II. Formation of an Ethanol-Soluble Product by Tobacco Cell Extract . , 35 . Tobacco cell extract, incubated Wlth [ S]-sulfate and ATP in . 35 a complete reaction mixture, catalyzed the formation of a [ S]- product, which coelectrOphoresed with authentic APS (Figure 3). 66 Table 3. Precipitation of [BSsl-PAPS by ethanol [358], CPM recovered Total 257,200 Fraction ppT 1 253,500I99)a SUPERNATANT 3,700(l) aNumbers in parentheses are [358] recovered, as percent of total. Reaction mixture contained: 50 mM glycine-NaOH (pH 9.0); 2 mM ascorbic acid; 10 mM MgClz; 40 mM NaSO4; 7.2 units inorganic pyro- phosphatase; ca. 100 ug protein; and ca. 35 uM [35$]-PAPS. Two and five-tenths milliliters of ethanol were added to tubes maintained at 4 C, followed by centrifugation for 10 min at 45,000 xg, and separation of the precipitate from the supernatant. The [358] derived from [3SS]-PAPS was determined by scintillation counting of aliquots of the redissolved precipitate and the supernatant. Alternatively, if ethanol is added to such an incubated mixture, and the precipitation procedure outlined in Figure 1 is carried out, an ethanol-soluble [3SS]-product, which was formed during incubation, is found in the ethanol supernatant fraction (Table 4). Furthermore, the formation of this ethanol-soluble [358] with time was completely dependentthnithe addition, to the incubated mixture, of native extract, ATP and magnesium ions (Table 5). Thus, the requirements for the enzymatic synthesis by tobacco cell extract of the [358]- product which is recovered in the ethanol supernatant after precipi- . 35 . . . . tation of [ S]—su1fate, is conSistent with APS formation by ATP 67 35 ' APS PAPS so..-2 ' ' i 30.- .. 25" d :7 E Q 20. 4 2 X g E < 3 I5- . Q C) '4 a: 'OT' . 75' D 2.. 5" a: V A H s . ‘ 2 x d s 8 1 410 G) MIGRATION DISTANCE (cm) (9 Figure 3. Radioelectrophoretogram of complete reaction mixture showing the [3SSJ-product synthesized by tobacco cell extract. Complete reaction assay, 0.5 ml, contained: 40 mM glycine-NaOH (pH 9.0); 2 mM ascorbate; 10 mM MgC12: 0.1 mM [3ssl-Na2804 (l mCi/ pmole); 10 mM ATP; 7.2 units inorganic pyrophosphatase: and 319 ug protein of tobacco cell extract. After 60 min incubation at 30 C, the reaction was stopped by placing the tube in ice, and 10 ul ali— quots were taken for electrophoresis analysis as described in Materials and Methods. Standard APS, [3551-504‘2, and [358]-PAPS are shown. 68 Table 4. Time dependent formation of ethanol-soluble [3SSI-product by tobacco cell extract cpnxlo'z Complete Complete 0-min 60—min Total added 1,009,370 1,011,955 Fraction ppT 1 647,970 501,340 PPT 11 357,900 498,600 PPT 111 3,428 5,704 pp'r IV 50 247 SUPERNATANT 22 6.064 Complete reaction mixtures as described in Figure 3 were incubated at 30 C for 0 and 60 min. The reactions were stOpped by the addie tion of 2.5 ml absolute ethanol, followed by the four precipitation steps described in Figure l. Aliquots of all fractions were analyzed for [3SSl-content by scintillation counting. sulfurylase. The relative ineffectiveness of the inorganic pyro- phosphatase on the accumulation of the [355] in the ethanol is not unexpected if the [358] that formed was synthesized by ATP sulfurylase at initial rates before equilibrium is approached. The indication that extract from tobacco cell incorporates [assl-sulfate into a single product (Figure 3), which can be recovered in the ethanolic fraction after the precipitation of [BBSl—sulfate by ethanol, prompted a detailed effort to characterize this product. 69 Table 5. Requirements for the enzymatic synthesis of the ethanol- soluble [3SSI-product by tobacco cell extract Incubation [355] in supernatant Reaction mixture time (min) (CPM) CGVIPLETE 0 0 (ma COVPLE'I'E 20 9,872 (100) boiled extract 20 0 (0) minus extract 20 0 (0) minus ATP 20 0 (0) minus Mg+2 20 992 (10) minus pyrophosphatase 20 7,104 (72) COMPLETE, 3X pyrophosphatase 20 9,440 (96) . 3 aNumbers in parentheses represent [ 5S], as percent of complete assay. Assays were performed as described in Table l. The formation of the [358]-product was proportional to time of incubation. III. Characterization and Identification of the Ethanol-Soluble [35S]-Product Formed py Tobacco Cell Extract as APS The objectives of the following studies were to determine whether the ethanol-soluble [358] formed by tobacco cell extract is APS, and if it is the only product formed during the incubation. (a) Electrophoretic Analysis The ethanol-soluble [355] formed by incubation of tobacco cell extract in complete reaction mixture (see Table 5) 70 coelectrophoresed as a single peak with authentic APS, but not with 3 [ 5S]--PAPS or [3SSJ-su1fate (Figure 4). The [BSSJ-sulfate and [358]- PAPS standards each electrophoresed as a single peak during separate runs, indicating radiochemical purity and lack of breakdown products. (b) Thin Layer Chromatography This ethanol-soluble [3SSI-product appears as a single peak during thin layer chromatography as well. It cochromatographed with authentic APS, but separated from [3ssl-PAPS (Figure 5), and from [3SS]-sulfate (Figure 6). The purity and stability of the [35S] in the standards PAPS and sulfate during the analysis was determined in separate runs. When the ethanol—soluble [355] was eluted from the thin layer plate (Figures 5-A and 6-A) and was subjected to a second analysis by either thin layer chromatography or electrophoresis, the same Rf values were obtained. (c) Charcoal Adsorption This ethanol-soluble [358] was adsorbed by charcoal and could be eluted by a mixture of ethanol:ammonia:water, which is consistent with its being a sulfur-nucleotide. The [358] eluted from charcoal coelectrophoresed and cochromatographed with authentic APS, exactly as did the ethanol-soluble [358] before the treatment with charcoal (see Figures 4, 5 and 6). (d) Acid Lability Upon incubation with 1.0 N HCl at 37 C for 60 min, all the [355] derived from the ethanol-soluble product appeared as sulfate 71 Figure 4. Radioelectrophoretograms showing the ethanol- soluble [358]-product derived from assay mixture with tobacco cell extract after precipitation with ethanol. The ethanol supernatant fractions of complete reaction mixtures incubated for 60 min (see Table 4) were combined, concentrated by evaporating the ethanol, and aliquots were subjected to elec- trophoretic analysis (see Materials and Methods). 35 . . The [ Sl-product recovered in the ethanol supernatant fraction. A with standard [355]-PAPS. A with standard [3531-804-2. —2 A with authentic APS, [35S]-PAPS, and [3SSI-SO4 as standards. [ S] RADIOACTIVITY (0pm I: IO'Z) 80L so, 20- 60- 40- 20- ”30-- ‘3 0| T 5()P 25- origin IO J j 50 MIGRATION DISTANCE (cm) 20 Figure 4 73 A 63.5 30- " a? 'o " 20- . I E 3 l0# ‘ >. p. S; C)? ‘ E; £3 IAPS 2 30- 3 0: 20f 1 '7n' I) d :2... W | 0+ ‘ 1 A L A ‘ 0 l 5 IO I5 120 MIGRATION DISTANCE (cm) Figure 5. Radiochromatograms showing absence of PAPS in the ethanol-soluble [3SSJ-product formed by tobacco cell extract. Aliquots of the concentrated ethanol supernatant fractions (see Figure 4) were chromatographed on polyamdde 6 precoated thin layer plates (Baker-flex) and developed in propanol:ammonia:water (8:2:3), as described in Materials and Methods. 35 . . A. The [ S]-product recovered in the supernatant fraction, run with standard APS. B. A with standard [358]-PAPS. 74 "'T- I 1 I I A APS l5- r—--—4 1 :7 9 '0’ -I X E s 5' i E: E 0‘ ‘ p. o 3 APS 3 60b O-—O ‘l C3 <1 C 40- .I '77)" 3 l—J 20? " Op-o—o—o—o—o—o-OJ a O l. . 5 IO I5 (20 CHOU" front MIGRATION DISTANCE (cm) Figure 6. Radiochromatograms showing absence of sulfate in the ethanol-soluble [35$]-product synthesized by extract from tobacco cells. Aliquots of the evaporated supernatant fractions (see Figure 4) were subjected to chromatographic analysis on cellulose pre- coated thin layer plates (Baker-flex) and developed in propanol: ammonia:water (8:2:3), as described in Materials and Methods. A. The ethanol—soluble [BSSl—product, run with APS standard. B. A with [3SS]-Na2504 standard. 75 (Figure 7). These conditions are known to hydrolyze authentic APS to sulfate and AMP (163). (e) Attempted Identification of the Ethanol- Soluble [35SI-Product as APS by the Reverse Reaction of ATP Sulfurylase Extracts of tobacco cells catalyzed the formation of [358]— sulfate in an enzyme-dependent reaction upon incubation with the isolated ethanol-soluble [3SS]-product, pyrophosphate and Mg+2 (Figure 8). However, because a crude extract was used, this reaction could not be rigorously attributed to ATP sulfurylase. Consequently, the experiment was repeated with comercial ATP sulfurylase purified from yeast, and the formation of both [BSSl-sulfate and ATP from the ethanol-soluble [BSSl-product was studied. Sulfate was determined as the [358] which precipitated by ethanol, added at 5:1. In con- trast to the assay which is described for the forward reaction of APS formation, the reverse ATP sulfurylase‘assay employed here does not have a huge excess of [3SS]-sulfate. Therefore, two precipitation steps after ethanol was added were sufficient to separate the sulfate from APS. ATP was assayed by the luciferin-luciferase enzyme system, with the assistance of Mr. B. Whitaker in this laboratory. Incubation of the purified ATP sulfurylase with the ethanol- soluble [3581 resulted in the formation of [3ssl-su1fate which was dependent upon addition Of native enzyme (Table 6). However, a simple interpretation of these results as being due solely to ATP sulfurylase is difficult because: (a) the formation of sulfate was independent of the pyrophosphate present, and (b) ATP could not be detected in the complete reaction mixture (Table 6). Because the original 76 Figure 7. Radioelectrophoretograms of the acid hydrolysis product of the ethanol-soluble [3581-product. The ethanol supernatant fractions (see Figure 4) were evaporated to dryness and redissolved in water. One hundred microliter ali- quots, containing approximately 90,000 cpm of the [3SSJ-product, were incubated with 1.0 N HCl at 37 C for 60 min in a total volume of 125 pl. Control tubes containing standard [3581-Na2804 were treated in the same way. After the inCubation, the HCl was evapo- rated, samples were redissolved in 50 ul water, and aliquots of 25 ul were subjected to paper electrophoresis as described in the text. 3 A. The [ 5Sl-product before acid hydrolysis, run with authentic APS. B. The [3SSJ-product after acid hydrolysis, run with authentic APS. C. B run with standard [3581—804-2. -2 . D. Standard [3581-804 after acid hydrolysis, run with authentic APS. The arrows indicate the Rf for PAPS and 804-2, determined separately. --..-_———‘ ~ ‘— d 60- APS [3’s] RADIOACTIVITY (cpm x IO'z) a m R3 9 9 9 9 60- APS 41 l l 1 I I0 20 MIGRATION DISTANCE (cm) Figure 7 40- 20- CL + 30 III I .‘ 'll‘i . 78 Figure 8. Radioelectrophoretograms showing enzymatic con- version of the [3SSI-product to [358]-sulfate by tobacco cell extract. Reaction mixtures, 125 pl, contained: 10 mM glycine-NaOH (pH 9.0); 2 mM ascorbate; 10 mM MgC12; 3 mM sodium pyorphosphate; [358]- product (see Figure 7), approximately 45,000 cpm; and 90 ug pro- tein of tobacco cell extract. Control assays containing standard [3SSI-PAPS were assayed in the same way. At the end of 60 min incubation at 30 C, 50 ul aliquots were analyzed by electrophoresis. A. [BSSl-product before incubation, run with standard APS. 3 O I I B. [ 5Sl-product after incubation with tobacco cell extract, run with standard APS. 3 C. [ 5S]--product after incubation with boiled tobacco cell extract, run with APS. D. Standard [3SSI-PAPS after incubation with tobacco cell extract, run with APS. Arrows indicate the Rf for standard PAPS and sulfate. [3’s] RADIOACTIVITY (cpm x IO'z) 60 4O 20 O 60 40 20 O 60 4O 20 O 30 20 l0 0 79 PAPS b h h _ D 21111 Tips APS APS APS p0 a I- ., I- -I .- origin O IOMIGRATION DlgTANCE (cm):50 Figure 8 OS 13"!!! 80 Table 6. Formation of sulfate from the ethanol-soluble [3ssl—product catalyzed by ATP sulfurylase purified from yeast [35$] APS Sulfate ATP Reaction mixture added recovered formed formed Complete 35.5 0.8I2)a 33.8(98) 1.1 boiled enzyme 35.5 26.7(74) 9.2(26) 5.0 minus enzyme 35.5 26.5(78) 7.5(22) 4.8 minus pp, 35.5 0.9(3) 32.9(97) 0.5 i . +2 minus Mg 35.5 0.4(1) 32.2(99) 12.4 aNumbers in parentheses are percent of total [358] recovered in each assay. Complete reaction mixture, 0.25 ml, contained: 20 mM glycine- NaOH (pH 9.0): 10 mM MgC12; 2 uM sodium perphosphate; ca. 0.14 uM [3SS]-product (see Figure 8), of specific activity of’ca. 560 cpm/pmole: and 1.5 units of commercial ATP sulfurylase purified from yeast. After incubation at 30 C for 30 min, reactions were stopped by adding 1.25 ml absolute ethanol, followed by two precipi- tation steps, with 10 and 50 pmoles carrier NazSOq, added in 50 ul, respectively. Precipitation fractions were dissolved in 1.5 m1 of 0.04 M glycylglycine, 3 mM MgC12 (pH 7.4). Three-tenths milliliter aliquots of all fractions were analyzed for [35$]—content by scin- tillation counting. ATP measurements, by the luciferin-luciferase assay described in the text, were conducted with 1.0 ml of the precipitate fractions and 0.1 ml of the ethanolic solution. 'l ( II.III 81 [35S1-product used as a substrate appeared to contain ca. 5 pmoles of ATP initially (Table 6), an actual net loss of ATP was observed in complete reaction mixture, indicating the presence of at least one ATP consuming enzyme in the commercial ATP sulfurylase prepara- tion. Because net formation of ATP was observed only when Mg+2 was omitted from the reaction mixture (Table 6), it may be assumed that the ATP consuming reaction was Mg+2 dependent. Nevertheless, even in the absence of Mg+2, the amount of ATP formed was considerably less than what was expected based on the formation of sulfate. Since the commercial ATP sulfurylase contained ca. 0.2% ATPase activity (Sigma specifications), the net loss of ATP observed could be accounted for, if one assumes a Km (ATP) for the ATPase reaction Of ca. 0.1 mM. Although the results of this section [(e)] do not strongly sup- port any single conclusion with regard to the chemical nature of the [3SSJ-product formed by tobacco cells, they are nevertheless worth noting because they shed some light on similar inconsistencies reported in the literature for ATP sulfurylases from other sources (see Discussion). (f) Characterization of the Chemical Composition of the [3SSl-Product An attempt to determine directly the molar ratio of adenine to sulfur in the ethanol-soluble [3SSJ—product, by a double- 1abe1 experiment with [3Hl-ATP and [3531-sulfate in the incubation mixture, was unsuccessful. Abundance of ethanol-soluble [3H]- derivatives was consistently found in the final supernatant fraction, in addition to the [3H],[3SS]-double labeled product. 82 A different approach was therefore attempted. A complete assay mixture containing the reaction product(s) formed by tobacco cell extract was chromatographed on a DEAE A-25 sephadex column, which has been shown to separate APS from sulfate, ATP, ADP, 5'AMP, 3'AMP and PAPS (171), prior to the chemical characterization. These studies, described below, were performed by Mr. G. Dilworth from the Department of Botany and Plant Pathology, Michigan State University. The elution profile of the complete reaction mixture from the DEAE A-25 sephadex column showed a single [353] containing product with a Rf characteristic of APS (fractions 13-16), well-separated from the [BSSI-sulfate which was eluted in Fractions 5-9 (results are not presented). When each of the fractions which contained the eluted [358]— product was analyzed for the content Of [358]-sulfur, adenine, and phosphorus (see Materials and Methods), a molar ratio of adenine: sulfur:phosphorus of 0.97:1:l.07 and 0.99:1:l.08 was obtained for Fractions l4 and 15, respectively (Figure 9), which is consistent with the 1:1:1 ratio expected for APS. (9) Determination of APS as the Only Sulfur- Containing Product Synthesized by, Tobacco Cell Extract The finding that the complete reaction mixture yielded a single ethanol-soluble [BSsl-product was not, however, sufficient for determining this product as the sole sulfur—containing product synthesized by the tobacco cell extract. Thus, the formation of [3SS]-derivative(s) which are insoluble in ethanol was conceivable. 83 IO- . nmohs/ml 0| I 1 () _ - 4 l L l J 8 K) I4 EB 20 ml Figure 9. Chemical composition of the [3SSJ-product eluted from DEAE sephadex column, showing the molar ratio of sulfur: adenine:phosphate. Complete reaction mixture (see Table 1) containing approximately 100 ug protein of tobacco cell extract, incubated at 30 C for 60 min, was chromatographed on DEAE A-25 sephadex column (see Materials and Methods). Fractions containing the eluted [355]- product (Fractions 12-17) were analyzed for [358] (CD); adenine content (I): and phosphate (A), as described in the text. 84 Such derivative(s), if formed by the conversion of APS, will result in an underestimation Of the amount of APS formed in the reaction catalyzed by ATP sulfurylase. Therefore, the identification of the [BSSJ-product(s) synthesized by tobacco cell extract was investigated. Electrophoresis (see Figure 3), as well as chromatography on DEAE sephadex, of the complete reaction mixture, indicated that APS was the only [BSSl-containing product synthesized by tobacco cell extract. Nevertheless, a third independent method was used to verify this conclusion. Chromatography on Dowex-l-nitrate resin is perhaps the most powerful method available to resolve the major sulfur-containing intermediates of sulfate reduction (146,175-176). When the incuba- tion of the complete reaction mixture was followed by chromatography on a Dowex-l-nitrate column, only one substantial [BSSJ-peak was found in addition to [3581-sulfate (Figure 10). The [358] in this major peak, which was eluted in the APS region, was verified as APS by electrophoretic analysis of the pooled fractions from the peak area. In addition to APS and sulfate, a trace amount of an unknown was found in a third peak (Figure 10). However, this peak contained only 1.6% as much [358] as the major APS peak and therefore would be a negligible factor in estimates of APS synthesis. (h) Summary of Conclusions Regarding the Measurement of Incorporation of [35S]-Sulfate by Tobacco Cell Extract Into Product It may be concluded from the results presented above that: 85 mums—4e MONO; ~ F—.07M—oIo——.I ———-I.—-.3 —-I.-3-24-2 (9" -2 Q _ . x 25 3 I5- ‘ .9 a3: . . i: .4. II I ) m’\ I l0" ‘ 9 x )4) E o. o v om 5. l) "1 l0 0 0 I50 300 450 ml Figure 10. Analysis Of the [35$]-derivatives formed by tobacco cell extract: chromatography on Dowex-l-nitrate column. Complete reaction mixture, 0.5 ml, as described in Table 1, was incubated with approximately 150 ug protein of tobacco cell extract, at 30 C for 30 min. Then the assayed mixture was diluted 1:10 with water and analyzed for [35$]-derivatives by chromatography on Dowex-l-nitrate column as described in the text. 86 (l) Cultured tobacco cells contain a soluble enzyme which catalyzes the synthesis of a single [3SS]-labe1ed compound from [assl-sulfate, in a reaction mixture that requires ATP and Mg+2. (2) The [3SS]—product has solubility properties, electro- phoretic and chromatographic behavior which are identical with those of APS, and distinct from those of sulfate and PAPS. (3) The compound is acid labile and adsorbs and elutes from charcoal in the same manner as authentic APS. (4) The enzymatic product contains adenine, sulfur, and phos- phate in a molar ratio of ca. 1:1:1, as expected for APS, and behaves as a substrate for ATP sulfurylase in the reverse reaction. (5) The [3SS]-product can be separated from the [35S]- precursor, sulfate, by quantitative precipitation of Na2804 in ethanol:water (5:1). Therefore, the product is APS and the enzyme is ATP sulfurylase. Moreover, the separation of APS from sulfate on the basis Of solu- bility in ethanol-water can be used for quantitative determination of the APS formed by the crude ATP sulfurylase of tobacco cells. IV. ATP Sulfurylase Assay Based on the Differen- tial Solubility of Sulfate and APS Conditions for assay of ATP sulfurylase activity, in the forward reaction, in which APS is quantitatively measured as the [358] recovered in the final ethanolic fraction (see Figure l), were readily established. With these standard assay conditions, the rate of APS synthesis by tobacco cell enzyme is constant with time (Figure ll—A) and proportional to enzyme concentration (Figure ll-B). APS ( p moles 1' 0.5 ml ) ,tase; and 6.3-25.2 ug protein of tobacco cell extract. 87 I00 500 50 RATE OF APS FORMATION (9 moles! mIn) l I A 20 4O TIME(mIn I IO 20 PROTEIN (pg/0.5 ml) 60 Figure 11. ATP sulfurylase assay based on APS formation: proportionality with time and enzyme concentration. Complete reaction mixtures, 0.5 ml, contained: 45 mM glycine- NaOH (pH 9.0); 2 mM ascorbate; 10 mM MgC12; 40 mM [35$]-Na2804 (2.5 uCi/umole); 10 mM ATP; 8.5 units of inorganic pyrophospha» After incubation at 30 C for the times indicated, the assays were stopped by 2.5 ml cold absolute ethanol. APS was assayed by determining the soluble [35$] remaining in the ethanol super- natant after precipitation of [358]-SO4'2. A. Proportionality of APS formation with time of incubation. B. Proportionality of the rate of APS formation with enzyme concentration. 88 When the concentrations of APS in the reaction mixture exceed 3 uM, a decrease in the rate of its synthesis was observed. This may be due to APS approaching the equilibrium concentration, but strict equilibrium was not reached even after 8 hr of incubation in which APS was at ca. 18 uM. Another possibility is that APS is an end- product inhibitor of the reaction. Extension of the proportional accumulation of APS (see Figure 11) beyond the limitation imposed by the equilibrium constant, which was expected to result from the presence of excess inorganic pyro- phosphatase in the assay mixture, was nevertheless not observed. Consequently, ATP sulfurylase activity was assayed under conditions confined to the range at which proportionality of APS formation occurs. This included 20 mM [BSSl-Nazso4 (5 uCi/umole), low amount of protein of ca. 1-40 ug, and short incubation times of 5-10 min. When [3SSI-APS of a high specific radioactivity was desired for analytical purposes, APS was allowed to be synthesized in a reaction mixture containing 0.1 mM [35$]-Na SO (1 mCi/umole), 2 4 excess of extract up to 400 ug of protein for 30-60 min incubation. Under these different conditions, summarized in Table 7, the forma- tion of APS had nevertheless the same dependencies as those described in Table 5. The validity of this new method for assay of APS formation in crude extracts was tested with extracts obtained from several higher plants. All species examined exhibited ATP sulfurylase activity similar to the tobacco cell extract. Thus, extracts pre- pared from seedlings of pea, tomato, cucumber and Lemna plants all 89 Table 7. APS formation during various conditions Total [35s1-SO4’2 Reaction added -6 [358] in superna- APS formed mixture (CPM x 10 ) tant (CPM x 10'2) (pmoles/0.5 m1) Completea 99.3 124 1,240 Completeb 101.2 6,040 302 aComplete reaction mixture, 0.5 m1, contained: 50 mM 35 glycine-NaOH (pH 9.0); 2.5 mM ascorbate; 10 mM MgC12; 20 mM [ S]- Na2804 (108 cpm per assay): 10 mM ATP; 7.2 units inorganic pyro- phosphatase; and 33 pg of protein from tobacco cell extract. Incu- bation time was 10 min at 30 C. Complete reaction mixture, 0.5 m1, prepared as described above for (a) except the following modifications: 0.1 mM [358]- Na2504 (108 cpm per assay); and 320 ug protein of tobacco cell extract. Incubation at 30 C lasted for 60 min. Both assays were stopped by 2.5 ml ethanol, and APS was determined, in the ethanol supernatant solution, after precipitation of [353]- so '2. 4 incorporated [3SSJ-sulfate into a single ethanol-soluble [358]— product verified as APS (Table 8). Therefore, APS formation can be generally assayed in plant extracts merely by determining the soluble [35$] remaining in the ethanol supernatant after precipitation of [35S]-su1fate. The assay is remarkably reproducible. The standard deviation within an experiment has not exceeded 2.5%. The high sensitivity of the assay is exemplified by the ability to detect easily as little as 20 pmoles of APS. Finally, the high suitability of this assay method for rapid performance of multiple assays was indispensable for the kinetic and regulatory studies described in the following 90 Table 8. APS formation catalyzed by extracts from higher plants Enzyme source nmole APS-.min‘E-g.f.wt.':I nmole AP’Svm:1n";[‘°mg"protein"I Peas 7.2 1.2 Tomato 7.6 1.2 Cucumber 7.1 1.7 Lemna 6.5 1.1 Tobacco cells 9.4 2.8 Reaction mixtures, 0.5 ml, contained: 45 mM glycine-NaOH (pH 9.0); 2 mM ascorbate; 10 mM MgC12; 40 mM [3581-Na2804 (2.5 uCi/umole): 10 mM ATP; 8.5 units of inorganic pyrophosphatase; and 0.2 m1 of crude extract. The protein content in each assay was: 248 pg for peas; 252 ug for tomato; 166 ug for cucumber; 228 119 for Lama; and 33 ug for tobacco cells. Assays were conducted at 30 C for 0, 15 and 30 min. Reactions were stopped by the addition of 2.5 m1 ethanol, and APS was determined from the soluble [35$] remaining in the supernatant after precipitation of [3581-504‘2. section of this dissertation. A Sorvall SS—34 centrifuge rotor with a capacity of 8 tubes was used in order to generate the centrifugal force needed to achieve tightly-packed [358]-sulfate precipitates. Allowing for ca. 30 min for each round of precipitation, 8 to 24 samples can be assayed in 4 and 8 hours, respectively, with a single rotor. It is noteworthy that, contrary to information in many commer- cial catalogs, the [3SS]-APS recovered in the ethanol supernatant fraction was rather stable. Thus, after storage Of the ethanolic fraction at -20 C for 10 days, no breakdown product could be 91 detected by electrophoresis. The APS was somewhat less stable when stored at -20 C in water solution. PART B: THE REGULATION OF ATP SULFURYLASE IN CULTURED TOBACCO CELLS B.l Regulation of ATP Sulfurylase by Sulfur Compounds Studies with bacteria and fungi indicated an apparent correla- tion between the ability of an organism to assimilate sulfate and the regulation of ATP sulfurylase by end-product repression (e.g., 24-27,66-67,126,129,185). The unsuccessful attempts to detect such a regulation of ATP sulfurylase in higher plants (104) were there- fore somewhat surprising. The finding that sulfate transport is subject to end-product regulation in cultured tobacco cells (123-124) further suggested that the development of ATP sulfurylase in plants is likely to be regulated. Therefore, a reexamination of the influence of end-products of the sulfate pathway on the development of ATP sulfurylase was undertaken in the tobacco cell system. Conditions in which the growth of the KB strain of cultured tobacco cells is dependent on the sulfur source added to the medium were determined earlier in this laboratory (123). This required the removal of contaminating anionic sulfur, of otherwise unknown chemistry, from reagent grade sucrose by means of an anion exchange resin. Under such conditions, 0.1 mM sulfate and 2.5 mM nitrate just meet the nitrogen and sulfur requirements of the tobacco cells to yield ca. a 30-fold increase in mass over ca. 14 days, up to ca. 30 g/l fresh weight of cells (179). Organic sulfur sources such as L—cysteine, 92 L-methionine, or reduced glutathione, at 0.1 mM each, can sustain growth of the tobacco cells when substituted for sulfate (123). The parallel utilization by the cells of nitrate and sulfate supplied at a molar ratio of 25 to 1 is consistent with the relative rates of uptake for nitrate and sulfate in the tobacco cell system of 1.5 and 0.05 umoles/hr/g.f.wt., respectively (70,123). The 25:1 ratio is also consistent with the relative abundance of nitrogen and sulfur in proteins (e.g., 199), and the fact that both nitrogen and sulfur are mostly (ca. 90%) assimilated into protein in plants (cf. 147,179). These correlations all suggest that the regulation of the rates of both nitrate and sulfate assimilation should be coupled to each other and to the rate of net protein synthesis. I. The Effects of Sulfur Compounds on Growth and ATP Sulfurylase Activity in Tobacco Cells Organic sulfur compounds vary in their relative effectiveness in supporting growth of the tobacco cells, as determined after 12 days of growth (Table 9). The utilization of either L—cysteine or L-methionine resulted in fresh weights similar to those obtained with sulfate. Glutathione and L-djenkolate also served as sulfur sources but resulted in lower growth rates. The elevated yield observed when cysteine and methionine were included with djenkolate in the medium (Table 9) suggested that the lower growth rate char- acteristic of djenkolate reflected its slow assimilation compared to L-cysteine, L-methionine or sulfate, rather than a toxic effect. The small increase in fresh weight above the initial inoculum observed in "sulfur-free" medium prepared with purified sucrose 92 L-methionine, or reduced glutathione, at 0.1 mM each, can sustain growth of the tobacco cells when substituted for sulfate (123). The parallel utilization by the cells of nitrate and sulfate supplied at a molar ratio of 25 to 1 is consistent with the relative rates of uptake for nitrate and sulfate in the tobacco cell system of 1.5 and 0.05 umoles/hr/g.f.wt., respectively (70,123). The 25:1 ratio is also consistent with the relative abundance of nitrogen and sulfur in proteins (e.g., 199), and the fact that both nitrogen and sulfur are mostly (ca. 90%) assimilated into protein in plants (cf. 147,179). These correlations all suggest that the regulation of the rates of both nitrate and sulfate assimilation should be coupled to each other and to the rate of net protein synthesis. I. The Effects of Sulfur Compounds on Growth and ATP Sulfurylase Activity in Tobacco Cells Organic sulfur compounds vary in their relative effectiveness in supporting growth Of the tobacco cells, as determined after 12 days of growth (Table 9). The utilization of either L—cysteine or L-methionine resulted in fresh weights similar to those obtained with sulfate. Glutathione and L-djenkolate also served as sulfur sources but resulted in lower growth rates. The elevated yield observed when cysteine and methionine were included with djenkolate in the medium (Table 9) suggested that the lower growth rate char- acteristic of djenkolate reflected its slow assimilation compared to L-cysteine, L-methionine or sulfate, rather than a toxic effect. The small increase in fresh weight above the initial inoculum observed in "sulfur-free" medium prepared with purified sucrose 93 Table 9. Growth of tobacco cells on various sulfur sources SULFUR SOURCE(S) YIELD (0.1 mM each) (9 fresh weight/l) Sulfate 25.0 L-cysteine 25.7 L-methionine 20.5 L-cysteine plus L-methionine 21.2 Reduced glutathione 11.5 L-djenkolate 9.9 L-djenkolate plus sulfate 23.9 L-djenkolate plus L-cysteine 24.2 L-djenkolate plus L-cysteine plus 20.5 L-methionine No sulfur added, purified sucrose 3.0 No sulfur added, reagent grade sucrosea 5.1 a(N,S)—MID in which reagent grade sucrose substitute for the purified sucrose. Stationary phase cells were subcultured into (N,S)- MID supplemented with 2.5 mM nitrate and 0.1 mM of sulfur source as indicated. Fresh weights were determined after 12 days, in duplicate cultures, and averaged. The initial inoculum was 1.7 g fresh weight of cells/l. 94 (Table 9), which is associated with a corresponding small increase in soluble protein, may mean that the anion exchange treatment did not remove all contaminating sulfur from the sucrose, or that other reagents were contaminated with sulfur. Regardless of its source, or chemical nature, this sulfur contaminant was sufficient to support ca. one doubling of the cells (Table 9). Consequently, in experiments in which strict dependence on an added sulfur source was desired, large initial inocula were used to rapidly exhaust the contaminating sulfur and thereby minimize its interference in experiments designed to determine the effects of specific sulfur sources on ATP sulfurylase development. The various sulfur sources supporting growth have a profound influence on the level of ATP sulfurylase in the cells (Table 10). Thus, exponentially multiplying tobacco cells yield low ATP sulfurylase activity when grown on those sulfur sources which support rapid growth (see Table 9), while a strikingly higher activity is found in cells growing slowly on the sulfur of djenkolate or in "sulfur-free" medium (Table 10). These results also show that ATP sulfurylase of the XD cells is not induced by its substrate sulfate, which is in agreement with data reported on the enzyme isolated from other sources (e.g., 96,104,126). In contrast, sulfate represses the level of enzyme relative to the level found in sulfur starved cells (Table 10). Neither cysteine nor methionine, nor the combination of both (see Figure 12), result in complete repression of the enzyme in the tobacco cells (Table 10). Nevertheless, within any given experiment, cells utilizing either cysteine or methionine consistently 95 Table 10. ATP sulfurylase activity of tobacco cells grown on various sulfur sources Sulfur source ATP sulfurylasea Sulfate 4.6 L-cysteine 3.0 L-methionine 2.7 L-djenkolate 20.1 No sulfur added 22.4 _ . -1 anmoles APS x min 1 x mg protein . Stationary phase cells were subcultured into (N,S) MID supplemented with 2.5 mM nitrate and 0.1 mM of the sulfur source indicated. After 5 days of growth, ATP sulfurylase was extracted and assayed (see Materials and Methods). The initial ATP sulfurylase activity was 2.1 nmoles APS x min"1 x mg protein’l. have a somewhat lower level of ATP sulfurylase compared with cells growing on sulfate. Therefore, the tobacco cell ATP sulfurylase is highly, but not completely, repressed under optimal growth conditions. Derepressed levels appear when the sulfur supply falls short of supporting the optimal growth rate possible. The possibility that these changes in ATP sulfurylase activities resulted by direct interactions of the sulfur compounds with the enzyme molecules was examined by determining the effects of the various sulfur sources on ATP sulfurylase activity in vitro. Neither cysteine nor methionine inhibited the rate of APS formation catalyzed by the tobacco cell enzyme, nor did djenkolate activate it (Table 11). 96 Table 11. Lack of effects of the sulfur amino acids on APS formation in vitro Assay mixture nmoles APS/10 min nmoles APS x min"I x mg-I Complete 823 5.5 Complete plus 904 6.0 L-cysteine Complete plus a 808 5.4 L-methionine Complete plus 800 5.3 L—cysteine plus L-methionine Complete plus 758 5.1 L-djenkolate aEither with or without 1.0 mM L-arginine. Complete reaction mixture, 1.0 ml, contained: 20 mM glycine-NaOH (pH 9.0); 0.5 mM ascorbate; 10 mM MgC12; 20 mM [3SSI-Nazso4 (5 uCi/ nmole); 10 mM ATP; 7.2 units of inorganic pyrophosphatase; and 0.015 mg protein of tobacco cell extract. Sulfur amino acids were added to a final concentration of 1.0 mM each. Extract, prepared from exponential cells grown on MID, dialyzed against 0.1 M glycine-NaOH buffer (pH 9.0) containing 5 mM ascorbate, for 7 hr at 4 C. Solutions of the sulfur amino acids were prepared in the assay buffer, and the pH was adjusted to ca. 9.0. After incubation for 10 min at 30 C, 5 m1 of absolute ethanol were added, and APS was determined as described in Materials and Methods. The forma- tion of APS was proportional with time and enzyme concentration. 97 A slight stimulation by L-cysteine of APS formation in vitro has been observed consistently. This phenomenon has not been investi- gated further. The above results lead to the conclusion that the changes in the level of extractable ATP sulfurylase activity which develop during growth on the various sulfur sources result from the repression-derepression type of regulation rather than from the feedback inhibition type of regulation. II. Dependence of the Regulation of ATP Sulfurylase on Growth Rate, as Determined by Nitrogen Source The results described above further indicated that the ability of a sulfur compound to bring about changes in ATP sulfurylase level was linked to its effectiveness in supporting growth. The growth rate of cells, however, can be manipulated independently of the sulfur source by means of the nitrogen supply. It was, therefore, desirable to investigate the dependency of the regulation of ATP sulfurylase on the growth rate as determined by various nitrogen conditions. Tobacco cells can satisfy their nitrogen requirements with urea, but the growth rate on urea is slower than on nitrate (70). Since the lower growth rate on urea should result in a lower rate of net protein synthesis and hence a lower rate of assimilation of sulfur, this raised the question of whether the ATP sulfurylase level in tobacco cells growing on urea nitrogen would also be lower than in cells grown on nitrate nitrogen. If the regulation of ATP sulfurylase is governed by the rate of sulfur assimilation in relation to protein 98 synthesis, slower growth on urea should yield a concomitant lower level Of the enzyme. If, however, the enzyme level is determined solely by the sulfur source, independent Of the rate of protein synthesis, activities on urea should be comparable to those on nitrate. Thus, experiments were performed to test these ideas. Indeed, cells grown on urea exhibited ca. a 2-fold decrease in growth rate concomitantly with a ca. 2-fold decrease in soluble protein compared with cells grown on nitrate (Table 12). This trend was found under all sulfur conditions. Thus, both growth rate and total protein are governed predominantly (although not exclusively) by the nitrogen supply. In contrast, the specific activity of ATP sulfurylase was rather independent of the nitrogen source (Table 12). This is a reflection of the fact that there was ca. a 2-fold decrease of total activity in cells grown on urea, which parallels the decrease in protein. Nevertheless, regardless of the nitrogen source, sulfate, cysteine and methionine resulted in.repressed levels of ATP sulfurylase, while cells grown on djenkolate, or starved for sulfur, have the characteristic dere- pressed activity (Table 12). Thus, the regulation of ATP sulfurylase, though governed primarily by the sulfur source, appeared to be coordinated in some way as well with the rates of nitrogen assimi- lation into protein. III. Kinetics of the Develgpment of ATP Sulfuryl- ase as a Function of the Sulfur Source The finding of different levels of ATP sulfurylase in cells grown on various sulfur sources prompted studies of the kinetics of the development of these differences. 99 no nusono mo mzeo v Hound no ouenuwn SE m.~ nonufle oneneeunoo on .nusonm Heflunenooxe mo m>eo b one m neesuen munmflez nmoum mo mnOwuenflEueueo on» no oemen one mouen np3on0 .mHHoo nzonm eeun one eueuuen Mom Hlnfiouonm me x HInflE x mod meaoen h.m one H.N mes muHDHuoe Heaven“ one .oenweneueo me3 >ufi>fiuoe omeahunmanm man one .oeume>uen oue3 mHHeo .eoun no m>eo m one eueupfln .>He>euoemmeu .eounOm Humane on» mo 25 H.o one mounOm nemonuen enu me eons SE o.m Am.zv nmeum Oune oewnuanonnm euez maaeo emenm auenOwueum o.vH o.a Ha ooooe unmanm on eeuo o.mH v.m om oeooe humane on eueuuwz m.mH m.H om eueaoxnonoIq eons m.ha o.m mm eueaoxnenoIA eueuuwz ¢.m m.v em enanoflnuoEIA eon: h.m m.n om eanOwnueeIA euenufiz o.m o.m mm eneeum>OIq eons m.m o.o mm oneoomSOIo ooouoez m.m h.m mm enemanm eons n.m N.@ No enemanm euenuflz AHInHeuOnm me x Aunmflez nmoum m\oEV AHImeo x we eonnow OOHnOm HInaE x moo moaoenv nweuoum emeouone unmwe3 nmonm m unwanm nemouuwz omeflsusmgm A: g meounom nemonuen one Romano noon no >Oneonemeo "sue>euoo omoasuseHSm Ase one spaces .NH oases 100 Stationary phase tobacco cells which have consumed all of the nitrogen and sulfur initially included in the medium have low levels of ATP sulfurylase. Because such cells have access to a minimal amount of free sulfur compounds, and contain a minimal amount of the enzyme, they are particularly suitable for use in studies of the kinetics of ATP sulfurylase development as a function of exogenously supplied sulfur. When these cells are transferred to fresh medium, ATP sulfurylase develops with kinetics that depend on the sulfur source. In the presence of readily assimilated sulfur sources such as sulfate, cysteine, or cysteine plus methionine, ATP sulfurylase specific activity remained continuously repressed at close to the initial level for 6 days (Figures 12 and 13). During this period, however, there was an increase of over 3-fold in total soluble pro- tein in the culture (Table 13). On the other hand, in cells slowly assimilating djenkolate sulfur, as judged by the slow increase of growth and soluble protein (Tables 13 and 14), the specific activity of ATP sulfurylase began to rise within 12 hr (Figure 12) and increased steadily during the subsequent 4 days, up to 10- to 25- fold above the initial level (Figures 12 and 13). After 4 days, when the growth rate of the cells on djenkolate declined, ATP sulfurylase specific activity decayed (Figure 13, Table 14). Subse- quently, when growth ceased, the level of ATP sulfurylase in cells grown on djenkolate returned to the low initial level, which is about the same as the repressed level found in cells grown on sulfate or cysteine. 101 o G: I I O l l (D r 1 n moles APS x min" a: mg protein" O 48 96 I44 Time (hr ) Figure 12. Kinetics of the development of ATP sulfurylase as a function of the sulfur source. Stationary phase cells grown on (N,S)- MID supplemented with 2.5 mM nitrate and 0.1 mM sulfate were aseptically harvested, washed with (N,S)’ MID and resuspended in (N,S)’ MID containing 2.5 mM nitrate and 0.1 mM of each of the sulfur sources: sulfate (O) , cysteine plus methionine (A ) , and djenkolate (O ) . At the times indicated, aliquots were aseptically removed for determinations of fresh weight, soluble protein and enzyme activity. 102 45 O) C) CD 1 I C) DD 0 I 1 n moles APS x min'I 1: mg protein" A! .. T‘{J 2 JH 7‘4. 0 O 214 ‘ 48 72 96 I20 Time (hr) Figure 13. Kinetics of the development of ATP sulfurylase: derepression by djenkolate and repression by sulfate or cysteine. Stationary phase cells grown for 10 days on (N,S)- MID supple- mented with 2.5 mM nitrate and 0.1 mM djenkolate were aseptically harvested, washed with (N,S)" MID, and resuspended in fresh (N,S)’ MID containing 2.5 mM nitrate and 0.1 mM of the sulfur source: sulfate (0), L-cysteine (D), and L-djenkolate (0). At the times indicated, aliquots were taken from each culture for determinations of fresh weight, soluble protein and ATP sulfurylase activity. 103 . nae cum me x nHE x we 05: HI . u HI . mac H n .NH enough nw oenwuomoo pnoswuemxo oEem scum eueoe o.me m.mo o.mm m.H ~.om o.me e.H «.me m.mo eon e.ee e.om o.m~ o.H ”.me m.~m m.~ ~.eo o.~e owe H.ee m.om o.o~ o.~ m.mo «.mm e.m m.om ~.em om H.mH e.He o.mm m.~ o.~oa o.e~ o.~ m.eoa m.mm me m.HH o.om o.m~ o.~ e.om o.m~ ~.m m.oe o.mm mo m.oa e.em e.on A.m o.ee m.om o.m o.me e.m~ om e.m m.no m.ea o.~ e.oe m.H~ e.m o.mm o.o~ om e.~ m.~m o.eH o.H o.~m ~.oe o.H H.om o.oe me o.H 4.6m o.oH m.H m.e~ «.mA e.e m.om m.on o oomoHsASMHom AH\oso 1H\oo nomonsnseesm 1H\oso Anxoo nooonsuoeeom lexoso Ae\oo inns nee someone unoeos nee aeouonm ozone: nee someone Drones ones nmeum nmeum nmeum IIIIImHjomzmauAIll 32% e255 emeonnow unmade mnoane> no nusoum mnflnoo nfiououm mannaom one unmfles nmenm ou uneemoae>oo omea>unmmnm mac m0 mwnmnOeueHOm .ma manea 104 These results suggest that under repressive conditions ATP sul- furylase was formed at ca. the same rate as total protein, while during sulfur-limited growth, an active enzyme accumulated at a much higher rate than that at which the average protein accumulated in the cells (see Table 13). Furthermore, the regulation of ATP sulfurylase during growth on slowly assimilated sulfur involved both derepression during active growth and a subsequent decay. Derepression of ATP sulfurylase also occurs in cells utilizing the slowly assimilated sulfur of glutathione, or in cells starved for sulfur in a "sulfur-free" medium containing a nitrogen source. Similar results are obtained with nitrate (Figure 14) or urea (Figure 15). Moreover, the rate of the derepression is inversely related to the growth rate, i.e., the lower the growth rate the higher the rate of development of ATP sulfurylase (Figures 14 and 15, see Table 9). If sulfate is included with djenkolate in the growth media, the level of ATP sulfurylase remains repressed (Figures 14 and 15). The derepression Of the enzyme is also prevented by the presence of cysteine with djenkolate (Figure 15) or cysteine plus methionine with djenkolate, indicating that it is the absence of the readily assimilated sulfur, rather than the presence of djenkolate which results in the derepression. Furthermore, the addition of sulfate to sulfur-starved cells in which ATP sulfurylase has been derepressed results in a rapid decline in the enzyme specific activity (Figure 16), which coincides with the resumption of net protein synthesis in response to the 105 H nweuo no os x H nee x mod meaoen n .MH eunmem no oenwnomeo unoEHnemxe ween Eonm eueoe o.mm 0.0a I I H.m 0.vm oma m.mo 0.va n.m m.mm m.m 0.m~ om v.mm m.ma e.m 0.mH v.v m.n~ we H.>m 0.ma e.m 0.HH >.v m.HH we m.o m.m m.H 0.HH m.v m.m em o.m 0.6 o.m 0.n o.m 0.5 0 nemeaaunmanm Aa\mv nemeaxunmanm AH\00 omeaxnnmanm AH\00 Anni PE 2vo3 e2 2vo3 o one 23oz ofie nmenm nmeum nmeum uH¢_dmzu1aI_ IMZHMHm»UI4 lukemnnm eonfloumao NO enemanm up nowmmeumeu on» one eueaoxneno an nowmmoumeueo on» mnfiuno zufl>fiuoe emeawunmanm man one nu3ono .va wanes 106 “3 C) T 5 l n moles APS x min'I I: mg protein" O 24 48 72 96 I20 Time (hr) Figure 14. Derepression of ATP sulfurylase during growth on glutathione or during sulfur starvation on nitrate. Stationary phase cells maintained continuously on (N,SI— MID sup— plemented with 2.5 mM nitrate and 0.1 mM sulfate were subcultured into (N,S)- MID containing 1.25 mM nitrate and 0.05 mM sulfate. After 9 days, cultures which contained ca. 13 g/l fresh weight were resupplemented with 2.5 mM nitrate and 0.1 mM of each of the sulfur sources: sulfate (0), sulfate plus djenkolate (I), reduced glutathione (A) , djenkolate (O), and no sulfur added (A). At the times indicated, aliquots were aseptically removed for determinations of fresh weight, protein and ATP sulfurylase activity. 107 5 n moles APS x min" I: mg protein" C” O0 23 48 7‘2 96 Time (hr) Figure 15. Kinetics of the repression-derepression develop— ment of ATP sulfurylase during growth on urea. Stationary phase cells maintained continuously on (N,S)- MID supplemented with 3.0 mM urea and 0.1 mM sulfate were subcul- tured into (N,S)’ MID containing 0.75 mM urea and 0.025 mM sulfate. After 7 days, cells were aseptically harvested, washed with (N,S)- MID and resuspended in (N,S)' MID supplemented with 3.0 mM urea and 0.1 mM of each of the sulfur sources: sulfate (O), sulfate plus djenkolate (D ) , cysteine (A), cysteine plus djenkolate (I), djenkolate (O) or no sulfur added (A). At the times indicated, aliquots were aseptically removed for determinations of fresh weight, protein and ATP sulfurylase activity. Initial inocula were 5.5 g/l of fresh weight cells. 108 30-1 I T T 1— fi— T5 1 ' 2 o 8 CA 0 ,. .-I s 20 X E I s “ x a . U) 0 0L 4 IO, 4 0’ CD '5 s C O O 24 38 72 98 E0 Time (hr) Figure 16. Kinetics of the decline of ATP sulfurylase activity upon addition of sulfate to sulfur-starved cells. Sulfur-starved cells on nitrate (O) or urea (0) ware described in the legends to Figures 14 and 15, respectively. After 60 hr (arrows), 0.1 mM of sulfate was added to aliquots of the sulfur— starved cells on nitrate (O) or urea (I). In addition, 0.1 mM djenkolate was also added to an aliquot of the sulfur-starved cells on urea ((3). At subsequent times, aliquots from each culture were removed for determinations of fresh weight, soluble protein, and ATP sulfurylase activity. 109 added sulfate (Table 15). Similar results are obtained when sulfate is added to cells that have been derepressed on djenkolate (see Figure 25-C). This decline in ATP sulfurylase specific activity upon addition of a repressing sulfur source to derepressed cells reflects a decay of enzyme rather than dilution as a result of increased total protein (Table 16). Thus, enzyme inactivation or degradation also plays a role in regulation of ATP sulfurylase. Djenkolate, however, is much less effective than sulfate in pro- moting both the decay of ATP sulfurylase and the resumption of net protein synthesis, when added to cells derepressed by sulfur star- vation (Figure 16; Table 15). The results are, therefore, leading to the conclusion that a repressing sulfur source is effective in both the repression of ATP sulfurylase as well as the decay of the enzyme. On the other hand, a derepressing sulfur source is effective neither in repression nor in the decay of ATP sulfurylase. IV. Specificity of the ATP Sulfurylase Repression- Derepression Mechanisms for Sulfur Amino Acids The activity of the sulfur amino acids cysteine, methionine, and djenkolate in the regulation of ATP sulfurylase is hypothesized to be due to their sulfur content. Nevertheless, alternative hypo- theses, e.g., that the a-amino group or carboxyl group determines activity, were conceivable. In an attempt to assess the specificity for the sulfur amino acids in the repression-derepression regulation, the effects of a mixture of non-sulfur amino acids on the development of ATP sulfurylase was studied. 110 . naeuonm on x nee x men medoan .H- . a- n .Aea unseen eemv “Snow ueume eHHeo oe>ueumrunmanm en» Scum oe>OEeu muonoflae Ou oeooe me3 eounom unmade one .nemonuen eenn no mHHeO oe>neum.nnmanm How ma eunoflh nw oenwuomeo unefieuemxe eEem scum eueoe m.~a 0.mm m.v m.ov om 0.ma >.ma om m.mH m.mm m.0H h.em NH m.oa o.Hm me I I I I I m.o 0.mm we I I I I I w.H e.ma 0 nemeamunmanm AH\oEv nemeawunmanm AH\mEV Anewueooe emeaawnmanm Aa\mav Anni naeuoum naeuoum Hoodoo ueume a man nweuoum eEHB nudge uH¢_amzutq auaqm mH¢m_:m .5: menu amaam m:m_:m dz Iueueuunmanm on enemane mo nowueooe nomn He>eH emeaaunmanm man no eneaoeo ecu nuw3 zdunequOOnoo memenunam nweuoum uen m0 nodumaneem .mH eases 111 .eunano hem H nHE x mm< meHoan me >DH>Huoe Heuoan , .unwoo ueume oeooe mes eueanm .enHer manHeunoo eueHan no nOHue>ueum uannm manno emeH>NDMHnm man no nOHmmeumeueo you .oH ennon nH oeanomeo uneEHuemxe eaem Bonn eueoe v.m0v ~.mb 0o h.mmo m.vm 0NH H.mmv . m.vo on h.mmh o.mm om o.mmb v.~m NH m.hmm o.~m me I I I m.mwo H.Hv we I I I n.mmH H.5v em I I I o.bm m.mm 0 HeunuHDO\mannnv HeunanO\mEv AnoHuHooe euem Heunan0\mannnV HewnuHDO\mEv Anne neeeHhunMHom nHeuoum IHnm neume any nemeHhuannm nHeuOHm eEHB mfid eEHB mad IllldmddmluHfi—JHVIIII IIIIdmdadlmgamlqzlll MHueumIeueHUHn nH oenHeuoO eue3 manmeH HeHHEHm . nHeuoum as x H H nHE x mod eeHOEn n :2 oo none... oeooe me3 eeuD .mHHeo oe>neumneenn How mH eHanm nH oenHHomeo uneEHnemxe eeem Edam eueoe o.v 0.0m om m.H v.0H om m.H ®.mN NH ©.H m.®H Nb I I I h.H o.hH we I I I m.H m.©H o nemeHawnMHnm HH\mE. Anny emeH>MSMHnm HH\oEv Hnnv Ase 530.8 2 oo note a use 5398 ones Illa: .I .I e82. lfladijmulzmudeHdl Inga—Mall IIIIImHfla—zmallll eneoouan m0 noHuHooe noon nOHumEnmen euH one mHHeO oe>ueuminemONan nH mHmenunwm nHeuOnm uen m0 eonemnd .RH eHneB 116 evident and continues with a rate characteristic of the derepression (Figure 18). The derepression of ATP sulfurylase level initiated by the addition of urea occurs concomitantly with the resumption of net protein synthesis (Table 17). Similar results are obtained upon addition of nitrate. Thus, active assimilation of nitrogen is required for the derepression of ATP sulfurylase. The requirement for nitrogen assimilation is also consistent with the finding of low ATP sulfurylase level in stationary phase (nitrogen starved) cells, regardless of the sulfur source. VI. The Effect of Inhibition of Protein Synthesis on the Derepgession of ATP Sulfurylase In an attempt to approach the molecular events underlying the derepression of ATP sulfurylase, the effect of inhibition of protein synthesis by cycloheximide on the development of ATP sulfurylase was investigated in cells grown on djenkolate. Cycloheximide, at 4 ug/ml, is an effective inhibitor of protein synthesis in the tobacco cells (70). The effect of cycloheximide on the development of ATP sulfurylase was studied in cells utilizing urea nitrogen in order to avoid the inhibition by cycloheximide of the induced development of nitrate reductase (181), which is essential for assimilation of nitrate nitrogen. The effect of cycloheximide on the derepression by dkenkolate sulfur of ATP sulfurylase was studied and compared with its effect on the enzyme level in control cells grown on djenkolate plus sulfate. Cycloheximide inhibited the rate of [14C]-arginine incorporation by the tobacco cells into protein by 95- 97% within 1 hr (Figure 19—B). 117 '5 -§ ‘5. E .:: zor - I0 3 5' E X a '5’ ° " Q 'o x '3 E E g? IOL - 5 § '3. '7} 2 ° 2 1—0 O—l O 4 1 . O O I 3 5 Time (hr) Time (hr) Figure 19. Inhibition by cycloheximide of the uptake and incorporation into protein Of [14C]-L—arginine in the tobacco cells. The effectiveness of cycloheximide as inhibitor of protein synthesis in the tobacco cells was studied concomitantly with its effect on inhibition of the development of ATP sulfurylase, in cells grown on 3.0 mM urea and 0.1 mM djenkolate as described in the legend to Figure 20. After 48 hr, aliquots of 30 ml were aseptically removed and incubated with 0.01 mM L-arginine plus 2 uCi of uniformly labeled [14C1-L-arginine, with 4 ug/ml cycloheximide (O) or without it (0). At 1, 3 and 5 hr thereafter aliquots were removed for determinations of uptake (A) and incorporation into protein (B) of the radioactive arginine (see Materials and Methods). 118 This inhibition could not be attributed solely to the inhibition by cycloheximide of the uptake of arginine. The latter was inhibited by cycloheximide only by 26-39% within 1 hr (Figure 19-A). Inhibition by cycloheximide of the rates of uptake and incorporation of [14C]- arginine into protein in cells grown on djenkolate (Figure 19) was identical to the control cells utilizing djenkolate plus sulfate. The cycloheximide effects are, therefore, independent of the sulfur source available to the cells. Cycloheximide, added to cells that have been derepressed for 48 hr, results in a complete inhibition of the development of ATP sul- furylase, observed within 1 hr after its addition (Figure 20). The inhibited level of enzyme in treated cells, however, remained constant during the subsequent 24 hr. If added at 72 hr, cycloheximide like- wise prevented the continuation of enzyme development. Cycloheximide has little or no effect on the repressed level of ATP sulfurylase in cultures grown on djenkolate plus sulfate (Figure 20). These findings indicate that the mechanism of the derepression of ATP sulfurylase in the tobacco cells depends in some way upon protein synthesis. B.2 Regulation of ATP Sulfurylase by Group VI Anions The physiological and biochemical studies described in the pre- ceding section indicated that the availability of sulfur to support growth of the tobacco cells governs repression-derepression of ATP sulfurylase. In efforts to further analyze this regulatory system, use was made of the observation that Group VI anions are structural analogs 119 I5 . 5 (fl n moles APS x min" x mg protein" 0L 1 1 1 l O 24 48 72 96 Time (hr) Figure 20. Inhibition by cycloheximide of the development of ATP sulfurylase during derepression on djenkolate. Stationary phase cells maintained on (N,S)- MID supplemented with 3.0 mM urea and 0.1 mM sulfate were subcultured into (N,S)‘ MID containing 0.75 mM urea and 0.025 mM sulfate. After 7 days, cells were aseptically harvested, washed, and resuspended in (N,S)” MID containing 3.0 mM urea and sulfur as either djenkolate ((3) or djenkolate plus sulfate (Ci), each at 0.1 mM. At thé times indicated, aliquots were aseptically removed for determinations of fresh weight, soluble protein and enzyme activity. After 48 and 72 hr (arrows), aliquots were aseptically removed and incu- bated with 4 ug/ml of cycloheximide. Aliquots from the cyclohexi- mide treated cells were assayed after Band 24 hr following the addition of the inhibitor. Open symbols are control untreated cells, closed symbols are cycloheximide treated cells. The initial inocula contained 5.5 g/l of fresh weight cells. 120 of sulfate in the ATP sulfurylase reactions in vitro. Logically, if these analogs of sulfate can also inhibit ATP sulfurylase activity in vivo, a specific perturbation of the sulfate assimilation system could be achieved. Such a perturbation could ultimately alter the level of sulfur-containing metabolites in the cells, which in turn could bring about changes in the level at which ATP sulfurylase is regulated. The specific prediction is that if ATP sulfurylase is derepressed by a decrease in the concentration of a sulfur-containing compound synthesized via ATP sulfurylase, then the Group VI anions, which are known to inhibit ATP sulfurylase activity in vitro, should derepress the enzyme in cells growing on sulfate, but not on cysteine or methionine. No previous attempt appears to have been made in any system to detect changes in extractable ATP sulfurylase activity as a result of growth in the presence of Group VI anions. I. Group VI Anions as Inhibitors of APS Formation in vitro The synthesis of APS, catalyzed by the tobacco cell ATP sul- furylase, is inhibited by Group VI anions in Vitro (Table 18), as has been found before for other ATP sulfurylases (47,104,107) and for other reactions catalyzed by ATP sulfurylase (e.g., 45,102). Molybdate, tungstate and selenate inhibited the incorporation of [3SS]-sulfate into APS in a concentration dependent manner, while chromate inhibited completely even at the lowest concentration tested (Table 18). Although the chromate result is in agreement with the high potency of chromate found by others in viva (72,74,76), it may not be inhibiting as a sulfate analog but rather in a 121 Table 18. Inhibition of APS formation in vitro by Group VI anions Anion concentration BIB 5m EHBXI ASE “11%le (BMQ'ES AESZE MIN) (mM) Molybdatea Tungstate Selenate Chromate o 560 448 448 443 20 280(50)C 193(43) 121(27) o 50 125(22) 134(30) 54(12) 0 100 56(10) 54(12) 21(5) 0 a,b Complete reaction mixtures of 0.5 ml (see Materials and Methods) contained 20 mM sulfate and either molybdate, tungstate, selenate or chromate at the indicated concentrations. Extracts were prepared from exponential tobacco cells, grown on MID for (a) 4 days or (b) 3 days and added at (a) 29 ug protein/assay, and (b) 35 ug protein/assay. Assays were conducted at 30 C for 5 min, and APS was determined as described in Materials and Methods. c . . . Numbers in parentheses represent percent of activ1ty observed with no anion added. nonspecific manner, e.g., as a protein denaturant. Tellurate, at 10 mM, did not inhibit APS formation (data not shown). Higher con- centrations, however, were difficult to obtain because of the insolu- bility of sodium tellurate in water. This could account for its rare use in biological studies involving Group VI anions. The kinetics of inhibition of APS formation were examined in detail for selenate, the only Group VI anion which is thought to be incorporated in plants into analogs of sulfur compounds via ATP | sulfurylase. 122 The inhibition of [3ssl—APS synthesis by selenate clearly exhibited competitive kinetics with respect to sulfate. The depen- dence of the rate of APS formation upon sulfate concentration followed Michaelis-Menten kinetics with an apparent Km for sulfate of 1.48 mM (Figure 21). The inhibition constant, Ki, of selenate ranged between 0.64 and 0.7 mM (Figures 22 and 23). Thus, selenate has a slightly greater affinity for the ATP sulfurylase of tobacco cells than the natural substrate, sulfate. II. The Effects of Group VI Anions on Growth and ATP Sulfurylase Level of Tobacco Cells The evidence pointing to a role for ATP sulfurylase in the toxic action of Group VI anions in vivo is rather limited: (a) growth of mutant strains of Salmonella (78), Aspergillus (76), and Penicillium (72) lacking ATP sulfurylase but functional in sulfate transport, exhibited resistance to molybdate (78) and selenate (72,76) under conditions in which the growth of the wild type was completely inhibited; (b) the inhibition of growth of E. coli by selenate or molybdate was completely prevented by conditions which were known to repress strongly the ATP sulfurylase activity in the absence of the Group VI anions (121); and (c) preloading the red alga Porphgridium with a high concentration of molybdate inhibited the incorporation of [3SS]-sulfate into sulfate esters under con- ditions that did not inhibit sulfate transport (111). With these considerations in mind, and the availability of a convenient assay for APS formation, a study was undertaken to determine the effects of the sulfate analogs in vivo on growth and ATP sulfurylase in the tobacco cells. 123 Figure 21-A. Dependence of the rate of APS formation on sulfate concentration. Complete assay mixture prepared as described in Materials and Methods, with various concentrations of sulfate. Crude ATP sulfurylase was prepared from exponential tobacco cells grown on MID. The tubes were incubated for 5 min, and APS was determined as described before. Figure Zl-B. Double reciprocal plot (Lineweaver-Burk) of the rate of APS formation versus sulfate concentration. The apparent Km for sulfate (arrow) is 1.48 mM. n moles APS /5 min l/v (n moles APS/5 min)" 124 IO 20 SULFATE (mM) l l l l l 2 3 4 |/[s], (mM SULFATE)" Figure 21 125 l5 - . '3 E up ((D- ' \ 00 G. <3 ‘ 2 I O o E 5 > 5 - . > - :gfa““.p 4/ 1 n n 1 O 0.5 LO L5 2.0 '/[S] . (MM 304.2) -| Figure 22. Double reciprocal plot of the concentration- dependent inhibition of APS formation by selenate. Tobacco cell extracts from exponential cells grown on MID were incubated for 5 min in complete reaction mixtures with varying concentrations of sulfate, in the absence (I) or in the presence of selenate at 0.5 (0) mM, 1 M (A), 2 mM (0) and 5 mM (0). Km for sulfate (arrow) is ca. 1.48 mM. Ki is ca. 0.64 mM. 126 E7: I 5 l/v (n moles APS/5 min)" 01 i SELENATE, mM Figure 23. Dixon plot of the concentration-dependent inhi— bition of APS formation by selenate. Same data as in Figure 22, but plotted according to Dixon (127). Assay mixtures contain 0.5 mM (0), l 1114(0), 2 M (A), 5 mM (0), and 10 mM (I) sulfate, in the presence of various concen- trations of selenate. The inhibition constant, Ki, for selenate (arrow) is ca. 0.7 mM. 127 Molybdate or tellurate, at concentrations equimolar with sulfate, caused relatively small changes in either growth or ATP sulfurylase specific activity (Table 19). Higher concentrations of either analog gradually inhibited growth but the specific activity of the enzyme remained relatively unchanged. In contrast, selenate at a concentration equimolar with sulfate was toxic to the cells, but the ATP sulfurylase level increased as much as 3—fold compared with cells grown on sulfate only (Table 19). Moreover, the increase in ATP sulfurylase specific activity diminished at more toxic concentrations of selenate. Because the cells were grown in the presence of Group VI anions, there was the possibility that the ATP sulfurylase activities in extracts were affected by direct inhibition of the enzyme by high concentrations of anions in the extracts, rather than being a simple reflection of the amount of enzyme present. This possibility was tested and excluded in the following manner. The rate of APS forma— tion was measured at two dilutions of extract, different by a factor of 2, and found to be proportional to the dilution factor. This result means that the anions in the extracts were at too low a con- centration to measurably inhibit enzyme activity. An attempt was made also to see if an increase in activity resulted from dialysis of the extracts, which would be expected if dialyzable anions were appreciably inhibitory. No such increase was observed. However, these results were complicated by the fact that there was a substantial loss of activity after 7 hr of dialysis. This is in contrast to the complete stability of ATP sulfurylase 128 Table 19. Effects of Group VI anions on growth and ATP sulfurylase in vivo 613 DAY 1113 DAY Group VI anion Fresh weight ATP Fresh weight ATP added (in mM) (g/l) sulfurylasea (g/l) sulfurylasea None 8.9 3.0 26.5 2.1 Molybdate, 0.1 6.8 3.2 21.5 1.7 Molybdate, 0.5 4.6 3.3 - - Molybdate, 1.0 3.3 3.7 11.2 1.8 Tellurate, 0.1 5.0 3.4 20.0 2.6 Tellurate, 0.5 5.2 4.1 15.5 3.6 Tellurate, 1.0 4.6 4.3 - - Selenate, 0.1 3.1 8.1 5.7 6.8 Selenate, 0.5 1.5 4.3 - - Selenate, 1.0 0.6 - - - Tungstate, 0.1 0.1 - - - Chromate, 0.1 0.4 - - - anmoles APS x min.1 x mg protein-1. .I Stationary phase cells were subcultured into (N,S)- MID supplemented with 2.5 mM nitrate and 0.1 mM sulfate. Sodium salts of Group VI anions were added to the culture media at zero time. After 6 and 11 days, the cells were harvested, and fresh weights, soluble pro- tein and ATP sulfurylase activities were determined. The initial fresh weight was 1.5 g/l, and ATP sulfurylase activity was 2.0 nmoles APS x min"1 x mg protein-1. 129 activity found in undialyzed tobacco cell extracts kept frozen at -15 C for 3 days. Both tungstate and chromate, at 0.1 mM, were very toxic to the tobacco cells and resulted in complete inhibition of growth (Table 19). The tungstate effect is believed to reflect its interference with the development of functional nitrate reductase; chromate, on the other hand, is a nonspecific toxicant to many organisms. The enzyme assays presented in Table 19, however, were done after the cells had been growing for 6 days or more in the presence of the anions. The responses to the anions at earlier times were examined in separate experiments (Tables 20 and 21; Figure 24). In order to obtain enough cell material for multiple analyses, station- ary phase cells were resuspended in the test media, but with less dilution than in the previous experiment (see Table 19), so that aliquots could be taken from one culture at various times. A gradual increase in extractable ATP sulfurylase specific activity of up to ca. 2.5-fold above the control occurred over 4 days in cells supplied with molybdate, but only when the molar ratio of molybdate to sulfate in the medium approached 10 (Table 20). Under these conditions, the total soluble protein did not increase during the 4—day incubation with molybdate. Thus, the increase in enzyme specific activity observed upon incubation with molybdate (table 20) could have resulted from sulfate starvation imposed by the high concentration of the inhibitory sulfate analog. The effect of selenate on the ATP sulfurylase, however, became evident in cells grown on selenate at 1/10 the concentration of 130 muosvfiam .ooumoaocw mesa» on» ad .mHHoo unmflm3 nmoum mo H\m vm .mo omcwmucoo «HSUOCH Howuwcw one .auw>wuom mahnco use cwwuoum wannaom .unmwms nmoum mo mcoflumcwsuouoo new om>OEmH maamowummmm ouo3 .mumoQAHOE mo mcofluouucoocoo msOwum> pom mummasm 28 H.o .mumuuflc :2 m.~ mafiaflmucoo on -xm.zo as omocmmmsmmu 8:4 9H: -xm.zv suns omnmms .omumm>umn adamoaummmm mums mummasm 28 H.o one oumuuwc 2E m.~ spas omucmsmammsm 9H2 IAm.zv so csoum mHHwo omega aumcoflumum . owmuoum as x CHE nu ma mmflogfl H H H.m m.mm m.m w.eh m.m m.mm om m.m m.mm m.m m.vn m.m m.vm Nb m.m N.om m.m o.mo v.v m.mo we v.v n.Hm m.m m.ve o.m o.ov vm m.H v.am o.H o.mm o.H m.mm o nonnamusmasm AH\mEV ommoazuswflsm Aa\mfiv mmmahuSMHSm AH\OEV szv mam cfimuonm m9< cflououm mam aflououm mafia dz o .H has mica mdeHoo on» so mumonxaoe mo uoomum one .om wanes 131 8" -i ‘7 .5 2 O 5 CL 0 5. . E X T .5 E X a) 4L 4 GL ‘1 ‘ 2 O E M. f 2 O 1 n L 1 4 0 24 48 72 96 Time (hr) Figure 24. Derepression by selenate of ATP sulfurylase in cells grown on sulfate. Stationary phase cells grown on (N,S)- MID supplemented with 3.0 mM urea and 0.1 mM sulfate were subcultured into fresh (N,S)" MID supplemented with 0.75 mM urea and 0.025 mM sulfate. After 7 days, the cells were aseptically harvested, washed with (N,S)- MID and resuspended in (N,S)‘ MID containing 3.0 mM urea, 0.1 mM sulfate and either zero (0) or 0.01 mM (A) selenate. At the times indicated, aliquots were removed for determinations of fresh weight, soluble protein and enzyme activity (see Materials and Methods). The initial fresh weight was 5.5 g/l. After 4 days, fresh weight determinations for both cultures were ca 15 g/l. ‘ 132 Table 21. The effects of selenate on soluble protein and ATP sulfurylase in cells utilizing sulfate for growtha SELENATE CONCENTRATION, None 0.01 mM Time Protein ATP Protein ATP b (hr) (mg/l) sulfurylaseb (mg/l) sulfurylase 0 15.4 1.8 15.4 1.8 48 31.5 2.2 25.0 5.4 72 54.6 2.2 53.2 8.9 96 63.0 2.6 76.5 6.9 aData from same experiment as Figure 24. bnmoles APS x min"l x mg proteindl. sulfate in the medium; a 4-fold increase in enzyme specific activity developed within 3 days (Figure 24) with no apparent inhibitory effect on growth or net protein synthesis in the cells (Table 21). Moreover, in contrast to molybdate, selenate was most effective in derepressing ATP sulfurylase when it was at sub-toxic or incipiently toxic concentrations (see Tables 19 and 21). The differences in the derepression by selenate as compared with molybdate suggest that they act by somewhat different mechanisms. According to the published literature in a wide variety of organisms, the sulfate transport system does not discriminate 133 between selenate or molybdate, whereas ATP sulfurylase acts in dif- ferent ways with respect to selenate and molybdate (see Literature Review). These differences suggest that the derepression by selenate is due to the synthesis, via ATP sulfurylase, of a compound Sex, an analog of a natural metabolite, SX, with a regulatory function, while derepression by molybdate is due to sulfur deprivation via inhibition of sulfate assimilation at the ATP sulfurylase step. III. Derepression of ATP Sulfurylase by Selenate: Dependence on the Sulfur Source The effect of selenate on the development of ATP sulfurylase activity in cells utilizing sulfate for growth was compared with its effect on cells utilizing either cysteine or djenkolic acid. Addition of 0.03 mM selenate to a culture growing on 0.1 mM sulfate resulted in more than a 4-fold derepression of ATP sulfurylase (Figure 25-A), with little or no inhibition of growth. However, selenate at the same concentration, 0.03 mM, added to cells growing on 0.1 mM L-cysteine, did not cause derepression of the enzyme (Figure 25-B). Again, growth was not inhibited. When 0.03 mM selenate was added to a culture growing on 0.1 mM L-djenkolate, growth was totally inhibited and so was the 27-fold derepression observed in the absence of selenate (Figure 25-C). Selenate, at 0.1 mM, is more toxic to cells grown on sulfate, and resulted in much less derepression than at 0.03 mM (Figure 25-A). However, selenate at 0.1 mM was not toxic to cells grown on cysteine, nor was it effective in derepressing ATP sulfurylase in such cells (Figure 25-8). The ineffectiveness of selenate, in causing dere- pression when cysteine is the sulfur source, is not altered by the 134 .meeflu msofiue> ue nexeu muonoflae no oenfleueueo euez >ua>wuoe mewune one naeuoum wannaom Somme.» nmeum .ACV eveneaem ZS moomnfinfieunoo meguano one . ADV eueneaem on no”: mounuano mo muosofiae HE om on oeooe maeafiueum me3 enemanm SE mo.o .Amsouuev no N“. Hound .An: eueneaem 25 ad no .Adv eveneaem SE mo.o . AOV openeaem on oenaeunoo omae mounuHsU .eou90m unmade eHOm on» me ADV eueaoxnenouq SE H.o no .Amv enweumxolq :8 H.o .Aev eueMHSm SE H.o nonufie one meadow nemouufln won me eueuuan SE m.m mnanaeunoo OH: iAm.zv nmeuu no oeonemmnmeu one .on iAm.zv sues oenmez .oeume>uen waaeofiumeme ewes eueaoxneno :5 H.o one eueuufin SE m.~ news oeuneseammnm DH: Am.zv no mxeo 0H now nsoum maaeo emenm wuenoHueum .eueneaem xo emeaxnsmasm men mo nowmmeumeueo on» no moonnom unmade mnoflue> mo muoemwe one .mm ennmfim 135 00 mm madman cs 2.: .2. 2.: 3.2: 2. 9. 3 o 8. 8 fl. 2. ea c o c 1 o 4 no. .. . 2 a. a .o~ mu ‘4 “mound bus 8 ,-ogm a: say snow It 136 addition of 0.03 mM sulfate to the culture grown for 72 hr with 0.1 mM cysteine and 0.03 mM selenate (Figure 25-B). Thus, it is the presence of cysteine rather than the absence of sulfate which is responsible for this lack of a selenate effect. Also, the severe inhibitory effect of 0.03 mM selenate on cells utilizing djenkolate is not reversed upon a subsequent addition of 0.03 mM sulfate (Figure 25-C). Thus, there is more to selenate toxicity for cells growing on djenkolate than mere sulfur starvation, which would be overcome by addition of sulfate. Regardless of the final interpretation, the effect of selenate on ATP sulfurylase development is clearly a function of the sulfur source used by the cells. IV. Selenate Toxicity as a Function of the Sulfur Source Utilized for Growth The experiments on the derepression of ATP sulfurylase by selenate described in the previous section suggest that the toxic effect of selenate on growth was enhanced by derepression of ATP sulfurylase and diminished by repression of the enzyme in the tobacco cell system. In order to test the validity of this interpretation, a series of growth experiments were performed in which conditions favoring repression or antagonizing the repression were created, and the sensitivity of growth to selenate was determined. Table 22-A shows that selenate toxicity in tobacco cells is a function of: (a) the molar ratio of [selenatel/[sulfur]; and (b) the nature of the sulfur source. 137 Table 22-A. Inhibition by selenate of growth of tobacco cells on various sulfur sources FRESHIHEIGHT. G/L Selenate (uM) Sulfate Djenkolate Cysteine None 25.5 8.5 22.0 1 25.0(98)a 3.6(28)a 17.5(78)a 3 25.5(100) 1.1(<0)b 16.1(71) 10 9.7(34) o.7(<0)b 8.7(35) 3o 2.2(2) 0.7(<0)b 8.7(35) 100 0.4(<0)b o.6(<0)b 5.9(21) a . . . Numbers in parentheses are percent of increase in fresh weight observed with no selenate added (for each sulfur source used). b . . . . . Indicates an actual net loss of initial fresh weight. Stationary phase cell which had been maintained continuously on (N,S)- MID supplemented with 2.5 mM KN03 and 0.1 mM Na2804, were subcultured into fresh (N,S)' MID containing 2.5 mM KN03, 0.1 mM of the sulfur source indicated, and various concentrations of sodium selenate. After 12 days, cells were harvested and the fresh weights were determined in duplicate cultures, and averaged. Initial fresh weight was ca. 1.7-2.0 g/l. 138 Cells on cysteine exhibit resistance to selenate at concentra— tions which completely inhibit cells utilizing sulfate. Selenate present at 1/100 the concentration of the sulfur source has little or no effect on cysteine- or sulfate—dependent growth, but is strongly inhibitory to cells growing on djenkolate (Table 22—A). Hart and Filner (123) showed that L-arginine inhibited the uptake of L-cysteine by tobacco cells, and consequently acted as an antagonist to the inhibition by cysteine of sulfate uptake. If cysteine protects cells from selenate toxicity by repressing the sulfate uptake system and/or ATP sulfurylase, then arginine should antagonize this protective action. Accordingly, arginine was tested as an antagonist of the cysteine-dependent resistance to selenate toxicity. Arginine, supplied in equimolar concentrations with cysteine, has no effect on yield of cells after 12 days of growth. However, it reduces their resistance to selenate to a significant extent (Table 22-B). This reduction was partially overcome by including sulfate in the medium, as would be expected if the inhi- bition of growth were due to a greater influx of selenate when arginine inhibited cysteine uptake. Furthermore, cysteine protects against the severe toxicity by selenate encountered in cells grown on djenkolate (Table 22-C), and arginine is an antagonist of this protective effect of cysteine as well. Thus, the results in Tables 22 A-C support the hypothesis that selenate toxicity in tobacco cells is mediated by the steps of the normal sulfate pathway, and conditions which repress or inhibit one 139 Table 22-B. The effect of L-arginine on the protection by L-cysteine of tobacco cells from inhibition of growth by selenate FRESH HEIGHT. G/L Cysteine, Cysteine, arginine, Selenate (pM) Cysteinea arginine sulfate None 22.0 26.0 26.5 1 17.5(78)b 22.7(86)b 25.8(97)b 3 16.1(71) 12.5(44) 24.9(94) lO 8.7(35) 2.3(3) 21.7(81) 3o 8.7(35) 1.6(<0)C 7.8(25) 100 5.9(21) 1.5(<0)C 2.3(2) aData taken from Table 22-A. bNumbers in parentheses are percent of increase in fresh weight observed with no selenate added (for each sulfur source used). c . . . . . Indicates an actual net loss of initial fresh weight. The experiment was carried out as described in Table 22-A, except that in addition to L-cysteine as the sulfur source, L-arginine or L-arginine plus sulfate, each at 0.1 mM, were added at zero time. 140 Table 22-C. The effect of L-cysteine on the djenkolate-dependent susceptibility to growth inhibition by selenate FRESH mamHL G/L Djenkolate, Djenkolate, cysteine, Selenate (uM) Djenkolatea cysteine arginine None 8.5 21.1 25.7 1 ‘ 3.6(28)b 18.5(87)b 23.0(89)b 3 1.1(<0)C 14.6(67) 8.1(27) 10 o.7(<0)C A 5.1(18) 3.2(6) 3o o.7(<0)C 3.3(8) 1.8(0) 100 o.6(<0)C 2.9(6) o.7(PROTE I N SULFUR , CONTAINING / METABOLITES Such a model was originally proposed by Monod and Jacob (198) as one possible way by which two different inducible or repressible path- ways could be interconnected via regulatory elements. This concept appears to resolve the apparently contradictory observation that stationary phase tobacco cells, although starved for sulfur, contain a low level of ATP sulfurylase. However, under the culture conditions used, 2.5 mM nitrate and 0.1 mM sulfate, the tobacco cells are in fact starved for both nitrogen and sulfur in the stationary phase (123,179). Consequently, nitrogen starvation is overriding the signal of sulfur limitation and prevents the derepression of ATP sulfurylase. It is worth noting that possibly similar observations have been described previously in the literature. Kredich, in his studies with Salmonella (126), noted that derepressed cells on djenkolate 154 had a very low level of ATP sulfurylase activity if they were har- vested during the late log or stationary phases of growth. He could not explain the results. Our findings suggest that perhaps the bacterial cultures in the stationary phase of growth had also depleted their nitrogen supply. The low level of ATP sulfurylase in nitrogen-starved cells proved to be rather useful in the studies presented here. Nitrogen starvation was a convenient device for obtaining low enzyme levels for initial conditions in derepression studies. The parallel consumption by the tobacco cells of the nitrate and sulfate provided at 2.5 mM and 0.1 mM, respectively, suggested that the desired stationary phase cells could be obtained by main- taining the molar ratio of nitrogen:sulfur of 25:1, regardless of the absolute amounts. Indeed, cells grown for 9 days in media con- taining 1.25 mM nitrate and 0.05 mM sulfate exhibited the desired low level of ATP sulfurylase characteristic of stationary phase cells (Figure 14). Consequently, lower amounts of nitrogen and sulfur were provided at a molar ratio of 25:1 when cells were needed within a short period of time (e.g., Figure 15). This concept also predicts that if the nitrogen supply exceeds the molar ratio of 25:1, stationary phase cells should have a high level of ATP sulfurylase activity as a result of the sulfur limita- tion for growth. Derepression of ATP sulfurylase has been found in stationary phase tobacco cells utilizing casein hydrolysate, 0.5 g/l, as the sole source of both nitrogen and sulfur (Figure 17). These results are not surprising because 0.5 g/l of casein hydrolysate is calculated to contain 3.6 mM nitrogen (Difco specifications), and 155 0.06 mM of sulfur amino acids (183), which results in a molar ratio of nitrogen:sulfur of 60:1. The proposed influence of the molar ratio of nitrogen to sulfur on the assimilation of sulfate via ATP sulfurylase in the tobacco cell system is supported by an independent study on the sulfate transport system in the XD cells carried out by Smith (124). He observed an increase in the rate of uptake of sulfate in stationary phase cells utilizing either sulfate of cysteine as the sole sulfur source. Contrary to his interpretation that the intracellular sulfate pool is the regulating factor, I wish to suggest that his results reflect simply sulfur starvation, because the cells were grown on 2.5 mM nitrate and 0.05 mM sulfur as either sulfate or cysteine, resulting in a ratio of nitrogen:sulfur of 50:1. The molecular bases for the described biochemical changes in ATP sulfurylase specific activity in response to specific perturba- tions in environmental sulfur and nitrogen are not known. Several lines of evidence suggest, however, that these changes are due to changes in the rate of synthesis and/or the rate of degradation of the enzyme molecules rather than activation-inhibition of pre- existing enzyme molecules. The evidence consistent with this idea is as follows: (a) The sulfur sources which mediate the changes of enzyme activity in vivo do not affect the enzymatic activity in vitro (Table 11) . (b) Derepression of ATP sulfurylase is inhibited by cyclohexi- mide (Figure 20) at a concentration which strongly inhibits incorpora- tion of amino acids into protein (Figure 19). Previous studies with 156 the tobacco cells indicated that cycloheximide at such a concentration does not inhibit the conversion of inactive tungsto-nitrate reductase to active molybdo-nitrate reductase, which is a slow activation of a pre-existing inactive enzyme (70,122), suggesting that the inhibi- tion of protein synthesis by cycloheximide in the tobacco cells is relatively specific. The effect of cycloheximide added to derepressed cells growing on djenkolate sulfur (Figure 20) is rather distinct, kinetically, from the effect of adding sulfate to such derepressed cells (Figure 25-C). Thus, cycloheximide does not promote the decay of ATP sul- furylase, but sulfate does cause decay (Table 16). Presumably, this decay is part of the regulatory processes by which the level of ATP sulfurylase can be altered. The results of these investigations using Group VI anions demonstrated that either molybdate or selenate, when included with sulfate in the culture medium, can cause a derepression of ATP sulfurylase (Tables 20 and 21, Figure 24). Kinetically, the dere- pression by these Group VI anions was rather similar to the drepres- sion by sulfur starvation (e.g., Figure 15). Analysis of the molybdate-dependent derepression supported the idea that molybdate acted upon ATP sulfurylase in vivo as an inhibitor of APS formation, and thereby imposed sulfur starvation in the cells, which in turn initiated the development of the enzyme (Table 20). On the other hand, the mechanism of the selenate-dependent derepression of ATP sulfurylase appears to be other than a mere sulfur starvation. Selenate, which is a competitive inhibitor of the activation of sulfate by the tobacco cell ATP sulfurylase in 157 vitro (Figures 22 and 23), derepressed the enzyme in vivo only when present at such a low concentration that neither the growth of the cells, nor the assimilation of sulfate into protein, was affected (Table 21). At higher concentrations, at which selenate becomes toxic, the derepression of ATP sulfurylase does not occur (Figure 23-A). It is suggested, therefore, that unlike molybdate, the selenate-dependent derepression was mediated by a seleno-compound synthesized via ATP sulfurylase to yield the seleno-containing antimetabolite of the hypothetical regulatory sulfur compound SX, the predicted corepressor of the sulfate pathway. I wish to suggest that the selenium analog is acting as an anti-corepressor. The proposed mechanism for the selenate-dependent derepression response predicts that selenate is metabolized by the tobacco cells via ATP sulfurylase. This is supported by the correlation between the.level of ATP sulfurylase in the tobacco cells and the toxicity of selenate to growth. Thus, selenate is extremely toxic to cells with a derepressed level of ATP sulfurylase (Figure 25-C, Table 22-A). Both the derepression of ATP sulfurylase by selenate via Sex, and the toxicity of selenate due to the incorporation of Se- selenocysteine and Se-selenomethionine into protein would require assimilation of selenate via the enzymes of the sulfate pathway. The proposal that selenate is incorporated by the tobacco cells into a seleno—analog, Sex, of the natural sulfur-containing corepressor, SX, and that Sex interferes with the repressive response, should be put to experimental test. With the aid of [7SSeJ-SeO4-2, it might be possible to isolate and identify the selenium containing anti-corepressor. 158 Although the actual corepressor of the sulfate pathway has not been identified as yet in any system, it is thought to be a close derivative of cysteine or methionine. It is therefore conceivable that Se-selenocysteine and Se-selenomethionine, both of which are commercially available, can be used to trace the hypothetical anti- corepressor. Preliminary attempts to use these seleno-analogs in the tobacco cell system were unsuccessful. At 0.05 mM of the seleno- analog with 0.1 mM sulfate in the culture medium, both analogs were very toxic to the growth of the cells. Lower concentrations were not tested. Based on the studies with selenate (Figures 24 and 25), the seleno-analogs are predicted to cause derepression of ATP sul- furylase only when present at sub-toxic concentrations. 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