. .q .»n.n— o"‘—~-.~M—“‘ L ACTIVATION 0F ASPARTATE TRANiSCARBAMYLASE OF- 2.2V" ESCHERICHIA COLI’BY PURINE NUCLEOTIDES' ‘ n. REGULATION OF NITRITE REDUCTASE IN TOBACCOCELLS ‘ * Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY HANNA CHROBOCZEK KELKER 1969 ,HESIS __.— Raw LIB RA R Y M iii-1:311] S tai Uni r. ersiiy I vr-VV vw —— -*—— This is to certify that the thesis entitled l. Activation of Aspartate Transcarbamylase of Escherichia Coli by Purine Nucleotides. ll. Regulation of Nitrite Reductase in Tobacco Cells. presented by Hanna Chroboczek Keiker has been accepted towards fulfillment of the requirements for ,1) . n 1* . 9’ 0 na fl ‘1‘” Mdegree in {bin/w! C A“ Kama/W / Qarfifimimlg Major professor Date «(f/”u; ’7’. / (Z 6- ‘1 0-169 ABSTRACT I. ACTIVATION OF ASPARTATE TRANSCARBAMYLASE OF ESCHERICHIA COLI BY PURINE NUCLEOTIDES II. REGULATION OF NITRITE REDUCTASE IN TOBACCO CELLS BY Hanna Chroboczek Kelker I. The properties of an ig_vitro System from Escherichia 29;; were examined in which an increase in aspartate trans- carbamylase activity occurred upon incubation with adenosine 5'—triph05phate (ATP), guanosine 5'-triph05phate (GTP), phos— phoenolpyruvate (PEP), amino acids and magnesium ions. The objective of this study was to ascertain whether protein synthesis or activation is reSponsible for the increase in enzyme activity. Utilizing density labeling and subsequent density centrifugation it was demonstrated that the increase in aSpartate transcarbamylase was not due to ge_ggyg synthesis of this enzyme or to extensive completion of preexisting peptides. It was demonstrated by kinetic studies that incuba- tion of Escherichia coli homogenate with ATP, GTP, PEP, amino acids and magnesium ions or of crystalline aSpartate transcarbamylase with the same components, resulted in activation of the enzyme. Hanna Chroboczek Kelker ASpartate transcarbamylase was activated in a syner- gistic manner by ATP and GTP in the presence of magnesium ions. Previously published studies showed that ATP is an activator and GTP is an inhibitor of this enzyme. Magnesium ions modify the effect of purine nucleo- tides on aSpartate transcarbamylase activity. GTP in equi— molar concentration with magnesium ions had little effect on enzyme activity, while ATP and equimolar magnesium together were much better activator than ATP alone. The activation of aSpartate transcarbamylase by ATP and GTP in the presence of magnesium ions reported here might be of biological significance in equilibrating the pools of pyrimidine nucleotides and purine nucleotides of Escherichia coli. II. The nitrate reductase activity of tobacco cells in liquid culture has been reported to be regulated by both its substrate, nitrate, and by end products of the nitrate assim— ilatory pathway, amino acids. The objective of this study was to determine if nitrite reductase, the next enzyme of the nitrate assimilatory pathway, is similarly regulated. It was also of interest to determine whether nitrate has to be converted to nitrite before it can cause an increase in nitrite reductase activity. A modification of an assay of nitrite reductase activity utilizing methyl viologen reduced by sodium hydro- sulfite was developed and the optimum conditions for Hanna Chroboczek Kelker determination of nitrite reductase activity of tobacco cells were established. Nitrite reductase activity increased simultaneously with the activity of nitrate reductase in tObacco cells grown on nitrate. It was demonstrated that tungstate, which inhibits the development of nitrate reductase activity in tdbacco cells grown on nitrate, does not affect the develop- ment of nitrite reductase activity. It is possible, there- fore, that nitrate is directly reSponsible for the increase in nitrite reductase activity. However, 7% of nitrate reduc— tase activity of the control was detected in cells grown in the presence of tungstate; therefore, although the rate of nitrite formation must be greatly lowered, the possibility that nitrite reductase activity increases in reSponse to nitrite, and not nitrate, cannot be excluded. Addition of casein hydrolysate to medium containing nitrate inhibits the development of both nitrate reductase and nitrite reductase activities. The lower level of nitrite reductase activity in the presence of amino acids is apparently caused neither by an increase in the rate of decay nor by inhibition. The activities of both enzymes change in a similar manner in conditions of "induction” by nitrate and "repres- sion" by amino acids. However, nitrite reductase is the more stable of these two enzymes. Its activity decreases more slowly upon transfer of tobacco cells into nitrate-less medium. I. ACTIVATION OF ASPARTATE TRANSCARBAMYLASE OF ESCHERICHIA COLI BY PURINE NUCLEOTIDES II. REGULATION OF NITRITE REDUCTASE IN TOBACCO CELLS BY Hanna Chroboczek Kelker A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology and MSU/AEC Plant Research Laboratory 1969 *x ‘0 \a v i ACKNOWLEDGMENTS The author wishes to eXpress her sincere apprecia— tion and gratitude to Professor R. S. Bandurski for his guidance and encouragement during the course of these studies. Appreciation is also eXpressed to Dr. P. Filner for help and guidance in the studies reported in Part II of this thesis. The author is also grateful to the members of the guidance committee, Dr. W. B. Drew, Dr. C. J. Pollard and Dr. J. A. Boezi. Helpful suggestions and discussions with Dr. J. E. Reimann, Dr. R. A. Hertel, Dr. J. E. Varner and Dr. N. E. Good are gratefully acknowledged. ***** ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . PART I ACTIVATION OF ASPARTATE TRANSCARBAMYLASE OF ESCHERICHIA COLI BY PURINE NUCLEOTIDES INTRODUCTION . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . Role of ATCase in Regulation of Pyrimidine Biosynthesis in E, coli . . . . . . . . Properties of E, coli ATCase . . . . . . . Regulatory Properties of ATCase from Other Organisms . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . Bacterial Strain . . . . . . . . . . . . . Growth Media . . . . . . . . . . . . . . Maintenance, Growth of the Mutant and Prepara— tion of the 30,000 x g Supernatant Solution of E, coli . . . . . . . . . . . . . . . Assay of ATCase Activity . . . . . . . . . Assay of a-amylase Activity . . . . . . . Protein Determination . . . . . . . . . . Incubation of the E, coli Homogenate Leading to the Increase in ATCase Activity . . . Equilibrium Density Centrifugation of ATCase Studies of the Effect of Components of Incubation Mixture on ATCase Activity . RESULTS . . . . . . . . . . . . . . . . . . . Separation of (1H)ATCase and (2H)ATCase by Equilibrium Density Centrifugation . . . iii Page ii vi vii I2 18 18 18 19 20 21 22 22 23 27 3O 3O Test for gg novo Synthesis of ATCase . . . . . . Effect of Pretreatment with the Complete Incubation Mixture on the Properties of ATCase from the 30,000 x g Supernatant Solution . . . . . . . . . . . . . . . . . . . Effect of Components of the Incubation Mixture on Crystalline ATCase . . . . . . . . . . . Studies of the Effect of Purine Nucleotides on the Activity of ATCase from the 30,000 x g Supernatant Solution of E, coli . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . PART II REGULATION OF NITRITE REDUCTASE IN TOBACCO CELLS INTRODUCTION . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . Properties of Nitrite Reductase from Plants . . Regulation of Nitrite Reductase and Nitrate Reductase . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . Tobacco Cell Lines . . . . . . . . . . . . . . . Growth Media . . . . . . . . . . . . . . Maintenance and Growth of Cells . . . . . . . . Harvesting of the Cells and Preparation of the Tobacco Cell Homogenate for the Enzyme Assays Assay of Nitrate Reductase Activity . . . . . . Assay of Nitrite Reductase Activity . . . . . . Determination of Protein Content . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . Properties of Nitrite Reductase of Tobacco Cells . . . . . . . . . . . . . . . . . . Changes of Activity of Nitrate Reductase with Age of Culture Tobacco Cells . . . . . . . . The Effect of Casein Hydrolysate on the Formation of Nitrate Reductase and Nitrite Reductase . . . . . . . . . . . . . . . . . . iv Page 30 37 41 50 53 59 64 67 67 7O 74 74 74 75 76 77 77 79 8O 80 86 92 Stability of Nitrate Reductase and Nitrite Reductase ig_vivo . . . . Effect of Tungstate and Casein Hydrolysate on the Formation of Nitrate Reductase and Nitrite Reductase DISCUSSION . BIBLIOGRAPHY Page 95 97 100 107 LIST OF TABLES PART I Feedback inhibitors and some properties of ATCase from different sources . . . . . . Content of the tubes for equilibrifm density centrifugation of (2H)ATCase and ( H)ATCase . Content of the tubes prepared for equilib- rium density centrifugation of (2H)ATCase incubated with buffer (control) or with the complete incubation mixture . . . . . . . . . Activity of (2H)ATCase after preincubation; recovery of ATCase activity from the gradient . . . . . . . . . . . . . . . . . . PART II The effect of varying concentrations of sodium hydrosulfite on the activity of nitrite reductase from tobacco cells . . . . The effect of heating the tobacco cell homogenate and the requirement for methyl viologen for nitrite reductase activity . . . Activity of nitrate reductase and nitrite reductase after transfer of induced cells to nitrate-less M-lD, or nitrate—less M-lD sup- plemented with casein hydrolysate . . . . . . The effect of tungstate and casein hydrolysate on the formation of nitrate reductase and nitrite reductase . . . . . . . vi Page 16 25 26 33 81 82 96 98 Figure 1. LIST OF FIGURES PART I Distribution of ATCase activity from E. coli grown on (2 H)water and from E. coli grown on ( H)water after centrifugation on cesium formate gradient . . . . . . . . . . . . . Distribution of ATCase activity from E, coli grown on (2H)water after centrifugation on cesium formate gradient. (a) Enzyme prein- cubated with standard buffer. (b) Enzyme preincubated with the complete incubation mixture . . . . . . . . . . . . . . . . . . (a) Dependence of the reaction rate of ATCase of 30,000 x g supernatant solution of E, coli on aSpartate concentration. ATCase was pretreated with buffer (control) or with complete incubation mixture (com- plete); complete and control were heated at 60°C for 10 minutes. (b) A double reciprocal plot of complete and control . . Dependence of the reaction rate of crystal- line ATCase of E. coli on aSpartate concen- tration. ATCase was pretreated with buffer (control), or with complete incubation mixture (complete) . . . . . . . . . . . . Activation of crystalline ATCase by GTP in the presence of ATP and magnesium acetate (bath 2 mM) 0 O O O O O C O O O O O O O I O (a) Dependence of reaction rate of crystal- line ATCase on aSpartate concentration in the presence and absence of ATP, GTP and magnesium acetate (each at 2 mM). (b) A double reciprocal plot of the same data . . vii Page 31 35 38 42 45 47 Figure Page 7. The effect of GTP (a) or GTP and magnesium ions (b) on ATCase activity of 30,000 x g supernatant of E, coli in the presence and absence of 8 mM ATP . . . . .-. . . . . . . . . 51 PART II 1. Dependence of nitrite reductase activity on the concentration of tobacco cell homogenate . . . . . . . . . . . . . . . . . . 83 2. Appearance of nitrite and nitrate reductase activity after transfer of 12 day old parent culture into M—lD. (a) XD cell line. (b) R cell line . . . . . . . . . . . . . . . . 87 3. Changes in the activity of nitrite reduc— tase and nitrate reductase with age of tobacco cells. (a) Activity is expressed per liter of culture. (b) Activity is eXpressed per milligram of protein . . . . . . 89 4. The effect of casein hydrolysate on the formation of nitrate reductase and nitrite reductase activity. (a) Twelve day old cells of the XD line were inoculated into M-lD containing casein hydrolysate as indi- cated and harvested after 48 hours. (b) Fourteen day old cells of the R cell line were inoculated into M-lD containing casein hydrolysate as indicated and harvested after 48 hours . . . . . . . . . . . . . . . . 93 viii PART I ACTIVATION OF ASPARTATE TRANSCARBAMYLASE OF ESCHERICHIA COLI BY PURINE NUCLEOTIDES INTRODUCTION ASpartic acid transcarbamylase (ATCase) catalyzes the first reaction of pyrimidine biosynthesis: L-aspartate + carbamyl phOSphate —+> 3- N-carbamyl aSpartate + P04 The functioning of this enzyme in Escherichia coli is under control by repression (Yates and Pardee, 1957) and by feedback inhibition by end products of the biosynthetic pathway, cytosine derivatives (Yates and Pardee, 1956; Gerhardt and Pardee, 1962). Singh (1966) reported that if a cell free prepara— tion of E, g9li_was incubated with ATP, GTP, PEP, magnesium ions and amino acids a twofold increase in the ATCase activ— ity was observed. The cell free preparation was obtained from an E, EQEE strain selected for its ability to synthe- size large quantities of ATCase lg yiyg_and grown under conditions in which derepression of ATCase occurred. Pro- tein synthesis in this mutant at the time of harvesting, therefore, would be directed towards selective synthesis of ATCase. The requirements for the increase in ATCase activity resembled those for protein synthesis (Singh, 1966). This system also was able to support some incorporation of (14C)- amino acids into trichloroacetic acid (TCA) precipitable material. However, some of the observations indicated that the increase in ATCase activity might not result from syn— thesis of new enzyme molecules. These were: a. the increase in enzyme activity was not absolutely dependent on amino acids, b. incorporation of (14C)amino acids into TCA precipi- table material was small, c. puromycin had no effect on the increase in ATCase activity while it caused a 90% inhibition of (14C)leucine incorporation. The purpose of this study was to learn what process is reSponsible for the increase in enzyme activity. The possibility that gg_ggy2 synthesis of ATCase occurred was investigated by means of the density labeling technique (Hu §£_§;,, 1962). Although Singh (1966) had discounted the possibility that ATP, a known activator of ATCase (Gerhardt and Pardee, 1962), might be acting as an activator in this system, further studies were carried out to determine whether ATP or other components of the reaction mixture could be activating the enzyme. This was done by studying the effect of components of the incubation mixture on the kinetics of ATCase. LITERATURE REVIEW The functioning of biosynthetic pathways can be regulated by their reSpective end products. If end products of the biosynthetic pathway accumulate the flow of metabo- lites through this pathway can be stopped by cessation of synthesis of the enzymes involved in the formation of the end products,_i,e,, through repression. The functioning of the biosynthetic pathway can be stopped faster if the first enzyme or even enzymes of the entire pathway are inhibited by the end products, iuen, by feedback inhibition (Atkinson, 1965; Stadtman, 1966). Enzymes of a given pathway can be also a subject to activation by the end products of the related pathways, and thus operation of a given pathway can be adjusted to the whole metabolic network (Stadtman, 1966). ASpartic acid transcarbamylase (ATCase) is the first enzyme of pyrimidine biosynthesis and therefore it is at a strategic point for the regulation of the flux of carbamyl phosphate and aSpartic acid towards synthesis of pyrimidine nucleotides. The regularity role of ATCase in the control of pyrimidine biosynthesis in different organisms is dis— cussed below with Special consideration of E, 99;; ATCase and its properties. Role of ATCase in Regulation of Pyrimidine Biosynthesis in E. coli The first evidence for regulation of pyrimidine biosynthesis by feedback inhibition was reported for E, gel; by Yates and Pardee (1956). They reported that E, 92;; ATCase is inhibited lg_yl£gg by the end product, cytidine 5'-phosphate (CMP). The effect of pyrimidine nucleotides on E, ggii_ATCase purified to crystallinity was reexamined by Gerhardt and Pardee (1962). The inhibition of this enzyme is Specific for cytidine nucleotides and cytidine 5'-triphos— phate (CTP) is the best inhibitor of the enzyme. Uridine derivatives have no effect on enzyme activity; deoxythymidine 5'-triphosphate is only slightly inhibitory. The role of cytidine derivatives as inhibitors of ATCase in vivo was demonstrated by Gerhardt and Pardee (1964). Addition of uracil or of CMP into the medium immediately prevented func- tioning of ATCase in E, ggig. If 6-diazo-5-oxy—norleucine, an inhibitor of conversion of uridine triphOSphate (UTP) to CTP was added to the medium along with uracil, no inhibition of ATCase was observed. Purine nucleotides also affect ATCase activity (Gerhardt and Pardee, 1962). ATP activates the enzyme while GTP inhibits, although the inhibition is less than that observed for CTP. Gerhardt and Pardee (1962) postulated that the activation of ATCase by ATP might have biological importance in the equilibration of purine and pyrimidine pools of E, coli. Yates and Pardee (1957) reported that the enzymes of pyrimidine biosynthesis in E, ggii are also regulated by repression. The first three enzymes of pyrimidine biosyn- thesis, ATCase, dihydroorotase and dihydroorotic dehydro- genase became derepressed when E, ggll_was grown in medium lacking uracil. Derepression was eSpecially pronounced in the case of ATCase where activity increased 500 fold due to g§_novo synthesis of this enzyme. Properties of E. coli ATCase ATCase of E, 99;; is one of the most extensively studied regulatory enzymes. It has been demonstrated by Gerhardt and Pardee (1962) that the dependence of reaction rate on aSpartate concentration is sigmoid and, as recently demonstrated by Bethell t EL- (1968), the saturation curve for carbamyl phOSphate is also sigmoid. These characteris— tics indicate the existence of multiple binding Sites and suggest that the binding of one molecule of substrate facilitates binding of another (Gerhardt, 1963). The Specific inhibitor, CTP, causes an increase in the apparent Km for aSpartate while the activator, ATP, causes a decrease in the apparent Km' In both cases the Vfiax remained unchanged. These effects of nucleotides were measured at saturating concentrations of carbamyl phOSphate. CTP and ATP had the same qualitative effect on the interac- tion between carbamyl phosphate binding Sites when aSpartate was saturating (Bethell g; 3;., 1968). The degree of interaction between aSpartate binding sites can be altered. This has been demonstrated by the isolation of several mutants of E, ggli_in which dependence of reaction rate on aSpartate concentration varies from hyperbolic to highly sigmoid. The saturation curve of ATCase for aSpartate can also be altered by pH (Gerhardt and Pardee, 1964; Weitzman and Wilson, 1966). At pH 8.5, the optimum pH of the enzyme, the saturation curve of the enzyme is sigmoid. Lowering the pH to 6.0 leads to a change to a hyperbolic saturation curve (Gerhardt, 1963). At pH 10.2 ATCase exhibits another pH optimum and the saturation curve for aSpartate becomes hyperbolic. The effect of high pH on decreasing the interaction between catalytic sites is fully reversible (Weitzman and Wilson, 1966). The sigmoid kinetics of ATCase with reSpect to aSpartate are abolished by short heating at 60°C, treatment with urea and mercurials (Gerhardt and Pardee, 1962). According to Gerhardt and Pardee (1962) the loss of sigmoid kinetics is accompanied by a loss of sensitivity of ATCase to CTP. To explain these results Gerhardt and Pardee (1964) postulated a model for the structure of ATCase in which the enzyme was constructed of subunits and interactions between aSpartate binding sites located on different subunits accounted for sigmoid kinetics. Those interactions are strengthened by CTP and decreased by ATP. The native struc- ture of ATCase is necessary for both interactions between substrate binding Sites and CTP inhibition. Treatments with heat, urea and mercurials dissociate ATCase into subunits and result in a change to hyperbolic kinetics and also loss of inhibition by CTP. However the results of Weitzman and Wilson (1966) indicate that interaction between subunits of ATCase is more complex since in their hands heat and urea treatment resulted in a loss of interaction between cata- lytic sites but not in a loss of inhibition by CTP. The structure of ATCase is complex. Gerhardt and Schachman (1965) were able to demonstrate that in the pres- ence of mercurials the native enzyme dissociates into two types of subunits: one with catalytic activity and another devoid of catalytic activity but able to bind the analog of the regulatory nucleotide, bromocytidine 5'-triph05phate (BrCTP). The subunits were named catalytic and regulatory, reSpectively. The molecular weight of the native enzyme and of the subunits were determined to be: 3.1-105 for the native enzyme, 9.4-104 for the catalytic subunit, and 3.4°104 for the regulatory subunit. Treatment of ATCase with 8 M urea (Weber, 1968) causes dissociation of the enzyme into two types of subunits. It appears that the catalytic subunit of Gerhardt and Schachman (1965) is fur- ther Split by this treatment into two subunits Since the molecular weight of the catalytic subunit reported by Weber (1968) is 4.7-104, The molecular weight of the regulatory subunit is 2.5-104 and this is in agreement with the value reported by Gerhardt and Schachman (1965). Only two amino- terminal amino acids are reported for ATCase-alanine for the catalytic subunit and threonine for the regulatory sub— unit (Herve and Stark, 1967; Weber, 1968). The structure proposed for ATCase is therefore one consisting of four regulatory and four catalytic subunits with each subunit representing a single polypeptide. The native ATCase has four aSpartate binding sites (Changeux gg.§;,, 1968). Studies of binding of the sub- strate analog, succinate, confirmed conclusions from kinetic studies (Gerhardt and Pardee, 1962) that aSpartate binding sites interact. Cooperativity of binding of succinate was exhibited only by the native enzyme and not by the subunits. t 2;, (1968) native ATCase binds four According to Changeux molecules of BrCTP. This is in contrast to the previous report by Gerhardt and Schachman (1965) of eight binding sites for CTP. Changeux §E_§;, (1968) point out the possi- bility that there exist four additional sites with lower affinity for CTP. According to Changeux g; 3;. (1968) ATP binds at the same site as CTP since in the presence of ATP the binding of CTP is diminished. One of the models describing the action of regula- tory enzymes has been proposed by Monod §£_3L. (1963). This model postulates that the activity of these enzymes can be controlled by indirect interactions between distinct Sites (allosteric sites) maintained by the protein molecule through conformational transitions. Monod §£__l, (1963) dis— tinguished two types of allosteric interactions: interac- tions between identical ligands, termed homotropic, and 10 interactions between different ligands, termed heterotropic. The homotropic interactions between substrate binding sites are c00perative and they result in a sigmoidal plot of velocity versus substrate concentration. In heterotropic interactions substances unrelated structurally to the sub- strate, called allosteric effectors, cause a change in the apparent affinity of the substrate for the enzyme or in the turnover number (or both). Heterotropic interactions may result either in activation or inhibition of the enzyme and are either cooperative or antagonistic, reSpectively. The regulatory enzymes were postulated to be composed of identi- cal subunits, designated as protomers. Each protomer binds one Specific ligand. Allosteric enzymes can exist in a number of conformations differing in their affinity for the substrate. The most stable of these are symmetrical confor- mations. Cooperativity between ligand binding arises from coordinated transitions of all protomers to the conforma- tional state for which the ligand has greatest affinity. The simplest model (Monod §£_3E., 1965) postulates a pre— existing equilibrium of two conformational States of the allosteric enzymes differing in their affinity for the sub— strate and for the allosteric effectors. The kinetic and structural prOperties of ATCase described above are compatible with this model. Some evi— dence is being accumulated that the homotropic and hetero- tropic interactions of ATCase are indeed maintained through conformational changes of ATCase. 11 Dratz and Calvin (1966) estimated from measurements of optical rotatory dispersion of ATCase that a helix-coil transition occurs upon addition of succinate and carbamyl phOSphate while CTP causes a transition of the opposite Sign. Further evidence that the binding of substrate and regula- tory nucleotide results in a change of conformation of ATCase was reported by Gerhardt and Schachman (1968). The sedimentation coefficient of the native enzyme in the pres— ence of succinate and carbamyl phOSphate was decreased by 3.5%.while in the presence of CTP the sedimentation coeffi— cient was only slightly reduced. The change in conformation of ATCase was also evidenced by a sixfold increase in reac— tivity of sulfhydryl groups in the presence of carbamyl phosphate and succinate. The enhancement of sulfhydryl group reactivity was opposed by CTP. The results of the analysis of the data of Changeux 2E.il- (1968) and of Gerhardt and Schachman (1968) by Changeux and Rubin (1968) indicate that one of the two conformational states of ATCase has little affinity for the substrate and high affinity for the regulatory nucleotide, CTP. This conformational state predominates in the absence of succinate. Another of the conformational states has a high affinity for succinate. These facts point out clearly that both cooperative and antagonistic effects are maintained through changes in pro- tein conformation and that the change on addition of inhib- itor is opposite and approximately equal to the effect of the substrate. 12 Regulatogngroperties of ATCase from Other Organisms Examination of properties of ATCase in a number of bacteria by Neumann and Jones (1964) revealed that this enzyme also has a regulatory function in bacteria other than E, ggil. ATCase from Aerobacter aerogenes and Serratia marcescens is inhibited ig_vitro by both uridine monophos- phate (UMP) and CMP. In S. marcescens, which is less closely related to E, QQEE, UMP is a better inhibitor of ATCase. Heating of ATCase from E. marcescens at 60°C for 5 minutes abolished the ability to be inhibited by nucleotides while the enzymatic activity remained unchanged. This suggests that the catalytic and regulatory Sites are different in this system. ATCase from Bacillus subtilis is not inhibited by pyrimidines or purines. The functioning of ATCase in this organism is regulated by repression. ATCase from Pseudomonas fluorescens has a hyperbolic dependence of the reaction rate with reSpect to both aSpartate and carbamyl phOSphate. This enzyme is inhibited by UTP and to a lesser degree by ATP and GTP. In the presence of the inhibitor UTP the saturation curve of the enzyme with carbamyl phosphate becomes sigmoidal. Similarly, as in the case with E, 92;; ATCase, an increase in carbamyl phOSphate causes a decrease in inhibition by UTP. It was not possible to achieve in this system a selective destruction of the inhibitory site by treatment with mercurials and heat. 13 ATCase is also an important enzyme in regulation of pyrimidine biosynthesis in eucaryotic organisms. In the yeast, Saccharomyces cereviseae, ATCase is repressed by uracil or its derivative (Kaplan _E,§;., 1967). This enzyme is also subject to feedback inhibition. Of the nucleotides tested UTP was the strongest inhibitor while CTP, GTP and ATP had no effect on ATCase activity. However ATP antago- nized inhibition of this enzyme by UTP. The lg_ylyg.counter— action by adenine of inhibition of ATCase by uracil was demonstrated previously by Burns (1966). Homotropic inter— actions are absent in this enzyme, Since ATCase from yeast exhibits Michaelis-Menten kinetics with reSpect to both sub- strates. The existence of a regulatory site distinct from a catalytic Site was inferred from the fact that inhibition by UTP is noncompetitive with respect to both substrates and from selective destruction of inhibition by UTP by brief heating at 600C. ATCase from Neurospora crassa is also subject to feedback inhibition (Donachie, 1964). It is inhibited lg yi££2_by uridine analogs (uridine, uridylic acid, UTP), thymidine and orotidylic acid but not by cytidine analogs. In contrast to E, gQ;;_ATCase the enzyme from Neurospora is inhibited by pyrimidines only at high aSpartate concentra- tions. The concentration of carbamyl phoSphate has no effect on inhibition by nucleotides. Activity of ATCase is also regulated by repression although the change in ATCase 14 activity upon withdrawal of exogenous pyrimidines is only threefold as compared with a SOD-fold increase in E, coli. Tentative evidence for feedback inhibition of ATCase from Chlorella was reported by Cole and Schmidt (1964). They found that the extracts of synchronous cultures of Chlorella contained heat stable inhibitors of the enzyme ~after one cell division. No evidence for repression of ATCase in higher plants was reported. However the activity of this enzyme is changed with the physiological age of plant tissues (Stein and Cohen, 1965). Cotyledons of growing soybean .seedlings contained lower activity than the shoot and the -root. Activity of this enzyme is also highest in the grow— ing tip of bean plant which contains the highest number of dividing cells. Feedback inhibition of ATCase from lettuce seedlings was reported by Neumann and Jones (1962). The lettuce enzyme is strongly inhibited by uridine derivatives ‘with UMP being the best inhibitor. Cytidine derivatives ‘were inhibitory only at high concentration and purine nucle- iotides had no effect on enzyme activity. The catalytic site is probably different from the regulatory site since UMP is as noncompetitive inhibitor with reSpect to both substrates. {This is also supported by the fact, that ribose-S-phOSphate xxalieves the inhibition by UMP. Ribose-S—phosphate does not aaffect catalytic activity and it likely acts by competing .vvith UMP for the regulatory site. 15 No definite evidence is available with regard to regulation of ATCase in mammalian systems. As in plant systems, ATCase activity is highest in embryonic tissues (Kim and Cohen, 1965). ATCase activity was higher in the livers of fetal rats and guinea pigs than in the livers of adult animals. ATCase activity was also elevated in regen- erating livers of rats. Evidence for the Operation of feed- back inhibition of ATCase in animal tissues is not available. Pyrimidine nucleotides had no effect on 140-fold purified enzyme from rat liver (Bresnick and Mossé, 1966). This enzyme had Michaelis-Menten kinetics with reSpect to both substrates. Curci and Donachie (1964) failed to detect an inhibitory effect of pyrimidine nucleotides on ATCase activ— ity from rabbit reticulocytes. Comparison of the properties of ATCase from differ— ent organisms (on the basis of available data) in Table 1 reveals that the E, ggli_enzyme is the only one in which homotropic interactions are detected with reSpect to both substrates. Cytidine derivatives are the best inhibitors of the E, 99;; enzyme and of two other representatives of the Enterobacteriaceae while in the other bacteria, fungi and higher plants uridine derivatives are the best inhibitors of ATCase. It appears that the activity of mammalian ATCase is not regulated by end products. 16 Aaomsv mseomcoo cam AUHSO Aoomav Moacmmnm Aaomav meson cam damasoz Aaemsv munumcon QED .mBD .mZD whom massefluouo .mcacassnu .mep oumooucumno pannmm um>AH umm m>aumm am mmmMHU . ZI ||.|hsmmav me: an acapannacu .Hm um swammm ... mmNflcommucm me< mBD ommmfl>ouoo xm Lemmav mumndmond mew .me¢ meson cam unmasoz HAEmflumo ... mHO .mED mcmomouosam am Aaemav .I moCOb cam sunfisoz ... ... m2: .mzu monomoumm .< Aaomsc meson can camadmz ... ... mZD..mSO mcwummoume aw .I..||Am6mav .Hm um Hamnumm mumnmmosd new Amomav mwcnmm ASEmQHmU me<>xooc mZUmxooc .meomxooc .I can ucumnumw mumuummmm mB< mEO .mQO .mBO HHOU .m mocmummmm mmumuumasm mucum>fluo< muouHQHcGH memncm on» mo monsom uaHoumOHad mmUHSOm uconommac Eoum ommuefi m0 mmfluummoum QEOm can mucuwnwnsw somnommm .H magma 17 The regulatory Site appears to be distinct from the catalytic site in all the systems discussed, but only in E, coli and E, fluorescens does the feedback inhibitor cause an increase in homotropic interactions. It is tempting to Speculate that ATCase of eucaryotic organisms (discussed here are the fungi, lettuce and mammalian enzymes) resembles the subunit form of E, QQEE_ATCase but is endowed with a regulatory Site in the case of fungi and plant ATCase. However, a valid comparison of the properties of ATCase from various organisms will be possible only when the properties of this enzyme in many organisms have been better studied. MATERIALS AND METHODS I. Bacterial Strain A strain of E, 99;; K12 411—189 obtained from Dr. J. C. Gerhardt was used throughout this work. This mutant is diploid with reSpect to that portion of the genome (approximately one-third) containing the gene or genes cod- ing for ATCase production. It has an absolute requirement for histidine and a partial requirement for leucine. Uracil is required for fast growth since the mutant has a defect in orotidylate decarboxylase and grows slowly in the absence of exogenous pyrimidines. II. Growth Media Growth medium MS-56 was used. It was prepared by adding the following compounds in grams per liter of dis— tilled water: Na HPO 2 4, 4.4; KH P0 2.6; MgSO -7 Ho, 0.02; 2 4' 4 2 3)2°4 H20, 0.014; FeSO4°7 H20, 0.05; (NH4)2 804-4 H20, 4 and uracil, 0.008. This solution was autoclaved at 122°C and Ca(NO 15 psi for 20 minutes if the volume was 110 m1 while 11 liter volumes were autoclaved for an hour. Sterile solutions of glucose and amino acids were added immediately prior to use 18 19 to make the final concentration in grams per liter: D- glucose, 2; L-leucine, 0.05; L-histidine, 0.07. Solid medium was prepared by the addition of 2% agar to MS-56. ’ III. Maintenance, Growth of the Mutant and Preparation of the 30,000 x g Supernatant Solution of E. coli The cultures of E, ggil_were maintained on MS—56 agar supplemented with 60 mg of uracil per liter. They were transferred every three months. The inoculum for large volume cultures was prepared as follows: 110 ml of MS-56 media was inoculated from agar. The cells were grown at 37°C and aerated by swirling on a New Brunswick shaker. When Klett readings of the culture reached 160 (which corresponds to 2-109 cells per ml) 1.1 ml was transferred to 110 m1 of MS—56 media. This culture was grown for approximately 12 hours, until Klett readings reached 160-190. Large amounts of bacterial cells were prepared by growing E, 22;; at 37°C in 11 liters of MS-56 containing 0.6 ml of 1.66% Antifoam A (a silicone anti-foam agent Obtained from Dow Corning Corporation). Three 110 m1 flasks of the above described cultures were used as an inoculum. The medium was aerated under pressure with air sterilized by serial filtration through a charcoal filter and glass wool filter connected by sterile tubing with two immersion fil- ters reaching to the bottom of the carboy. The cells were 20 grown for approximately four hours until the Klett readings reached 70. Growth of the culture was Stopped by immersing the carboy in ice water for one hour. The cells were har— vested by continuous flow centrifugation with the flow rate maintained at 600 ml per hour. The harvested cells were suSpended in 40 m1 of 0.01 M phosphate buffer at pH 7.4 containing 0.06 M KCl and 0.14 M magnesium acetate. The suspension of cells was centrifuged for 20 minutes at 30,000 x g. The washed cells were suspended in 14 m1 of the above buffer and passed through a precooled French Press Cell at 16,000 psi. The broken cells were centrifuged for 30 minutes at 30,000 x g. The supernatant solution was dialyzed for 4 hours against 2 liters of the above described buffer with one change of buffer. The dialyzed solution was used as a source of ATCase and was able to support the incorporation of amino acids into TCA precipitable material (Singh, 1966). IV. Assay of ATCase Activity The activity of ATCase was assayed by formation of carbamyl aSpartate according to the method of Koritz and Cohen (1954) as modified by Gerhardt and Pardee (1962) for ATCase assay. The assay conditions unless otherwise Speci- fied were as follows: 3.6 mM carbamyl aSpartate, 5 mM aSpartate, 40 mM potassium phosphate buffer (pH 7.0) and the ATCase preparations were incubated in a volume of 0.5 ml for 30 minutes at 30°C. The reaction was stopped by addition of 21 2.5 ml of a cooled mixture of reagents a, b, and c mixed in a ratio of 3:1:1 before addition. The reagents were: a. 66% H2804, b. 22.5 mg of diacetyl monoxime dissolved per m1 of water, c. 114 mg of diphenyl—p-sulfonate dissolved in a 100 ml of 0.1 M HCl containing 0.4 g of detergent Atlas BRIJ. The contents of the tubes were mixed and heated at 600C for 30 minutes. Subsequently they were cooled in ice water and 0.5 m1 of K28208 solution (2.5 mg/ml of water) was added. Color was developed during incubation at 30°C for 20 minutes and after cooling to 40C the absorbancy was determined at 560 mu against a reagent blank. The activity of ATCase was eXpressed as mnmoles of carbamyl aSpartate synthesized per minute. V. Assgy of Okamylase Activity_ The activity of oramylase (obtained from Worthington) was assayed according to method of Shuster and Gifford (1962) as modified by ChriSpeels and Varner (1967). A solution of potato starch was prepared before each assay. Native starch (150 mg) was boiled in 100 m1 of a solution containing 600 mg 2. After cooling the starch solution was centrifuged at 20,000 x g for 10 minutes. The of KH2P04 and 200 umoles of CaCl clear supernatant was decanted and used for the assay. A 22 stock of iodine solution was prepared by dissolving 6 grams of KI and 600 mg of iodine in 100 m1 of water. Before use one m1 of this solution was diluted to 100 ml with 0.05 ml HCl. An aliquot of the enzyme was diluted with water to make the reaction volume one ml. The reaction was started by adding one m1 of starch solution and incubation was from 1 to 10 minutes at 30°C. The reaction was stopped by the addition of one m1 of iodine reagent. Subsequently 5 m1 of water were added to each tube and the absorbancy was read at 620 mg. The decrease in absorbancy at 620 mu as compared with the non enzymatic control was taken as a measure of a-amylase activity. VI. Protein Determination Protein content was determined according to Lowry ._E 1;. (1951). Egg albumin was used as a standard. VII. Incubation of the E. coli Homogenate Leading to the Increase in ATCase Activity Incubation of the E, ggll_homogenate which leads to an increase in ATCase activity was performed as described by Singh (1966). The "complete" System contained the following compounds in umoles: potassium phosphate buffer (pH 7.4), 50; magnesium acetate, 60; ATP, 10; GTP, 10; PEP, 5; 50 “grams of each of 20 amino acids, an aliquot of 30,000 x g supernatant solution of E, coli and enough distilled water 23 to make a volume of one m1. AS a control an equal aliquot of 30,000 x g supernatant solution was incubated with enough of 0.01 M phOSphate buffer (pH 7.4), containing 0.06 M KC1 (designated standard buffer) to make a volume of one ml. Incubation was for 10 minutes at 370C and it was terminated by placing the tubes in an ice bath. An aliquot of the con— trol and the "complete" System was dialyzed at 4°C for 3 hours against 125 m1 of Standard buffer with two changes of buffer. Subsequently the dialyzed solution was diluted with the standard buffer and assayed for ATCase activity. VII. Equilibrium Density Centrifugation of ATCase 1. Separation of (1H)ATCase and (2H)ATCase by equilibrium density centrifugation a. Preparation of 30,000 x g supernatant from cells grown on (2H)water containing medium (2H)water was purified after previous use by boiling with charcoal, two distillations with KMnO4, one from acidic and another from basic medium. The density of the distil— late was 1.10 g/cm3 while the density of the commercial 99% (2H)water was 1.105 g/cm3. MS-56 medium was prepared with the purified (2H)water, the salts, glucose and amino acids were added as aqueous solutions. The total amount of water added was 8% of 1100 ml total volume. The autoclaved medium brought to 37°C was inoculated with E, 99;; grown in 33 m1 of MS-56 containing (2H)water. Growth of the cells, 24 harvesting, breakage and preparation of 30,000 x g super— natant were carried out as described in section III of Materials and Methods. The protein content of the dialyzed preparation was 4.92 mg/ml. b. Preparation of the tubes for centrifugation A stock solution of cesium formate of density 1.72 g/cm3 was prepared by dissolving 9.6 grams of cesium formate in 6.4 ml of standard buffer. The volume of the stock solu~ tion needed for the gradient was calculated using the for- mula of Vinograd (1963): Vl, = (pC—pO)/(pC-0.997) where V1,w is the volume of water used to dilute a stock solution of CsCl of density pc to obtain one m1 of a solu- tion of the desired density p0 (pO is here equal to 1.295 g/cm3). The calculated volumes of stock solution and dilut— ing buffer were multiplied by 4.5 to obtain the apprOpriate volumes adding to 4.5 m1 of a final volume. The tubes were prepared for centrifugation as described below (see Table 2). The contents of the tube were mixed with a capillary and 0.3 ml of paraffin oil was layered on top. The tubes were placed in a precooled SW-39L rotor and centrifuged in Spinco Ultracentrifuge for 67 hours at 39,000 rpm° Subse- quently the tubes were punctured with a needle and one drop fractions were collected at 40C. The refractive index of every fifth fraction was determined with a Bausch and Lomb Abbe-type refractometer. ATCase and dramylase were assayed 25 Table 2. Content of the tubes for equilibripm density centrifugation of (2H)ATCase and ( H)ATCase Component Volume (m1) Protein (mg) (2H)ATCase 0.08 0.394 (1H)ATCase 0.02 0.500 d—amylase 0.005 0.095 standard buffer 2.4 ... cesium formate (1.70 g/cm3) 1.91 ... in the remaining alternate fractions. The correSponding densities were determined with the aid of a standard curve. The standard curve was prepared by measuring the refractive index of a series of cesium formate solutions of known den— sity. The densities were determined by weighing the cesium formate solutions in a 100 A pipette. 2. Test for gg_ggyg synthesis of ATCase Density labeling of preformed ATCase was achieved by growing E, 22;; on (2H)water as described in section I of Materials and Methods. The density of the (2H)water used was 1.08 g/cm3. The protein content of the 30,000 x g supernatant was 4.18 g/cm3. a. Incubation of 30,000 x g Supernatant solution from deuterated E, 92;; A 0.1 ml aliquot of the dialyzed supernatant of E, coli was incubated with the components of the "complete" 26 system containing (1H)amino acids, ATP, GTP, PEP, and magnesium ions as described in section VII of Materials and Methods. As a control 0.1 m1 of the same preparation was incubated with 0.9 m1 of standard buffer. After 3 hours of dialysis against standard buffer an aliquot was taken for assay of ATCase activity. The remaining solution was diluted with 1.5 m1 of standard buffer and the measured volume of the diluted enzyme was taken for equilibrium density centrifuga- tion. b. Equilibrium density centrifugation The solution of ATCase with the a-amylase marker in cesium formate of density 1.295 g/cm3 was prepared as described in Table 3. Table 3. Content of the tubes prepared for equilibrium den— sity centrifugation of ( H)ATCase incubated with buffer (control) or with the complete incubation mixture Control Complete Component Volume Protein Volume Protein (ml) (mg) (ml) (m9) Diluted (2H)ATCase 1.89 0.28 2.02 0.316 Cesium formate (1.70 g/cm3) 1.90 ... 1.91 ... standard buffer 0.69 ... 0.59 ... a-amylase 0.01 0.19 0.01 0.19 27 The tubes were covered with 0.3 m1 of paraffin oil and centrifuged as described above. One drop fractions were collected. The refractive index of every fifth fraction was determined and a-amylase and ATCase were assayed in alter- nate fractions of those remaining. IX. Studies of the Effect of Components of Incubation Mixture on ATCase Activigy 1. ATCase from 30,000 x g supernatant of E, 92;; pretreated with the incubation mixture An aliquot of the 30,000 x g supernatant of an E,‘EQ;E homogenate containing 8 mg of protein was preincu— bated either with the buffer (control) or with the complete incubation mixture (complete) as described in section VII of Materials and Methods. Aliquots of the dialyzed solution were diluted to a final protein concentration of 0.08 mg/ml. Some of the samples were heated for 10 minutes at 60°C in 0.01 M phosphate buffer (pH 7.0) and subsequently cooled on ice. ATCase activity was assayed as described in section IV Materials and Methods, but the aSpartate concentration was as indicated in Figure 3a. Inhibition of ATCase by CTP was measured utilizing 2 mM CTP and the assay of the enzyme activity was conducted as described in Materials and Methods, section IV. 28 2. Crystalline ATCase pretreated with the incubation mixture An aliquot of crystalline ATCase (obtained from Dr. J. C. Gerhardt) containing 2 pg of protein was preincu- bated with the buffer or complete incubation mixture as described in section VII of Materials and Methods. After incubation the enzyme was dialyzed against 20 mM phOSphate buffer (pH 7.0) 2 mM mercaptoethanol and 0.2 mM ethylenedia- minetetraacetic acid (EDTA). Aliquots of the diluted dia— lyzed solution containing 0.06 ng of protein were assayed for ATCase as described in section IV of Materials and Methods, but the aSpartate concentration was as indicated in Figure 4. 3. Effect of GTP on activity of crystalline ATCase assayed in presence of ATP and magnesium ions Crystalline ATCase (0.5 ug of protein) was incubated with 3.6 mM carbamyl phOSphate, 5 mM aSpartate, 0.1 mM EDTA, 40 mM MOPS (2-(N-morpholino)propanesulfonic acid, Dr. N. B. Good, unpublished) at pH 7.0, 2 mM ATP and 2 mM magnesium acetate. GTP concentration was as indicated in Figure 5. 4. Effect of ATP, GTP and magnesium ions on the dependence of reaction rate on aSpartate concentration Crystalline ATCase (0.08 ug of protein) was incu— bated with 3.6 mM carbamyl phOSphate, 40 mM MOPS (pH 7.0), ATP, GTP and magnesium acetate (each 2 mM). Aspartate con— centration was as indicated in Figure 6a. 29 5. Effect of GTP, and GTP plus magnesium ions on the activity of crude ATCase in the presence and absence of ATP The 30,000 x g supernatant solution of E, coli (0.054 mg of protein) was incubated with 14 mM carbamyl phosphate (purified according to Gerhardt and Pardee, 1962), 40 mM phosphate buffer (pH 7.0) and 5 mM aSpartate. ATP, when used, was 8 mM and magnesium acetate was equimolar to the indicated GTP concentration (Figure 7). RESULTS Separation of (1H)ATCase and (2H)ATCase by Equilibrium Density Centrifugation (1H)ATCase (obtained from E, 22;; grown on H20) and (2H)ATCase (from E, 99;; grown on (2H)20) were well resolved by equilibrium density centrifugation in a cesium formate gradient. As is shown in Figure 1 two major peaks of ATCase activity were observed which, while overlapping, differed in their density by 3.2%. The small middle peak at about frac- tion 60 is believed to be Spurious since it did not appear in subsequent eXperiments. The recovery of ATCase activity from the gradient was satisfactory. Approximately 105% of the activity of (2H)ATCase was recovered in the heavier peak and 88% of the activity of (1H)ATCase was recovered in the lighter peak. Test for de novo Synthesis of ATCase Density labeling and equilibrium density centrifuga— tion was introduced by Hu §E_§;, (1962) as a test for g;_ £222 synthesis of B-galactosidase. This technique, as described by Filner and Varner (1967) was utilized in this study as means of detecting newly synthesized ATCase. 30 Figure 1. 31 Distribution of ATCase activity from E, coli grown on (2H)water and from E, coli grown on (IH)water after centrifugation on cesium formate gradient. The direction of decreasing density is from left to right. Abbreviations used: CA = N-carbamyl aSpartate, A A 620/min = decrease in absorbancy at 620 mu taken as a measure of d-amylase activity. umoles CA/min x l034 , l . l l . l l l 0J5 (ATCase T ' \j ‘ "l \ O . -— 0.05 O i - \ \. cox , Que/I _ .___J. l l J\.°‘1_..J ' 40 so so 70 so 90. Fraction Number AA 820mm 33 For this purpose 30,000 x g supernatant solution obtained from E, 39;; grown on (2H)water and therefore containing (2H)ATCase was incubated under conditions in which the increase in ATCase activity occurs and then dialyzed for 3 hours (as described in Materials and Methods, section VII). (1H)amino acids were utilized as a component of the incuba- tion mixture so that newly formed ATCase would be of differ— ent density. Subsequent separation of proteins differing in their density by equilibrium density centrifugation enables one to detect newly synthesized enzyme. As demonstrated in Table 4 the activity of ATCase in a supernatant solution prepared from E, 29;; grown on (2H)— water was increased by 100% upon incubation with the com- plete incubation mixture, containing (1H)amino acids, ATP, GTP, magnesium ions and PEP as compared with the control. This is in agreement with the report of Singh (1966). Table 4. Activity of (2H)ATCase after preincubation; recovery of ATCase activity from the gradient umoles of 3 carbamyl aSpartate/min x 10 In 1 m1 of Put on the Recovered from System incubation mixture gradient the gradient Control 55.7 30.9 36.4 (109%) Complete 113.0 78.6 40.3 ( 51%) 34 Subsequent equilibrium density centrifugation of the preincubated (2H)ATCase revealed that the distribution of ATCase activity on the gradient remained the same relative to the a-amylase marker regardless of whether the enzyme was preincubated with the buffer or with the complete incubation mixture (Figure 2a and b). Since there was no change in the density or in the symmetry of the peak of ATCase upon incu- bation with (1H)amino acids neither appreciable finishing of preexisting polypeptide chains nor of g§_ggyg synthesis of ATCase occurred in this system. The equilibrium position of (2H)ATCase peak was at lower density, as compared with the d-amylase marker, than in Figure 1. This could be explained by the fact, that the density of (2H)water used in this experiment was 1.08 g/cm3, as compared with 1.10 g/cm3 in the previous experiment. Recovery of ATCase activity from the gradient was complete for the control (ATCase preincubated with buffer). However only half the activity was recovered after centifu— gation of ATCase preincubated with the complete incubation mixture. This eXperiment has been repeated with similar results. Previous results indicated that the recovery of both (1H)ATCase and (2H)ATCase from cesium formate gradient is good. Therefore, it can not be argued that the preferential loss of (1H)ATCase was reSponsible for this poor recovery. It is possible that if ATCase became activated by the undialyzed components of the incubation mixture (dialysis Figure 2. 35 Distribution of ATCase activity from E, coli grown on (2H)water after centrifugation on cesium formate gradient. a. Enzyme preincubated with standard buffer. b. Enzyme preincubated with the complete incuba- tion mixture. The arrows indicate the equilibrium position of (1H)ATCase. Sesame << _ ....Exomo <4. w m. w o w 0 .— ‘I e . 5 . m .. m. , I ..0 ... 7 r a O 0 e .D Full? 1 m 0 ..u I l6N . n .w 6 ll, 3 .I [mm r .e F s a m _. ... A .1“ I .....O 3 d ._ . _ .m I I”. I. _ . .. . _ _. _ O , 5 4. 3 2 I. 5 4 MW m N no. a 522.0 3.85: . no. x 5220 8.25 Fraction Number 37 was only for three hours) the loss of ATCase activity would be due to the dissociation of activating compounds during prolonged centrifugation. Effect of Pretreatment with the Complete Incubation Mixture on the Properties of ATCase from the 30,000 x g Supernatant Solution The kinetic properties of ATCase in this system were studied tn) examine the possibility of whether the enzyme became activated upon pretreatment with ATP, GTP, PEP, magnesium ions and amino acids (eXperimental details as in Materials and Methods, section IX, 1). The dependence of reaction velocity on aSpartate concentration was changed from a sigmoidal to a hyperbolic function as a result of preincubation (Figure 3a). The dif- ference in reaction rate of the control and of ATCase pre— treated with the complete incubation mixture was greatest at low aSpartate concentrations. At saturating concentrations the difference in reaction rates almost disappeared. The increase in.ATCase activity reported by Singh (1966) and in this study was measured at 5 mM aSpartate, the concentration at which the difference in the reaction rate was a 100%. The Lineweaver-Burke plot of the control and of ATCase pre— treated with the complete incubation mixture shows that preincubation leads to a change from an upward curving to a nearly linear plot (Figure 3b). These changes in the kinet- ics of ATCase indicate that the enzyme became activated as a result of preincubation. 38 Figure 3a. Dependence of the reaction rate of ATCase of 30,000 x g supernatant solution of E, coli on aSpartate concentration. ATCase was pretreated with buffer (control) or with complete incubation mixture (complete); complete and control were heated at 60°C for 10 min. b. A double reciprocal plot of complete and control. 39 a 2.5 22.642 9. on on mm on .... o. n u d u] d H d i q . so 0 x: s x x s X .m .o s s q. .x 4 \s .... 35.2.30 0 ss \ f \s . s \\ o\ UOfiUOS JOLhP—OO st . \\.\- \.. \\ \\ x \a. ..R . l. ‘J \ \ \ \o \‘F ”fig—GEO” 3 (fil| |AXl|l\\\|« ( vgfiumc nwfiw—QEOU “I‘ll llllllllll xi.“ 0 — P p — . p 5 ill I) 2 .om NO > NH nitrate reductase nitrite redutase Nitrate reductase catalyzes the reduction of nitrate to nitrite. The subsequent reduction of nitrite to ammonia, which involves a Six electron change, is catalyzed by nitrite reductase. A vast amount of evidence has been accumulated demonstrating that nitrate reductase is an adaptive enzyme. The activity of this enzyme appears in plants grown on nitrate as a nitrogen source and not in plants grown on ammonia or amino acids (Candela g; gl,,1957; Afridi and Hewitt, 1962; Beevers §E_g;” 1965). The increase in nitrate reductase activity in response to nitrate can be abolished by inhibitors of protein synthesis (Afridi and Hewitt, 1965; Beevers 2E.§1-: 1965) which suggests that the increase in nitrate reductase activity is due to synthesis of enzyme rather than activation. However, activation could also con- ceivably depend upon protein synthesis. It has been demon- strated in two systems that the increase in nitrate reduc- tase activity in the presence of nitrate is accompanied by 64 65 an increase in nitrite reductase activity (Ingle g£.§£., 1966; Stewart, 1968; Joy, 1968). The Studies reported in this thesis were carried out to examine the regulation of nitrite reductase in tobacco cells. Experiments were designed to answer the following questions: 1. Is the increase in nitrite reductase activity asso- ciated with the increase in nitrate reductase activ- ity in tobacco cells growing on nitrate? The in- crease in nitrate reductase activity in tObacco cells in reSponse to nitrate was demonstrated pre- viously by Filner (1966). 2. Do amino acids, which act as repressors of nitrate reductase activity in tobacco cells (Filner, 1966), also control the activity of nitrite reductase? 3. How is the regulation of nitrite reductase activity linked to the regulation of nitrate reductase activ— ity? In particular, can nitrate cause an increase in both enzyme activities or does it have to be converted to nitrite before it can cause an increase in nitrite reductase activity? In order to study the regulation of nitrite reduc- tase activity in tdbacco cells it was necessary to develop a simple assay for enzyme activity and to establish optimum conditions for determination of nitrite reductase activity. 66 To gain an understanding of the relationship between the processes which cause an increase in the activities of both enzymes the kinetics of appearance of activities of nitrate reductase and nitrite reductase were examined in tobacco cells grown on a nitrate containing medium. The end product regulation of nitrite reductase was studied by fol— lowing the effect of amino acids on the increase of activity of nitrite reductase and of nitrate reductase in tobacco cells grown on nitrate containing medium. The question of whether nitrate has to be reduced to nitrite before it can cause an increase in nitrite reductase activity in tobacco cells was studied utilizing tungsten. Tungsten is in the same group of elements as molybdenum and consequently may act as competitive inhibitor of molybdenum functions. Keeler and Varner (1958) Showed a competitive inhibition of molybdenum by tungstate in the growth of Azotobacter. Both (185W)tungstate anui (99Mo)molybdate were found to be incorporated into molybdo- and tungsto- proteins. Tungstate inhibits the appearance of nitrate reductase activity in tobacco cells (Heimer et al., unpub— lished) and therefore provides a valuable technique for the study of appearance of nitrite reductase activity in the absence of nitrate reductase activity. LI TE RATURE RE VIEW Properties of Nitrite Reductase from Plants It is known that nitrite is metabolized rapidly by green plants in the light (Vanecko and Varner, 1955; Kessler, 1955). One of the first reports of nitrite reductase activ- ity in an Eg_vitro system was by Huzisige and Satoh (1961). The authors demonstrated that nitrite was metabolized by a soluble enzyme from Spinach in the presence of an illumi- nated grana preparation. Subsequently, Sanderson and Cock— ing (1964) demonstrated, in a Similar system isolated from tomato leaves, that the disappearance of nitrite is accompa- nied by a quantitative accumulation of ammonia. It was later demonstrated that the reduction of nitrite can be carried out by plant enzymes in darkness in the presence of an appropriate reducing agent. Hageman 33 ,gl. (1962) reported a quantitative reduction of nitrite to ammonia using nitrite reductase isolated from Cucurbita pepo. The reducing agent affective as the electron donor for this enzyme could be either palladized asbestos under hydrogen or reduced nicotinamide adenine dinucleotide phOSphate (NADPH) with catalytic amounts of benzyl viologen. 67 68 In Clostridium pasteurianum ferredoxin participates in the reduction of nitrite to ammonia and can be replaced by methyl viologen, a dye of Similar oxidoreductive poten- tial (Mortenson $5.31., 1962). Plant nitrite reductase can also utilize reduced ferredoxin in addition to viologen dye t EE., 1963; Hewitt and Betts, as an electron donor (Losada 1963). Reduction of nitrite by NADPH in a chloroplast extract in darkness is ferredoxin dependent (Losada t 31-: 1963). Ferredoxin can also serve as an electron donor if it is reduced in the light in the presence of a Spinach grana preparation, or if it is reduced in darkness by hydrogen gas and hydrogenase.of Q, pasteurianum. The reduction of nitrite mediated by ferredoxin in light is accompanied by 02 evolu- tion and formation of high energy bonds of ATP (Paneque g5 .EE., 1964). Nitrite reductase has been purified from several plants. The best studied of these Systems is nitrite reduc- tase from spinach (Ramirez §p_gl., 1966; Joy and Hageman, 1966; Shin and Oda, 1966; Hewitt gE.gE., 1968). Huzisige _£.§;, (1963) reported that a preparation of nitrite reducé' tase from Spinach of Specific activity 0.45 nmoles NOE/min per mg protein showed the absorption Spectrum of a flav0pro- tein. However, preparations of nitrite reductase from Spinach of higher purity : 0.98 umoles NOEflmin per mg of protein (Shin and Oda, 1966) and 3.25 pmoles NOg/min per mg of protein (Ramirez §£_EE., 1966) did not contain a flavin component. NADPH can be utilized as an electron donor for 69 the enzyme only if the reaction mixture is supplemented with ferredoxin and NADP reductase (Ramirez g; al., 1966; Joy and Hageman, 1966). Hydroxylamine reductase activity was found to be associated with the purified nitrite reductase (Betts and Hewitt, 1966; Hewitt §£_§l,, 1968). Hydroxylamine is one of the postulated intermediates in the reduction of nitrite to ammonia. It has been demonstrated that nitrite reductase from Q, pgpp reduced hydroxylamine Slower than nitrite with either reduced benzyl viologen or reduced ferredoxin as t 21»: 1962; Betts and Hewitt, electron donor (Hageman 1966). Also hydroxylamine did not inhibit reduction of nitrite by the enzyme from g, p§p9_(Creswell gg_gi., 1965). Lazzarini and.Atkinson (1961) utilizing E, 99;; nitrite reductase showed that (lSN)NO2 can be reduced to ammonia without dilution by a hydroxylamine pool. Therefore, free hydroxylamine is not an intermediate in the reduction of nitrite to ammonia. The presence of a sulfhydryl protecting agent in the extraction medium was beneficial for extraction of nitrite reductase in an active state. The Optimum concentration of cysteine required for the extraction of this enzyme from 3 M (Sanderson and Cocking, 1964). The tomato leaves is 10- enzyme from maize had the highest activity if extracted with cysteine at concentrations between 10..3 M and 10.2 M (Joy and Hageman, 1966). 70 Measurements of the effect of pH on nitrite reduc— tase activity indicate that the pH optimum is dependent upon the assay utilized. Spinach nitrite reductase exhibited the highest activity between 7.1 and 7.8 if the assay was con- ducted with ferredoxin reduced by hydrosulfite (Ramirez g£_ _EE., 1966). A similar pH optimum for this enzyme was estab- lished if benzyl viologen reduced by sodium hydrosulfite was utilized as an electron donor but the pH optimum was between 6.0 and 7.0 if the reducing agent was NADPH (Joy and Hageman, 1966). In no case was a Sharp pH optimum for nitrite reduc- tase observed. The intracellular location of nitrite reductase has been Shown to be within the chloroplasts in green leaves t El,, 1966; Ramirez t 1., 1966). However, (Ritenour nitrite reductase activity has also been found in roots of tomato (Sanderson and Cocking, 1964), roots of barley (Miflin, 1967) and in etiolated radish cotyledons (Ingle t.21~: 1966). Regulation of Nitrite Reductase and Nitrate Reductase Nitrate reductase isolated from soybean was de- scribed as a flavoprotein by Evans and Nason (1953). Fluo- rometric analysis indicated that this enzyme contained flavin adenine dinucleotide (FAD) and that the boiled enzyme was active with D-amino acid oxidase Specific for FAD. Nitrate reductase is able to utilize reduced FAD or flavin 71 mononucleotide (FMN) as electron donors (Paneque _E al., 1965; Schrader 25 EE., 1968; Maretzki and Dela Cruz, 1967). Plant nitrate reductase could also be coupled to pyridine nucleotides. Soybean nitrate reductase can utilize both nicotinamide adenine dinucleotide (NADH) and NADPH (Evans and Nason, 1953) while the enzyme from other plants is specific for NADH (Beevers t §;,, 1964). Molybdenum was demonstrated as a component of nitrate reductase from fungi (Nicholas and Nason, 1954). There is no unequivocal evi- dence that molybdenum is a component of purified plant nitrate reductase but it has been shown to be associated with soybean nitrate reductase during four purification steps (Evans and Hall, 1955). Molybdenum deficient plants grow better on ammonia than on nitrate (Agarwala, 1952) and molybdenum was demonstrated to be essential for utilization of nitrate by intact plants (Spencer and Wood, 1954). Fur- thermore, molybdenum is necessary for induction of nitrate reductase in cauliflower leaves (Afridi and Hewitt, 1962). Nitrate reductase activity appears in higher plants in the presence of nitrate (as reviewed by Kessler, 1964). It was demonstrated that in the presence of nitrate an in- crease in activities of both nitrate reductase and nitrite t 3;,, 1966) reductase occurs in radish cotlyledons (Ingle and in Lemna (Joy, 1968; Stewart, 1968). Activities of these two enzymes started to increase in a sequential manner, .iae., the activity of nitrate reductase increased first and then after several hours lag nitrite reductase activity 72 started to increase. Ingle §£.§;, (1966) postulated that nitrate causes an increase in the activity of nitrate reduc- tase while nitrite reductase increases in response to nitrite, which was produced as a result of nitrate reductase action. Nitrite causes an increase in the activities of both enzymes in radish cotyledons (Ingle gglgi., 1966). Neither ammonia nor amino acids caused repression of the activities of both enzymes in radish cotyledons (Ingle ._E._E., 1966). In Lemna ammonia causes repression of the activities of both enzymes. Repression of nitrate reductase and nitrite reductase does not occur in a coordinate manner Since the ratio of nitrite reductase to nitrate reductase activities decreases with increasing amounts of ammonia added to the medium (Sims §E_EE., 1968). Nitrate assimilatory enzymes in the yeast, Candida utilis, are isolated as a large complex, designated the "nitrosome" (Sims S£.21-: 1968). Both enzymes are induced by nitrate and nitrite and they are repressed completely by ammonia and partially by glutamic acid. The ratio of activ- ities associated with nitrosome is constant regardless of growth conditions. This constant ratio of activities is characteristic of enzymes regulated in a coordinate manner. Pateman g; 3;. (1967) demonstrated that nitrate assimilation in Aspergillus is under the control of three genes. These genes are not linked and are as follows: a regulator gene, a structural gene for nitrate reductase 73 and a structural gene for nitrite reductase. Mutants in the regulatory gene loci either have constitutive nitrate reductase and nitrite reductase or lack both enzymes. Nitrate and nitrite can induce nitrate reductase and nitrite reductase. Pateman gp_§;, (1967) demonstrated unequivocally that nitrate directly induces nitrite reductase since mutants lacking nitrate reductase activity can induce nitrite reductase in reSponse to nitrate. MATERIALS AND METHODS Tobacco Cell Lines Tobacco cells grown in liquid culture were utilized as an eXperimental material. The cell line utilized through- out most of this study was the XD cell line isolated from tobacco stems by Filner (1965). This cell line was selected for its ability to grow on a defined liquid medium, M—lD, described below. Another cell line utilized was the R line which was selected from the XD line for its ability to grow on M-lD in the presence of the inhibitory amino acid, threo- nine (Heimer, unpublished). Growth Media Liquid medium M—lD described by Filner (1965), con— 5 tained the following compounds in moles x 10_ in a liter of distilled water: Ca(NO3)2'4 H O, 84.8; KNO 79.1; 2 141.0; MgSO 3I INaH PO - H '7 H 0, 146.0; KC1, 2 4 87.1; FeC 20, 11.9; NaSO4, 4 2 6H507-3 H20, 0.67; MnSO4'4 H20, 2.2; ZnSO4°7 H2O, 0.52; H B0 2.4; KI, 0.45; nicotinic acid, 0.41; pyridox- 3 3' ine-HCl, 0.049; thiamine-HCl, 0.03; 2,4-dichlorophenoxy acetic acid, 0.23; sucrose, 5,840. The pH of this solution was adjusted to 6.5 before autoclaving. 74 75 Nitrate-less M-lD was prepared by substituting the chlorides of potassium and calcium for nitrates. M-lD sup- plemented with tungstate was prepared by dissolving Na2W04-2 H20 in M-lD prior to sterilization. Casein hydrol- ysate solution was prepared by dissolving vitamin free casein hydrolysate (purchased from Difco) in distilled water in a concentration of 0.1 g per ml. The medium Supplemented with casein hydrolysate was prepared by adding a sterilized solution of casein hydrolysate to the previously sterilized medium. Maintenance and Growth of Cells Tobacco cells were grown in 500 m1 aliquots of M-lD. Stock cultures were transferred every 12 days to a new batch of medium by adding 25 ml of 12 day old parent culture to 500 m1 of M-lD. The cells were grown on a shaker with a horizontal displacement of 4 inches at 80 cycles per minute at 28°C. The growth eXperiments were started by inoculating media with cells from a 12 day old culture as described above. The cells were grown for the desired period of time and then harvested. 76 Harvesting of the Cells and Preparation of the Tobacco Cell Homogenate for the Enzyme Assays The cells from one liter of culture were harvested after the desired period of growth by filtration on Whatman paper No. l and their fresh weight was determined. The cells were suSpended in tris(hydroxymethyl)aminomethane (Tris)-HC1 (pH 7.5) containing 10_3 M cysteine (5 m1 of buffer was used for 1 gram of cells) and homogenized at 4°C with forty strokes of a motor driven teflon-glass homoge- nizer. The homogenate was centrifuged at 10,000 x g for 20 minutes. The supernatant solution was used as a source of nitrite reductase activity. Nitrate reductase was assayed in the fraction pre- cipitable by 50 per cent saturated ammonium sulfate. This fraction was prepared as follows: an aliquot of the 10,000 x g supernatant solution of tobacco cell homogenate was added to an equal volume of saturated ammonium sulfate (pH 7.5) containing 10'.3 M cysteine. After 1 hour at 40C this solution was centrifuged at 20,000 x g for 20 minutes. The precipitate was suspended to one-fifth of the original volume of the cell extract in 0.01 M phosphate buffer at pH 7.5 containing 10.3 M cysteine. An aliquot of this solu- tion was assayed for nitrate reductase activity. 77 Assay of Nitrate Reductase Activity Nitrate reductase activity was determined by a modi- fication of the method of Paneque _§_§l, (1965). The compo— sition of the incubation mixture was as follows: 50 umoles of phOSphate buffer (pH 7.5), 10 umoles of KNO 200 umoles 3. of FMN, 2.3 umoles of sodium hydrosulfite (added in 0.05 ml of 0.095 M NaHCO3), an aliquot of the enzyme extract and enough water to make the final volume 1 ml. After the addi— tion of enzyme extract the reaction mixture was incubated at 25°C for 15 minutes. The reaction was stopped by stirring on a Vortex mixer until the FMN was completely oxidized. The nitrite formed was determined by the colorimetric method of Snell and Snell (1949). To the contents of the tubes 1 ml of 1% sulfanilamide in 3 M HCl was added followed by 1 m1 of 0.02% N-l—naphtylethylenediamine dihydrochloride. The tubes were centrifuged in a clinical centrifuge for 5 minutes. The absorbancy of the supernatant at 540 mu was determined and compared to a zero time control. The activ- ity of nitrate reductase was eXpressed as mumoles of nitrite formed during one hour per gram of fresh weight of cells. Assay of Nitrite Reductase Activity Nitrite reductase was assayed according to the modi— fied assay of Ramirez £3.21- (1966). The assay was con- ducted in the following manner: 78 The reaction mixture was prepared by adding 100 umoles of phOSphate buffer (pH 7.5), 2 nmoles of methyl viologen, 5 umoles of KNO and enough distilled water to 2. make the final volume 3 ml. The tubes were flushed with nitrogen and covered with rubber stoppers. An aliquot of the cell homogenate was then added and the reaction was started by adding 40 nmoles of sodium hydrosulfite in 0.4 m1 of 0.29 M NaHCO (the sodium hydrosulfite solution was pre- 3 pared immediately before the assay). The tubes were incu- bated at 30°C for the desired period of time. The reaction was stopped by shaking the tubes vigorously on a Vortex mixer until methyl viologen was completely oxidized. An aliquot of the reaction mixture was taken for nitrite deter— mination. It was diluted 30-fold with distilled water and 1 ml of 1% sulfanilamide in 3 M HCl was added followed by 1 ml of 0.02% N—l-naphtylethylenediamine dihydrochloride. After 10 minutes the absorbancy was determined at 540 mu. Controls from which cell homogenate was omitted were done with each assay. The average absorbancy at 540 mn of those controls was taken as a measure of initial absorbancy of nitrite under the conditions of the assay. The decrease in the absorbancy at 540 mg was taken as a measure of nitrite reductase activity. The activity of nitrite reductase was eXpressed as “moles of nitrite utilized per hour per gram of cells. 79 Determination of Protein Content Protein content of the 10,000 x g supernatant solu— tion of tobacco cell homogenate was determined in the frac- tion precipitable by 10% trichloroacetic acid (TCA). To 0.5 m1 of the 10,000 x g supernatant solution an equal volume of 20% TCA was added. After 12 hours enough 10% TCA was added to make the final volume 10 ml. The tubes were centrifuged in the clinical centrifuge at 3,000 rpm for 10 minutes. The precipitate was resuSpended in 10 ml of 95% ethanol and sub- sequently centrifuged as described above. The supernatant solution was decanted and the pellet was dried in the air stream. The pellet was dissolved in 1 m1 of 1.0 M NaOH and its protein content was determined by the procedure of Lowry §£_g;, (1951). Bovine serum albumin dissolved in 1.0 M NaOH at a concentration of 1 mg/ml was used as a stan- dard. RESULTS Properties of Nitrite Reductase of Tobacco Cells One of the objectives of this study was the develop- ment of a reliable and Simple assay for the nitrite reduc- tase of tobacco cells. The assays, utilizing benzyl viologen or methyl viologen reduced by sodium hydrosulfite as electron donors, were considered eSpecially suitable Since they can be carried out aerobically. The assay of nitrite reductase activity was first carried out using benzyl viologen reduced by sodium hydro- sulfite as the electron donor, as described by Joy and Hageman (1966). Nitrite reductase activity could be detected using this assay. However, this assay did not prove satis- factory. A number of different concentrations of both benzyl viologen and sodium hydrosulfite were tested, but in no case was it possible to determine a ratio of concentrations of these two reagents which gave reproducible conditions of reduction of benzyl viologen. The assay of nitrite reductase activity, utilizing methyl viologen reduced by sodium hydrosulfite as an electron donor (Ramirez g3,§E., 1966), was subsequently utilized. 80 81 As demonstrated in Table l, the activity of nitrite reduc- tase, measured as disappearance of nitrite, was not detected in the absence of hydrosulfite. An increase in the amount of sodium hydrosulfite up to 20 umoles in the assay caused an increase in the amount of nitrite utilized. Table 1. The effect of varying concentrations of sodium hydrosulfite on the activity of nitrite reductase from tobacco cells* N05 reduced Sodium hydrosulfite “moles/h per g System umoles .of cells Complete 0 0.0 5 5.6 10 14.0 20 13.5 30 16.4 40 18.5 50 15.5 Complete minus cell homogenate 30 0.0 40 0.5 *The complete system contained in a final volume of 3 ml the following: 100 umoles of Tris-HCl (pH 7.5), 4 umoles of KNOz, 2 umoles of methyl viologen and 0.8 ml of homogenate of 2 day old tobacco cells. Tobacco cell homoge- nate was prepared by homogenizing 1 g of tobacco cells in.2 ml of 0.1 MTris-HCl (pH 7.5) containing 10-3 M cysteine. Hydrosulfite solution was prepared by dissolving 100 nmoles of Na2S204 per m1 of 0.29 M NaHCO3 and was added as indi- cated. Hydrosulfite in a 20 fold excess over methyl viologen did not cause inhibition of nitrite reductase activity and nitrite reductase activity was dependent upon methyl viologen as an electron donor (Table 2). 82 Table 2. The effect of heating the tobacco cell homogenate and the requirement for methyl viologen for nitrite reductase activity* N02 reduced umoles/h per g System of cells Complete 8.9 minus methyl viologen 1.1 Complete, cell homogenate heated at 1000C for 10 minutes 0.2 *The complete system contained in a final volume of 3 ml the following: 100 nmoles of TriS°HC1 (pH 7.5), 4 umoles of KNO , 2 pmoles of methyl viologen, 40 umoles of sodium hydrosulfite added in 0.4 ml of 0.29 M NaHCO3 and 0.8 ml of homogenate of 5 day old tobacco cells. Tobacco cell homogenate was prepared by homogenizing 1 g of tobacco cells in 2 m1 of 0.1 M Tris-HCl (pH 7.5) containing 10'3 M cyste- 1ne. The activity observed in the absence of methyl viologen is within limits of eXperimental error. The fact that no dis- appearance of nitrite was detected in the absence of tobacco cell homogenate (Table 1) indicates that no chemical reduc— tion of nitrite occurred under the conditions of the assay. The nitrite reductase activity of the tobacco cell homoge— nate was destroyed by boiling (Table 2). The activity of nitrite reductase was linear with incubation time during 1 hour. As demonstrated in Figure 1, nitrite reductase activity was also linear with the amount of tdbacco cell homogenate. The estimation of nitrite reductase activity was more accurate if the decrease in the nitrite content was higher than 10% of the initial value. Figure 1. 83 Dependence of nitrite reductase activity on the concentration of tObacco cell homogenate. Nitrite reductase was assayed as described in Materials and Methods. The incubation time was 14 minutes. The homogenate of 3% day old tObacco cells was added as indicated. Nitrite reductase activity is eXpressed as “moles of nitrite dis- appeared from the incubation mixture under condi- tions of the assay. pmoles NO'Z reduced 3.0 LC 84' l l l . /. O -- -1 O l y ' I - l o _ 0.2 . 0.4 0.6 ml Amount of tobacco cell homogenate in the assay 85 For routine estimations of nitrite reductase activity the time of incubation and the amount of enzyme extract were ap- propriately adjusted so that the decrease in nitrite content during the assay was at least 30%. Nitrite reductase of tobacco cells does not demon- strate a sharp pH optimum but it appears to be approximately 7.5. A variety of buffers were found to affect the activity of nitrite reductase. In the standard conditions of the assay and at pH 7.5 the nitrite reductase activity was the highest in phOSphate buffer, while in Tris and Hepps (N-2- hydroxyethylpiperazine-N'-3-propanesulfonic acid, Dr. N. B. Good, unpublished) the rate was 33% and 50% lower, reSpec— tively. The dependence of nitrite reductase activity from tObacco cells on nitrite concentration was Studied. The increase in nitrite concentration up to 1.6 mM caused an increase in the reaction rate but nitrite was inhibitory above 2 mM. The inhibition of nitrite reductase activity of illuminated grana preparation from tomato by nitrite at con- centrations above 0.25 mM was reported by Sanderson and Cocking (1964). Substrate inhibition of purified nitrite reductase from Spinach was not observed (Ramirez 2£.21-: 1966). Nitrite reductase from tobacco cells resembles other plant nitrite reductases described in the literature. Its activity is dependent upon reduced viologens which can sub- Stitute for ferredoxin, a likely physiological electron for 86 nitrite reductase. Addition of cysteine at 10_3 M causes an increase in the activity of nitrite reductase extracted from tobacco cells. The pH optimum of this enzyme is approxi— mately 7.5. An important characteristic of nitrite reduc- tase that was not demonstrated in the present study is a quantitative relationship between nitrite utilized and ammonia formed. Preliminary experiments indicate that ammonia was formed upon incubation of tobacco cell homoge- nate in the conditions of this assay. However, the stoi- chiometric relationship remains to be determined. Changes of Activity of Nitrate Reductase with Age of Culture Tobacco Cells Parent cultures of tobacco cells grown on M—lD do not contain any nitrate reductase activity but do have detectable levels of nitrite reductase activity. The activ— ities of both enzymes increased simultaneously after 12 day old tObacco cells of XD and R line were inoculated into fresh M-lD (Figure 2a and b). The increase in the activity of both enzymes was observed after 2 hours. No apparent lag ,in the appearance of nitrite reductase was observed as com- pared to nitrate reductase. Tobacco cells inoculated into M—lD deplete the medium of nitrate after 10 days (Filner, 1966). Figure 3a demon- strates that, following inoculation, the total content of soluble protein increases, reaches a peak after 8 days of growth, and subsequently declines. The activities of nitrate 87 Figure 2. Appearance of nitrite and nitrate reductase activity after transfer of 12 day old parent culture into M—lD. a. XD cell line. b. R cell line. Activities of both enzymes were assayed as described in Materials and Methods. sueo 10.61190. u/peonp 88 . - 91 EON lo seloumu: O 3 8 8 3 8 3 .. 9 co o v N - l . l l I I I n - 0 we 2 .... 20 ea 2 . , ’89: 3 3 4v .1 0" 3 tr .20 ,, :3 - ~~‘~ '52 I” l l o o o ‘0 V N sueo 10 6 18d u/peonpel EON 10' $810er 81180 )0 6 ed u/peonpea EON 40 seloumui~ O O O 3 § § § 9 a l l l l l l O 0‘ \\ \\ 3 \\ 2g ...... O o :3 m m ... q) :2 O c: L O m "' o .;.-. g . e s. o :8 l X I: s 0‘ \‘\ x ‘\ l l l O O O 0 " V N sneo to b .180 u/peonpeJ SON 10 I seloum 20 IO 30 20 IO Time (hoursl Time (hour‘s) 89 Figure 3. Changes in the activity of nitrite reductase and nitrate reductase with age of tobacco cells. a. Activity is eXpressed per liter of culture. b. Activity is eXpressed per milligram of protein. Ten day old XD cells were inoculated into M-lD and harvested as indicated. Activities of nitrate reductase and nitrite reductase were determined as described in Materials and Methods. 90 22.3 to ..2:\c_29_n_ oE O O O O 8 6 4 2 _ _ _ _ m _ _ m. . 82.3 .6 ..2: .3 530.33.. .02 .6 $681 5205 gunman 225268 -02 .6 $683: w o m m , o w W. 8 4 5 w 3 2 w I _ _ _ m _ I ... a. _ _ _ _ I s ..0 mm o l a w m \ b w .I . nw \o . mm o M I r 1.x R I r U I, W. .o ) nd/ . o ) . W n m p .r . s \m 0 o s V. \ IAIN I s a“ I. \o I 8 II 0\ \o.| 8 !\ e s . e a . \ m a m o \\\0\»\ m at ..I. |‘| e A,” W T \ II.\\° e“ n ..I- ’IIII‘Q" t I dl |‘ t l ../n m. 4 [nit test“ mm 4 all, 0.. I'I . 5' l Inn... IO'I'IIIO' .md J.,? /wl.-!i!.-odL m _ _ _ _ ML _ _ 9],? 0711.011. 0 o o o o 6 m 8 4 O O O 0 2 l 8 6 4. 2 95:3 Lo ..2: 8a 5826?. Nuoz .6 $68: 5296 oE Log 5326?. moz Lo 3681 91 reductase and nitrite reductase (eXpressed per liter of culture) reach a peak after 4 and 7 days of growth, respec- tively. The disappearance of the activities of both enzymes from the culture is faster than that of total protein. This is eSpecially true in the case of nitrate reductase, which declines to a undetectable level after 12 days of growth, while some nitrite reductase activity was still detectable in 15 day old tobacco cells. The specific activities of the two enzymes increased during the first 2% days of growth on M-lD and during subsequent growth the Specific activity of nitrite reductase declined at a slower rate than that of nitrate reductase (Figure 3b). This increase in the activity of nitrite reductase was observed only in tobacco cells grown in medium contain- ing nitrate as the only nitrogen source. If tobacco cells were grown for 15 days in medium containing casein hydroly- sate and nitrate, the activity of nitrite reductase was negligible at all times. The absence of nitrite reductase activity in cells grown on medium supplemented with casein hydrolysate apparently was not caused by inhibition of nitrite reductase by amino acids, since the nitrite reduc- tase activity of the mixture of extracts of tObacco cells grown on M-lD and grown on M-lD supplemented with amino acids was equal to the sum of activities of nitrite reduc— tase in the two extracts. 92 The Effect of Casein Hydrolysate on the Formation of Nitrate Reductase and Nitrite Reductase Casein hydrolysate inhibits the formation of nitrate reductase in tobacco cells grown on medium containing nitrate (Filner, 1966). It was therefore of interest to determine whether the formation of nitrite reductase is similarly affected. To test this, 12 day old XD cells and 14 day old R cells were grown for 48 hours on M-lD supple— mented with different concentrations of casein hydrolysate. The activities of nitrate reductase and nitrite reductase were then estimated in the homogenates. As demonstrated in Figure 4a and b, casein hydroly- sate inhibits the appearance of both enzymes in R and XD cell lines in a strikingly similar manner. The inhibitory effect of casein hydrolysate was not proportional to its concentration in the medium. Addition of casein hydrolysate at a concentration of 0.1 gram/liter caused 75%}inhibition of nitrate reductase activity and 70% inhibition of nitrite reductase activity in XD cells. Raising the concentration of casein hydrolysate 10 fold caused only a few per cent higher inhibition of both enzymes. The R cell line was selected from the XD line for its ability to grow on nitrate containing medium in presence of 10"4 M threonine, an amino acid which inhibits growth apparently by preventing the development of nitrate reduc- tase activity. Both the nitrate accumulation and the Figure 4. 93 The effect of casein hydrolysate on the formation of nitrate reductase and nitrite reductase activity. a. Twelve day old cells of the XD line were inoculated into M-lD containing casein hydrolysate as indicated and harvested after 48 hours. b. Fourteen day old cells of the R cell line were inoculated into M-lD containing casein hydrolysate as indicated and harvested after 48 hours. Activity of nitrate reductase was assayed as described in Materials and Methods. The reaction mixture for determination of nitrite reductase activity included in a final volume of 3 ml 100 umoles of Tris-HCl (pH 7.5), 4 umoles of KNOZ, 2 umoles of methyl viologen and 40 umoles of sodium hydrosulfite, and tobacco cell homogenate prepared in 5 ml per gram of tissue of Tris-HCl (pH 7.5) containing 10‘3 M cysteine. 94 suao £0. 6 - Jed q}paonp91 EON. w éa’loww' 0. § 0 .88.. com 2253 . 23:22.”: £300 . QN 0.. o . _ . _ $2262 22:: 32033.. 2.3%.. w. A _ O N ‘0. ¢ 0 . 0 O O snao ;o 5 19d q/peonpaJ 3_0N JO saloum sum 40 6 19d u/peonpaJ gON 4o salowmn, . 3233 2329.21 5360, QN 0.. O § 0 O Q’ I 000. com I. 3...... _. _ 322.89. 22...... 2.: ox _ _ cmnmfl. O. zfikl In 0 N II Infiflhfifid on Int-II ”\fi 0? bllflfl 95 activity of nitrate reductase are higher in R cells than in the XD cells (Heimer, unpublished). As demonstrated in Figure 4, the activity of nitrite reductase is also higher in the R cells. The ratio of activities of nitrate reductase and nitrite reductase was not constant in XD and in R lines of tObacco cells grown in the media supplemented with different amounts of casein hydrolysate. This indicates that regula— tion of nitrate reductase and nitrite reductase is not executed in a coordinate manner. Stability of Nitrate Reductase and Nitrite Reductase in vivo Knowledge of the stabilities of both nitrate reduc- tase and nitrite reductase is important for comparing the levels of their activities in different growth conditions and consequently in evaluating the means of their regulation. The effect of casein hydrolysate on the stability of nitrite reductase was also estimated to ascertain whether the de— crease in the activity of nitrite reductase in tobacco cells grown in the presence of casein hydrolysate is caused by a decrease in the formation of the enzyme or by an increase in its breakdown. The stability of both enzymes was examined in the following eXperiment. A 12 day old parent culture of tobacco cells was subcultured into M-lD for 24 hours. The cells were then transferred sterilely to nitrate-less M—lD 96 or nitrate-less M-lD supplemented with 3.0 g/liter of casein hydrolysate. .As demonstrated in Table 3, the activity of nitrite reductase increased during the first 8 hours after transfer into nitrate-less medium and after an additional 16 hours only a 16% decrease in nitrite reductase activity from its highest value was observed. Table 3. Activity of nitrate reductase and nitrite reductase after transfer of induced cells to nitrate-less M-lD, or nitrate—less M-lD supplemented with casein hydrolysate* Time after Nitrate reductase Nitrite reductase transfer mumoles NOE/h umoles of NOE/h (hours) per g of cells per g of cells _ NOE-less + _ NOS-less + NOB-less casein hydr. NO3-less casein hydr. O 320 21.15 4 350 320 23.4 32.7 8 260 325 33.8 27.9 12 375 255 29.7 33.6 24 42 150 28.5 32.7 *Twelve day old cells of the XD line were subcultured into M-lD for 24 hours. Subsequently they were transferred sterilely to nitrate-less M—lD, or nitrate-less M-lD supple- mented with casein hydrolysate at 3.0/g liter. Activities of both enzymes were assayed as described in Materials and Methods. 97 Addition of casein hydrolysate to the nitrate-less medium did not cause an increase in the disappearance of nitrite reductase activity from the cells. On the contrary, after the initial increase in nitrite reductase during the 4 hours after transfer of the cells to the medium supplemented with casein hydrolysate enzyme activity remained virtually unchanged. Nitrate reductase is less stable under the same conditions. Twenty—four hours after transfer of tobacco cells to nitrate-less M-lD nitrate reductase activity de- creased by 87% from its highest value. Casein hydrolysate does not increase the decay rate of nitrate reductase in tobacco cells. Effect of Tungstate and Casein Hydrolysate on the Formation of Nitrate Reductase and Nitrite Reductase Tungstate causes a decrease of nitrate reductase activity in tObacco cells grown on M—lD, prObably by compet- ing with molybdate and thus causing formation of a nonfunc- tional enzyme (Heimer §£_§l,, unpublished). Therefore, the use of tungstate makes it possible to determine whether nitrite formation is necessary for the induction of nitrite reductase, or whether nitrate can induce this enzyme directly. Table 4 demonstrates that tungstate at 2-10—5 M and 4°lO_5 M causes reSpectively, 82 and 92% inhibition of nitrate reductase activity. Tungstate at 2-10.5 M has no 98 Table 4. The effect of tungstate and casein hydrolysate on the formation of nitrate reductase and nitrite reductase* Nitrate Nitrite reductase reductase mumoles N03/h umoles NOZ/h Growth medium per g of cells per g of cells M-lD 528 29.7 M-lD + 2-10_5 M tungstate 95 35.7 M-lD + 4-10_5 M tungstate 40 24.9 M-lD + casein hydrolysate 84 10.0 M—lD + 2-10-5 M tungstate + casein hydrolysate 16 15.3 M—lD + 4°10-5 M tungstate + casein hydrolysate 10 11.7 *Twelve day old tobacco cells of XD line were sub- cultured into growth media described in Table 4. Casein hydrolysate, when used, was at 3.0 g/1iter. The cells were harvested after 24 hours of growth and the activities of nitrate reductase and nitrite reductase were assayed as described in Materials and Methods. effect on the activity of nitrite reductase, while at 4-10'5 M it causes a 17% decrease in the activity of nitrite reductase. Addition of casein hydrolysate to M—lD has a greater effect on nitrate reductase activity than the addition of tungstate. Casein hydrolysate at 3.0 g/liter causes 84% .inhibition of nitrate reductase activity and 66% of nitrite reductase activity. 99 The fact that tungstate has little or no effect on the activity of nitrite reductase does not prove that ni- trate is the inducer of nitrite reductase. The low level of nitrate reductase observed in the cells grown in these con- ditions may provide enough nitrite to induce fully nitrite reductase. However, when the same level of nitrate reduc— tase is present in casein hydrolysate grown cells, nitrite reductase activity is reduced to one-third as compared with its level in the cells grown on M-lD. One of the effects of the addition of casein hydrolysate to M—lD is a decrease in the accumulation of nitrate by tobacco cells, while tung- state does not affect nitrate accumulation by tobacco cells (Heimer §£_§1,, unpublished). It appears, therefore, that there is a better correlation between nitrate content of the cells and the activity of nitrite reductase than between the activities of nitrate reductase and nitrite reductase. How- ever, since casein hydrolysate lowers the nitrate content and decreases the activity of nitrate reductase, it also causes a decrease in nitrite formation. The question of whether nitrate or nitrite is the inducer of nitrite reduc- tase remains Open. DISCUSSION A reproducible and convenient assay was developed for nitrite reductase of tObacco cells by modification of a method of Ramirez gt_al. (1966). The reaction rate measured by this assay is proportionate to both time and amount of cell homogenate in the assay. Therefore, this assay is suitable for quantitative studies of changes in nitrite reductase activity with the physiological state of the cul- tured cells. The nitrite reductase activity estimated in tobacco cells utilizing this assay is higher than that of nitrate reductase in tobacco cells. The ratio of the activ- ities of the two enzymes vary, depending on the physiolog- ical state of the cells, but nitrite reductase activities are at least 40 fold higher. It appears that tObacco cells have a very efficient mechanism for metabolizing nitrite which is toxic. The nitrite reductase of tobacco cells has been found to be an adaptive enzyme. The appearance of this enzyme activity in tobacco cells depends on the nitrogen source present in the medium. Nitrite reductase activity is not detected in cells grown on a medium containing amino acids as the sole nitrogen source but it is Observed in 100 101 cells grown on nitrate containing medium. It is tempting to Speculate that nitrite reductase synthesis is induced in the presence of nitrate, but it is not possible to distinguish on the basis of available data whether nitrate causes activa- tion of the preexisting enzyme or its gg_ngyg synthesis. It is also possible that nitrite reductase is derepressed upon utilization of repressive amino acids after the transfer of the cells to the fresh medium. The increase in nitrite reductase and nitrate reduc- tase activities occurs simultaneously in tobacco cells grown on nitrate containing medium and continues roughly until the bulk of the nitrate is utilized from the medium. This sug— gests that the mechanism governing the appearance of the two enzymes is similar. When the activities of both enzymes were followed in tobacco cells during 16 days of growth nitrite reductase activity persisted after nitrate reductase decreased to an undetectable level. The difference in the levels of activ- ities of the two enzymes in older cells can be eXplained by the Observation that nitrite reductase is more stable than nitrate reductase upon transfer of induced cells to nitrate free medium. The difference in the stability of the two enzymes has been observed also in other plants. The decay rate of nitrate reductase in the absence of substrate is twice as fast as that of nitrite reductase in the wheat seedlings (Schrader §£_§1,, 1968). Nitrite reductase in radish cotyledons is also more stable than nitrate reductase 102 (Ingle 23 al., 1966). The fact that the first enzyme of assimilatory nitrate reduction is eSpecially labile might be of biological importance for the efficient regulation of nitrate assimilation. The rate of nitrite reductase formation decreases if amino acids are added to growth medium containing nitrate. The decrease in nitrite reductase activity could be caused by inhibition of the enzyme by amino acids. However, there was no decrease in nitrite reductase activity in a mixture of homogenates of cells grown on nitrate and cells grown on nitrate plus casein hydrolysate indicating that an effective concentration of inhibitor is not present in cells grown in the presence of amino acids. The possibility that amino acids act by increasing the rate of decay of nitrite reduc- tase has to be excluded since, as demonstrated in this study, amino acids do not enhance the decay of nitrite reductase activity. It appears, therefore, that amino acids act by preventing synthesis or activation of nitrite reductase. Data reported in this study do not provide a full understanding of regulation of nitrate reductase and nitrite reductase in tobacco cells. Studies of regulation of meta- bolic pathways in bacteria were advanced by combining data on enzyme activities in conditions of induction and repres- sion, and genetic studies. These data made it possible to distinguish several regulatory systems. It can not be assumed in the absence of any conclusive evidence that the 103 same regulatory mechanisms operate in bacteria and in higher plants, but it might be informative to compare the regula- tion of nitrate assimilatory enzymes with the different mechanisms of regulation of synthesis of bacterial enzymes described below: 1. The enzymes of a metabolic pathway, i.§,, enzymes reSponsible for utilization of exogenous lactose in E, 991;, are coded for by a cluster of structural genes and are under control by regulator molecules coded for by dis- tinct regulatory gene. This cluster of genes and the associated operator region is designated "operon" (Jacob and Monod, 1961). The genes of the operon are eXpressed in a coordinate manner, i.e., the ratio of the amount of any enzyme to the amount of another enzyme in the same operon remains constant regardless of its induction or repression. The activities of the enzymes of a given operon increase simultaneously; however, it has been demonstrated that the genes of lactose operon appear in temporal sequence (Alpers and Tomkins, 1966). In certain conditions the genes of the histidine operon are translated in a sequential manner (Berberich §£_§l., 1967). 2. Enzymes of a metabolic pathway are coded for by structural genes not adjacent to each other. Such a system consists of several operons and is under control of a single regulatory gene, and has been designated "regulon" (Maas and Clark, 1964). The genes of the regulon show similar, but 104 not coordinated, expression as described for arginine bio- synthetic enzymes by Maas and Clark (1964). 3. Enzymes of a metabolic pathway are induced coor- dinately in groups, as in the pathway of degradation of mandelate (Stevenson and Mandelstam, 1965; Mandelstam and Jacoby, 1965). In this pathway the first three enzymes are coordinately induced by mandelate, the fourth enzyme is induced by its substrate and its product, in turn, coordi- nately induces a group of remaining enzymes. A considerable lag (40 minutes) is Observed between the appearance of the enzymes of the first and the third Operon. This mode of induction is termed "sequential." Each of the three operons can be repressed. The product of the last enzyme of each operon can repress the enzymes of this operon and also inde— pendently, the enzymes of the operons coding for enzymes catalyzing earlier steps in the mandelate pathway. This type of repression is called "multisensitive." No information is available with respect to the linkage of the genes coding for nitrate reductase and ni- trite reductase in plants. It has been demonstrated that nitrite reductase is a chloroplast enzyme while nitrate reductase has been characterized as a soluble enzyme t al., 1967). However, localization of the (Ritenour enzyme in cell organelles does not exclude the possibility that it is coded for by nuclear genetic material. Utiliz- ing genetic methods it has been demonstrated that a plant 105 mitochondrial enzyme is coded for by nuclear genes (Longo and Scandalios, 1969). The fact that ratios of nitrate reductase to nitrite reductase activity in tobacco cells grown on media contain- ing different amounts of amino acids is not constant indi- cates that the regulation of these two enzymes in tobacco cells, unlike yeast (Sims g£_gl,, 1968), is not coordinate. The fact that there is no lag in the appearance of nitrite reductase activity in cells grown on nitrate does not support the idea advanced by Ingle gt a1. (1966) that the induction of nitrate reductase and nitrite reductase is sequential. Also inconsistent with both the sequential model of induction and the postulate that nitrite and not nitrate is the inducer Of nitrite reductase activity is the fact that tungstate had little effect on the formation of nitrite reductase activity, while it caused a considerable decrease in the activity of nitrate reductase. Nitrate reductase and nitrite reductase were induced by nitrite in radish cotyledons (Ingle gt al., 1966) and nitrite induces nitrate reductase in cauliflower (Candela §£_gl., 1957). Nitrite, therefore, may be an inducer of both enzymes in higher plants. However, nitrate might be contaminated with sufficient nitrate or it may be oxidized to nitrate either in the medium or by the plant. 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