. .1,I1o:- .w~o ha»-'l\..‘fl«..3,dld. s ‘ Juli-II {5. .Jlnl. .' . v , “Hill. - 1 b. {I .rQa.‘ ‘ ABSTRACT A STUDY OF THE ENZYMES IN LEAF PEBOXISOMES by Russell K. Yamazaki Peroxisomes have been isolated from leaf homogenates by isopycnic centrifugation. An analysis of the enzymic content of the leaf peroxisomes has revealed the presence of a serine-pyruvate aminotransferase and isozymes of NAD- malate dehydrogenase. NADP-isocitrate dehydrogenase, and AaSpartate-a-ketoglutarate aminotransferase in addition to catalase, glycolate oxidase, NAD-glycerate dehydrogenase and glutamate-glyoxylate aminotransferase which have been described in previous publications by our laboratory. Characteristics of the NAD-malate dehydrogenase activities of Spinach leaves were studied. The peroxisomal enzyme, Which was assayed by following reduced pyridine nucleotide oxidation, had a broad pH optimum from 6.4 to 7.4 and was Specific for NADH. The peroxisomal and mito- chondrial forms of NAD-malate dehydrogenase were differen- tiated by their kinetic and electrophoretic behavior. The mitochondrial form had a Km (oxalacetate) of 5.7 x 10‘6M and was inhibited by oxalacetate concentrations above 7 X 10'5M. The peroxisomal form had a Km (oxalacetate) of 1.4 x 10'5M and was inhibited by oxalacetate concentrations in 8X 88 th 0 Hi 10 any Ch C8. *1 Q5 an: Elf" ‘I( the Russell K. Yamazaki - 2 excess of 2 x 10'4M. The supernatant NAD—malate dehydrogen- ase showed kinetic characteristics intermediate to those of the peroxisomal and mitochondrial forms. Starch-gel elec- trophoresis of the supernatant fraction showed the presence of both the peroxisomal and mitochondrial forms together with another isozyme. The serine-pyruvate aminotransferase was found to be localized Specifically in the peroxisomes. No activity was detected in mitochondria or chloroplasts. Low but significant levels of NADP-isocitrate dehyd- rogenase were found in peroxisomes and mitochondria. Most of the activity in leaf homogenates was associated with the supernatant fraction. Whether or not this supernatant activity was due to leakage from particles could not be assessed. ‘ASpartate aminotransferase activity was found in chlorOplasts, mitochondria. and peroxisomes. In the last case, it is assumed that the enzyme Operates in conjunction with the NAD-malate dehydrogenase also present. Other enzymes of the tricarboxylic acid cycle (NAD- isocitrate dehydrogenase, fumarase, citrate synthetase, and aconitase) were localized in the mitochondrial fraction after separation of cellular organelles. Malic enzyme and formate dehydrogenase were also found to be localized in the mitochondrial fraction. The diversity of enzymic content of the microbodies from various sources is suggested to be an indication of dive thi: late act: of ' str: ham SOIL! km to‘ Russell K. Yamazaki - 3 diversity of function. In the case of the leaf peroxisomes, this function is suggested to be the conversion of glyco- late to glycerate. ;,g,, the glycolate pathway. All enzyme activities necessary for this pathway, with the exception of the conversion of glycine to serine, have been demon- strated in the peroxisome. These peroxisomal activities have been shown to be sufficient to convert glycolate to glycerate. in the intact lean assuming 50% of the carbon fixed during photosynthesis passes through glycolate. [A unified scheme for the function of leaf peroxi- somes has been suggested. This scheme, which utilizes known peroxisomal enzymes, assumes the peroxisomal membrane to be impermeable to pyridine nucleotide. The consequences of such a scheme are discussed in terms of metabolic con- trol in the plant. A STUDY OF THE ENZYMES IN LEAF PEBOXISOMES BY Russell K. Yamazaki A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 ACKNOWLEDGMENTS I thank Dr. N. E. Tolbert for his guidance, encouragement. and support during the course of this work. Support from the National Institutes of Health is also gratefully acknowledged. I eSpecially thank Miss Angelika 0eser whose tech- nical assistance has been invaluable to this thesis. I also thank my fellow graduate students for stimulating discussions which have contributed to much of this work. Finally I think my wife, Jane, whose contribution in help and encouragement have made much of this possible. 11 BIT LI: 1/1. 41% m TABLE OF CON TENTS INEODUCTION O O O O O O O O O O O O O O O LITERATIJBE REV IEw O O O O O O O O O O O 0 General Comments About Enzymes of the Tricarboxylic Acid Cycle . . . . . NAD-Malate Dehydrogenase . . . . Citrate Synthetase . . . . . . . Aconitase . . . . NAD- and NADP- Isocitrate Dehydrogenase Fumarase . . . . Enzyme Activities Related to the Tri- carboxylic Acid Cycle . . . . . . Malia Enzyme . . . . . . . . . . ASpartate.Aminotransferase . . . Formate and Formate Dehydrogenase PhOSphoenolpyruvate Carboxylase Cycle . . . . . . . . . . The Glycolate Pathway . . . . . . . . Peroxisomes . . . . . . . . . . . . . Glyoxysomes and Enzymes of the Glyoxylate MATERIALS AND METHODS . . . . . . . . . . Plants . . . Preparation of Fractions by Differential Centrifugation . . . Sucrose Density Gradient Centrifugation Assay methOdS o o o o o o o e o Malate Dehydrogenase . . . Glycolate Oxidase . . . . Cytochrome c Oxidase . . . ASpartate Aminotransferase . Isocitrate Dehydrogenase, NAD and Hello Enzyme . . . . . . PEP Carboxylase . . . Citrate Synthetase . . Malate Synthetase . . Isocitrate Lyase . . . Aconitase and Fumarase Formate Dehydrogenase . . . Serine-Pyruvate Aminotransferas Starch-Gel Electrophoresis . . . . O O O O O O 0 e O 111 booooooo N .‘Uooooooo D Page BESL Page Protein and Chlorophyll Determinations . . . . . 40 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . #1 NAD-Malate Dehydrogenase . . . . . . . . . . . . 41 Aminotransferase Activities . . . . . . . . . . 51 NADP-Isocitrate Dehydrogenase . . . . . . . . . 62 NAB-Isocitrate Dehydrogenase, Fumarase, Citrate Synthetase, and Aconitase . MalicEnZyme.......oo.... PEP Carboxylase . . . . . . . . . . . Formate Dehydrogenase . . . . . . . . Glyoxylate Dehydrogenase . . . Malate Synthetase and Isocitrate Lyase B-OXIdatlon o o o o o o o o o o e o o a-Oxidation . . . . Other Enzyme Activities Not Found in PerOXISomeS o o o o o o o o o o o 0 6n 69 7o 71 76 77 78 79 Gm EBAL D ISCUSS ION O O O O O C O C C O C C O O O O C 8 2 O O O I O O O I O O O O O O O O O O 0 O O O O O O O O O O O I O O O C O O I O O ‘3 :- Activities Not Found in Leaf Peroxisomes . . . . 83 Peroxisomal.ActivitieS . . . . . . . . . . . . . 89 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 112 BIBLIOGRAPHY O O O O O O O O O O O O O O O C O O O 0 111+ iv LIST OF TABLES Table no. Page 1 Kinetic PrOperties of Malate Dehydro- genaSeFomsoooooooooooooo “’7 2 Aminotransferase Activity of Leaf Peroxisomes as Measured by Coup- ling to Endogenous Dehydrogenases . . . 53 3 a-Keto Acid Specificity of Citrate Synthetaseooooéoooooooooo 66 h Effects of Adenine Nucleotides and Palmityl-CoA on "Malate Synthetase" Activity of Citrate Synthetase . . . . . 67 5 Summary of Intracellular Localization in Spinach Leaves of Enzymes Studied . . 84 6 Enzyme.Activities of Peroxisomes from Various Sources and Glyoxysomes . . . . 90 7 Calculated Specific Activities of Peroxisomal Enzyme Activities . . . . . 106 LIST OF FIGURES Figure no. Page 1 Distribution of NAD-Malate Dehydrogenase and Marker Activities after Sucrose Density Gradient Centrifugation of Spinach Broken ChlorOplast Fraction . . 44 2 Distribution of NAD-Malate Dehydrogenase and Marker Activities after Sucrose Density Gradient Centrifugation of Spinach Broken Chloroplast Fraction (cont'd). O O O O O O O O O O O O O O O “6 3 Schematic Representation of Starch-Gel Electrophoresis of the Various NAD- Malate mhydrogenases o o o o o o o o o 50 h Distribution of ASpartate Aminotrans- ferase and PEP Carboxylase Activ- ities Among Differential and Sucrose Density Gradient Fractions . . . . . . 56 5 Distribution of Serine-Pyruvate Amino- transferase, NADP-Isocitrate Dehydro- genase, NAB-Isocitrate Dehydrogenase, Fumarase, Citrate Synthetase, Aconi- tase, Malic Enzyme, and Marker Activ- ities after Sucrose Density Gradient Centrifugation of Spinach Broken Chloroplast Fraction mno . .'. . . . . 6O 6 Distribution of Formate Dehydrogenase and Marker.ActivitieS after Sucrose Density Gradient Centrifugation . . . . 73 7 Enzyme Reactions of Spinach Leaf Peroxi- somesooo'oooooooeoooooo101 vi bioine CoA CoA-SH DCPIP DTNB HEPES a-KG MES a-NPO 0AA PEP PHMS PPO TCA cycle TES tricine LIST OF ABBREVIATIONS N,N-bis(2-hydroxyethyl)g1ycine Coenzyme A Coenzyme A, free reduced form 2,6-dichlorOphenolindOphenol 5,5'-dithiobis(Z-nitrobenzoic acid) (Ethylenedinitrilo)tetraacetic acid N-Z-hydroxyethylpiperazine-N'-2— ethanesulfonic acid a-ketoglutarate 2-(N-morpholino)ethanesulfonic acid a-naphthylphenyloxazole oxalacetate phOSphoenolpyruvate 2~pyridine-hydroxymethanesulfonate 2,5-diphenyloxazole tricarboxylic acid cycle N-tris(hydroxymethyl)methylamino- ethanesulfonic acid N-tris(hydroxymethyl)methylglycine vii INTRODUCTION The study of the intracellular localization of enzymes has seen much progress within the last twenty years. Before this period, it was felt necessary to remove particulate material from extracts in order to demonstrate enzyme action - thus the term "cell-free extract." This removal effectively silenced the propo- nents of vitalism who maintained that the biochemical reactions observed in extracts were due to the presence of whole cells. In the early 1950's, it became evident that the enzyme activities of the Tricarboxylic Acid Cycle were localized in particulate material. Work from several laboratories established that the mitochondrion, a distinct cellular organelle, was the site of TCA Cycle activity and energy production. Later. photosynthetic carbon dioxide fixation and ATP formation were demon- strated to take place in another distinct cellular organ- elle; the chloroplast of green plants. All enzyme activ- ities necessary for photosynthetic carbon dioxide fixa- tion have Since been demonstrated to be present in the Chloroplast. Since many of the above activities were originally thought to be "soluble," it became evident that intracellular localization of enzyme activities Should be studied. and the iti 011' 113 Per tis 2 Use of the differential centrifugation techniques for cell fractionation as develOped by Claude, Hogeboom, and Schneider led to the discovery of another organelle, the lysosome. Through the work of de Duve and coworkers, it was demonstrated that the lysosomes were the site of hydrolase activity in liver cells. Application of the technique of iSOpycnic centrifugation to rat liver homog- enates revealed the presence in the cell of still another organelle. Because of the presence of flavoprotein oxidases and catalase and the implied role in 3202 metabo- lism, these particles were termed "peroxisomes" by de Duve. Peroxisomes were found to be limited to liver and kidney tissue in the animal. In the case of plant tissue, the issue of the intra- cellular localization of glycolate oxidase has been confused. This flavoprotein oxidase, which catalyzes the oxidation of glycolate to glyoxylate as the first step of the glycolate pathway, was first assumed to be soluble. Later reports located activity in chlorOplastS and in mitochondria. Application of iSOpycnic centrifugation to particulate frac- tions as done in our laboratory has now revealed that gly- colate oxidase, NAD-glyoxylate reductase, and catalase are localized in microbodies in leaf tissue (97). Because of the similarity in enzyme content of the plant particles with liver peroxisomes (2h), these particles have been terlned "leaf peroxisomes." Further work has demonstrated 3 the presence of a glutamate-glyoxylate aminotransferase activity in the leaf peroxisomes (#7). I Concurrent work on microbodies from various sources indicate that, although all the microbodies characterized to date contain an a-hydroxy acid oxidase and catalase, diversity with reSpect to other enZyme activities exists. The glyoxysomes as isolated from germinating castor bean endoSperm contain all the enzymes of the glyoxylate cycle and B-oxidation (10, 18). Peroxisomes from rat liver con- tain D-amino acid oxidase, urate oxidase, and.NADP-iso- citrate dehydrogenase (23). Peroxisomes from Tetrahymena contain D-amino acidoxidase, isocitrate lyase, malate synthetase, and NADP-isocitrate dehydrogenase (67). The purpose of this thesis was to study the enzymic composition of the leaf peroxisomes. Large parts of this thesis have already been published (97, 98, 103) and will only be summarized at this time. Comparisons with the microbodies from other sources have been drawn where pos- sible. A unified scheme for the function of leaf peroxi- somes has been proposed on the basis of demonstrated enzyme activities. Acid p1an+ most What to h anim Synt Phat aeor anir appe 911a] LITERATURE REVIEW General Comments About Enzymes of the Tricarboxylic Acid Cycle It is now generally assumed that the Tricarboxylic Acid Cycle (TCA Cycle) is operative in the mitochondria of plant cells just as it is in animal mitochondria where most of the component enzymes have been studied in some- what greater detail. A few of the enzymatic steps appear to have different Specificity than the correSponding animal enzyme (21). For instance, the plant succinyl-CoA synthetase utilizes ADP rather than IDP or GDP as the phos- phate acceptor. Suggestions have been made that the plant aconitase dehydrates citric acid differently than the animal enZyme. But the functioning of the total TCA cycle appears to be the same in plants and animals. Plants have long been known to accumulate large quantities of acids (Ref. 79). Some plants are utilized (commercially for production of acids; in many cases these acids are acids of the TCA cycle. AS Banson (79) has [pointed out, the TCA cycle cannot be used for the accumula- ‘bion of any of its intermediates or metabolites thereof itithout arresting the action of the cycle. But, accumula- illon of any of the cycle acids may be accomplished if there exists an enzymic mechanism for the synthesis of one of the 4 acin cit rea con of dri 88E en: 130‘ Ch in ca of 5 acids independent Of the TCA cycle. PEP Carboxylase may be cited as one such example. Following the same line Of reasoning, we might also eXpect to find enzymes involved in conversion Of the cycle intermediates Operating independently Of the TCA cycle. Putting this another way, we now have established a rationale for the existence of extramitochon- drial forms of TCA cycle enzymes. In animal systems which have been studied, it is assumed that the extramitochondrial forms of TCA cycle enzymes exist to provide for the tranSporting of reducing power into and out of the mitochondria (for example, see Ref. 78). Several roles can be postulated for the extramito- chondrial TCA cycle enzymes in plants. They may be involved in the storage Of organic acids for reducing power and/or carbon Skeletons. Or they may be involved in the formation of amino acids. The existence Of extramitochondrial forms Of the TCA cycle enZymes has been inferred in plants from the presence Of multiple isozymes Of various enzymes (§.g,. Ref. 8“), but the locations and prOperties Of these isozymes have not been studied extensively because of the problems involved in the separation Of plant cell organelles. Thus malate dehydrO-> genase activity has been described as residing in chloro- plast fractions from differential centrifugation (109), but actual proof of the existence of a chloroplast malate dehyd- rogenase could not be Obtained because Of the presence of ll' Illlll mit: It : as ; tha thu 2e: abt the is at- (11 Dr be or he 6 mitochondria and peroxisomes in these chloroplast fractions. It is only through the application Of other techniQueS such as nonaqueous isolation or density gradient centrifugation that satisfactory separation of organelles can be attained, thus allowing further study. NAD-Malate Dehydrogenase In the following review, discussion will be limited primarily to the enzymes as studied in plant sources. With reference to Specific enzymes, the case of malate dehydro- genase stands out in confusion. Reference has been made above to a few reports as to the intracellular location Of the extramitochondrial forms. A somewhat more complete list is given in Reference 103. Many of these references attribute malate dehydrogenase activity to the chloroplast, but, as will be discussed later, in all of these reports, differential centrifugation was used for the chloroplast preparations. Use Of differential centrifugation alone has been shown to be inadequate for separation of cellular organelles (98). By use Of DEAE chromatography, Davies (20) separated two malate dehydrogenase activities from pea epicotyl homogenates. One activity was attributed to mitochondrial enzyme and the other, to a supernatant enZyme. Yue (10h), utilizing polyacrylamide gel electrophoresis, was able to demonstrate the existence of three malate dehydrogenase iso and ste VeI the 101 7 isozymes from barley. Two of these were termed supernatant, and the third was termed the mitochondrial activity. C trate Synthetase —_ Citrate synthetase or condensing enzyme has been studied in homogenates of peanut cotyledons by Marcus and Velasco (61). The enzyme was found to be associated with the particulate fractions and was inhibited somewhat by Mg ions. The enzyme was further Shown to be Specific with reSpect to the COA ester, reacting with only acetyl-COA ester, at.a significant rate. Aconitase Aconitase localization has been studied in tobacco by Pierpoint (7#) who concluded that although most of the activity was found in supernatant fractions, some of this activity could be attributed to leaching Of the enzyme from the mitochondria. The particulate activity was found in the mitochondrial fraction. NAD— and NADP-Isocitrate Dehydrogenases The cases of the plant NAD- and NADP-linked iso- citrate dehydrogenases appear to be Similar to those found in animal tissues. The MAD-Specific isocitrate dehydrogen- ase activity is associated exclusively with the mitochondria (21). The NADP-linked activity has been found. both in the mite pari ani of der aci W11 Che W0: Fee. 8 mitochondria and in supernatant fractions, with the major part Of the activity residing in the supernatant (21, 61). In two cases (56, 71) it has been suggested that chloro- plasts contain an NADP-Specific isocitrate dehydrogenase activity. The MAD-linked isocitrate dehydrogenase has been found to be irreversible (21), a situation also found with the animal NAB-linked activity. AS to NADP-Specific activ- ity, reversibility can be achieved in both the plant and animal enzymes. Cleland (16) has suggested on the basis of kinetic evidence that the NADP-Specific isocitrate dehydrogenase of the cytoplasm in animal tissues may be acting in the direction Of the reductive carboxylation Of a-ketoglutarate. Such studies have not been carried out with the enzyme from plant sources, but comparative bio- chemistry might lead one tO suggest that the plant enzyme works in a similar manner. Fumarase The distribution of fumarase in tobacco homogenates was studied by Pierpoint (73) who found that 90% Of the total fumarase activity was associated with the mitochon- dria. Pierpoint, however, was not able to state unequivo- cally that all of the cellular fumarase activity was mito- chondrial because of the presence of nuclear material in his particulate fractions. He thought it possible that an 9 enzyme such as fumarase might become bound to the nucleic acids upon homogenization. thus producing an artifactual distribution Of the activity. Enzyme Activities Related to the Tricarboxylic Acid Cycle Malic Enzyme In 1949, Conn, Vennesland, and Kraemer (17) studied the occurrence Of malic enzyme and found this activity to be wideSpread among higher plants. More recently the intra- cellular distribution of malic enzyme has been studied in quntia phylloclades by Mukerji and Ting (65) and in maize leaves by Slack and Batch (87). In both cases the particu- late activity was found tO be localized in the chloroplasts, suggesting that malic enzyme in these plants is active in the photosynthetic fixation of carbon dioxide. The intra- cellular localization Of malic enzyme was also studied in corn root tips by Banner and Ting (19) who concluded that the activity was nonparticulate, i,g,. nonmitochondrial. In later work by MukerJi and Ting (66), three isozymes of malic enzyme were found in Opuntia stem tissue. These were designated as mitochondrial, chlorOplastic, and soluble and were separated by DEAE cellulose chromatography and electro- phoresis. The three isozymes were found to differ with reSpect to kinetic and physical properties. NO conclusions could be drawn. however, concerning the metabolic functions of th is 8!. 10 of each Of the isozymes. Aspartate: a-Ketoglutarate Aminotransferase ASpartate aminotransferase is an apparently ubiqui- tous enzyme (for example, Ref. 8) which catalyzes the reversible reaction shown in Equation I. The enzyme is L-aSpartate' + a-ketoglutarate" = L-glutamate' + oxalacetate“ (I) thought to function in tranSporting Of oxalacetate and in the biosynthetic formation of aSpartate from oxalacetate. As isolated from all sources to date, the enzyme contains pyridoxal phOSphate and can be resolved in an apoenzyme and the pyridoxal phOSphate. ASpartate aminotransferase activity as isolated in animal systems has been found in both mitochondrial mem- branes and in the cytosol (53). These two isozymes are thought to mediate tranSport Of the oxalacetate carbon skeleton from inside the mitochondria to the cytosol. Because of the high reactivity of the a-keto acid oxalace- tate, aSpartate is assumed to be a tranSport form along with malic acid. Thus it might be eXpected that aSpartate aminotransferase activity would be associated with systems involved in oxalacetate metabolism. This may be the case with peroxisomes, mitochondria, and chlorOplastS. ASpartate aminotransferase and malate dehydrogenase are dure put wit! thei acie 11 are Often found to be associated during purification proce- dures (2.5,. Ref. 20), and in fact the suggestion has been put forth that in NeurosROra these activities are associated with the same protein (68). This suggestion was based upon their physical properties, immunochemical behavior, amino acid composition, tryptic maps, and genetic behavior. Kisaki and Tolbert (#7) found aSpartate aminotrans— ferase activity in leaf mitochondria and peroxisomes. The latter activity was attributed to nonSpecificity of the glutamate-glyoxylate aminotransferase. Investigation Of the amino group donor and acceptor Specificity, however. indicates that there is more aSpartate aminotransferase activity present than can be accounted for by the gluta- mate-glyoxylate aminotransferase. ASpartate aminotrans- feraSe activity has also been reported in chloroplast preparations from Triticum vulgare and Y$£$3.222E by Heber (#0) and from Opuntia by Mukerji and Ting (65). Formic Acid and Formate Dehydrogenase The roles for formic acid and formate dehydrogenase in plant metabolism are not completely understood. Tolbert (9h) has Shown that when inc-labeled formic acid is fed to barley leaves, the distribution of labeled products indicate entry of formic acid into both the C1 pool and intermediates Of the Photosynthetic Carbon Reduc- tion Cycle. The entry of formic acid into the Ci pool is 12 probably mediated by the formate-activating enzyme, thus leading to 1Ll'C-label in tetrahydrofolate derivatives. This label in turn Spreads into products of the glycolate path- way via the B-carbon Of serine through serine transhydroxy- methylase action. Radioactive formic acid was also found to label malic, aSpartic and glutamic acids in the light but not in the dark. This difference may be eXplained if both formate dehydrogenase and one or more Of the B-carboxylating enzymes are present in the mitochondria. Labeled 002 released by formate dehydrogenase might thus be preferentially utilized by the B-carboxylating enzyme(s) Of the mitochondria rather than being fixed via the Photosynthetic Carbon Reduction Cycle. The light-dependence may simply be a reflection Of the need Of the B-carboxylating enzymes for PEP or pyruvate which presumably would be in greater abundance during photo- synthesis. It has been suggested that formic acid is an intermediate in the reductive fixation Of carbon dioxide, but the above Observations tend to rule out such theories. Early work on plant formate dehydrogenase was limited to seedlings. The enzyme was shown to be present in pea (22) and bean (101) seedlings but was found to dis- appear after the seventh day of germination. The pea epicotyl enzyme was shown by Davies (21) to be localized in the mitochondrial fraction. Mazelis (63) later demon- Strated the presence of MAD-formate dehydrogenase in leaf (n In 1*" I 13 tissue from several plants including Spinach. In all the tissues studied. the activity was associated primarily with the cytoplasmic particles, i,g,. mitochondria. No sugges- tion has been brought forth as to the function Of the enzyme, but studies of the reaction Show that the equilib- rium lies far toward the direction of the oxidation of formate. This suggests the function probably does not reside in formate formation. The source in the plant of formic acid, if indeed there is one, is unknown at present. Ig 31332 studies Of glycolate oxidase (#6) demonstrated that formate could be produced by nonenzymatic oxidation Of glyoxylate by hydro- gen peroxide (Equation II), but the in_zi12 significance Of 3202 320 02 (II) glycolate MglyoxylatM formate 4- CO2 this Observation must be questioned in light Of the finding Of Kisaki and Talbert (#7) that labeled 002 is not released uC-glycolate. For- during the peroxisomal oxidation of 1-1 mate might arise from aldehyde oxidase or dehydrogenase action upon formaldehyde. Little knowledge is available concerning the metabolism of formaldehyde in plants. PhosEhoenolpyruvate Carboxylase The enzyme PEP carboxylase was first described by JBandurski and Greiner (6) in 1953 in spinach leaf homogen- of of th! the Do in er ho l# ates. The enzyme irreversibly catalyzes the reaction shown in Equation III. If lac-labeled bicarbonate is used, all _ Mg++ PEP + HCO3 -———9eoxalacetate + P1 (III) of the label incorporated is found in the B-carboxyl group of the oxalacetate. The enzyme is probably reSponsible for the major part Of the dark fixation in plants possessing the Photosynthetic Carbon Reduction Cycle. In plants such as sugarcane which possess the Cu-dicarboxylic acid pathway, PEP carboxylase iS apparently the major photosynthetic carbon-dioxide-fixing enzyme (87). In such plants the PEP carboxylase activity is associated with the chloroplasts. The location of PEP carboxylase in plants utilizing the Photosynthetic Carbon Reduction Cycle has never been rigorously determined. Rosenberg, Capindale and Whatley (82) found activity in chloroplast extracts Of Spinach leaves. Mukerji and Ting (65) found PEP carboxylase activ- ity in chlorOplasts of Opuntia, a plant which probably possesses Crassulacean Acid Metabolism. PEP Carboxykinase activity has also been reported in Spinach leaf preparations, but anomalies with two differ- ent assay procedures have cast some doubt as to whether or not carboxykinase activity is present (6#). Glyoxysomes and Enzymes Of the Glyoxylate Cycle The glyoxylate cycle by which acetate is converted to 15 carbohydrate was first elucidated in bacteria by Kornberg and Krebs (50). In 1957 Kornberg and Beevers (#9) demon- strated the presence in castor bean endOSperm extracts Of isocitrate lyase and malate synthase, two enzyme activities exclusive to the glyoxylate cycle. as well as NADP-iso- citrate dehydrogenase. NADP-glyoxylate reductase, citrate synthetase, and malate dehydrogenase. They were also able to demonstrate the net conversion Of exogenously-supplied labeled acetate to labeled sucrose in the endOSperm. Later studies Of the distribution Of isocitrate lyase and malate synthase Showed that these key enzymes were present only in those plant tissues actively convert- ing lipid storage material into carbohydrate (7). Thus these enzyme activities were found only in those germinat- ing seeds which contain a high proportion of lipid. Marcus and Velasco (61) studied the intracellular localization of the glyoxylate cycle enzyme activities in germinating peanuts and castor beans and suggested that they were mito- chondrial. In 1963, Rogg and Kornberg (#2) concluded that the enzymes Of the glyoxylate cycle in Tetrahymena pyriformis, a protozoan, were localized in a Specialized mitochondrial fraction. More recent work of Muller, Rogg and de Duve (67) identified this Special mitochondrial fraction as actually being a peroxisomal fraction. Harrop and Kornberg (36) have also demonstrated the presence of isocitrate 16 lyase in a dense particulate fraction from Chlorella with a functional glyoxylate cycle. In the case Of higher plants, the association of the glyoxylate cycle activity with a subcellular particle dis- tinct from the mitochondrion was first shown by Breidenbach and Beevers (10). These particles, because Of the presence Of the glyoxylate cycle, were termed glyoxysomes and were shown to contain isocitrate lyase, malate synthetase. citrate synthetase, and malate dehydrogenase. They were devoid Of cytochromes, fumarase, NADR oxidase, and succinic dehydro- genase which were localized in the mitochondrial fraction. Further characterization of the glyoxysomes by this group (11) indicated that glycolate oxidase and catalase were also present in the particles. By using sulfhydryl-protect- ing reagents in the gradients, the authors were also able to demonstrate the presence of aconitase in the glyoxysomes, thus completing the sequence of enzymatic activities required in the glyoxylate cycle. The problem of the source of the acetyl-COA utilized in the glyoxylate cycle has been clarified with the finding Of the presence Of the complete B-oxidation system in the glyoxysomes (18). More than 80% of the particulate B-oxida- tion activity was found to be localized in the glyoxysomes. Addition of palmityl-COA to the glyoxysomes were Shown to produce oxygen uptake, NADH accumulation and acetyl—COA production in a ézizi stoichiometry. The addition of 17 cyanide doubled the oxygen uptake, but had no effect on the NADR accumulation. These data were interpreted as indicat- ing the presence of an oxygen-requiring acyl-COA dehydro- genase which yields hydrogen peroxide which in turn is decomposed by catalase. One of the problems remaining to be solved is the fate Of the NADH generated during Opera- tion Of the 8-Oxidation pathway and the action Of the malate dehydrogenase of the glyoxylate cycle. It may be that NADH generated internally must pass outside of the glyoxysome to be oxidized. Nothing is known at the present concerning the permeability Of the glyoxysomal membrane with reSpect to organic acids, pyridine nucleotides and cofactors. The presence of the B-oxidation system in these dense particles from castor bean endOSperm has been con- firmed by Rutton and Stumpf (##). These authors found ricinoleate to be oxidized most rapidly by the particles from the maturing seeds whereas the particles from the germinating seed were found to utilize palmitate and linoleate most rapidly. Possible control mechanisms with reSpect to the gly- oxylate cycle have been demonstrated by several workers. One of the problems concerned with the operation of the glyoxylate cycle is how is it that the isocitrate in the tissue is metabolized primarily by the isocitrate lyase rather than being used by the mitochondrial isocitrate oxidizing system. Tanner and Beevers (92) studied this l8 competition and found that the isocitrate lyase activity was three-fold higher than the isocitrate oxidizing system, thus providing a possible eXplanation for the low TCA cycle oxidation activity in castor bean endOSperm. Rock and Beevers (#1) studied the develOpment and decline of the glyoxylate cycle enzymes and the effect Of Dactinomycin and cycloheximide in watermelon seedlings. They concluded that the increase in activity of isocitrate lyase and malate synthetase was due to reutilization Of a relatively stable m-RNA which is produced only during the first day Of germination. The later decline in activity Of the enzymes was attributed to the limited half-life (2-3 days) Of the enzymes. Control at the feedback inhibition level has been suggested by Nagamachi, Fujii, and Honda (69) who found that the isocitrate lyase of the germinating castor bean endOSperm was uncompetitively inhibited by glucose-6- phOSphate. Competitive inhibition has been demonstrated by Kornberg (#8) in the case Of isocitrate lyase from Escherichia 22;; upon the addition of PEP. Similarly John and Syrett (#5) have shown that oxalacetate and pyruvate inhibited competitively the isocitrate lyase Of Chlorella pyrenoidosa grown on acetate. Control of the glyoxylate cycle activity at the hormonal level has been suggested by Penner and Ashton (72) who found that benzyladenine and cytokinin promoted isociti They a the ac been : repcr' taken the 3 $1111] bios; 19 isocitrate lyase activity in squash and peanut cotyledons. They also found that puromycin inhibited the formation of the activity and so suggested that the activity arose from 92.2212 synthesis. Pinfield (75) found that the addition of gibberellin increased the isocitrate lyase activity in hazel cotyledons. Repression Of isocitrate lyase synthesis in castor bean seeds germinated in the presence Of glucose has been reported by Lado, Schwendimann and Marre (52). These data taken with the reports of hormonal effects indicate that the glyoxylate cycle activity is under metabolic control Similar to that found in the case of bacterial pyrimidine biosynthesis. Feedback inhibition Of an early enzyme in the system is seen. Further control at the nucleic acid level is evidenced in the hormonal reSponseS and repression by one Of the products of the pathway. Recent studies by Longo (58) and Gientka-Rychter and Cherry (32) utilizing the technique Of density labeling have indicated unequivocally that isocitrate lyase and malate synthetase are synthesized gg_ngzg in the germinat- ing peanut cotyledon. The increases in buoyant densities Of the proteins upon germination in either H218 0 or D20 were larger than could be accounted for by the introduc- tion Of heavy isotOpeS due to hydrolysis of reserve pro- tein. This suggests that the amino acids utilized in the synthesis of these enzymes are derived from other sources 20 than reserve protein. Investigations of the development of glyoxysomes in germinating peanut and castor bean have revealed that the glyoxysomes as a whole are under the same type of control as found in the cases Of the individual enZymeS of the cycle. Longo (59) was able to demonstrate that culturing of isolated peanut cotyledons in a glucose solution reduced both the isocitrate lyase activity and the total amount of glyoxysomes. Gerhardt and Beevers (31) have reported that the density Of the glyoxysomes from castor bean endOSperm remains at 1.25 g/cm3 at all stages Of germination. They also reported the puzzling observation that, at the earlier stages Of germination. only 20-30% Of the isocitrate lyase activity was associated with the particulate fractions. After day # Of germination. 70-80% Of the isocitrate lyase was associated with the particulate fractions. They were able to rule out breakage of the glyoxysomes as the cause for the soluble isocitrate lyase by showing that other enzymes Of the glyoxysome remained in the particulate frac- tions. The process by which the isocitrate lyase activity becomes particle-bound is not known at present. This Situ- ation Of the anomalous behavior Of isocitrate lyase is Similar to that found by RarrOp and Kornberg (36). They found the Brannon NO. 1 strain Of Chlorella vulgaris to be constitutive for isocitrate lyase activity but also found 21 that the glyoxylate cycle was not functional as a whole unless the isocitrate lyase was incorporated into a dense particle. The Glycolate Pathway During photosynthesis with 1” C02. glycolic acid becomes labeled at early times. Feeding eXperiments have established that glycolate may be metabolized in plants via glycine. serine and glycerate to give sucrose in the light. This series of reactions has been termed the gly- colate pathway (95). The glycolate formed during photo- synthesis is uniformly labeled and the same has been found to be true of the intermediates Of the glycolate pathway, thus establishing evidence for Operation of the pathway in the plant. Estimates Of the magnitude Of the pathway have indicated that 50% or more Of the carbon fixed during photosynthesis may be passing through this route. The origin Of the glycolate is unknown at present, but it has been suggested that it arises from the oxidation Of sugar phosphates in the chloroplast. The initial product Of this oxidation is presumed to be P-glycolate, which is then dephOSphorylated by a Specific phOSphatase to give glycolate (81). Glycolate oxidase, the first enzyme in the pathway starting from glycolate, was first described by Claggett, Tolbert and Burris (15). The enzyme as isolated from 22 tobacco and barley was characterized as an c-hydroxy acid oxidase which was Specific for the L-isomers. Glycolate was found to be the best substrate. Glycolate oxidase was later shown by Kenton and Mann (#6) and Zelitch and Ochoa (113) to be a flavoprotein. The enzyme was crystallized from Spinach by Frigerio and Harbury (30). These workers found that the enzyme had a minimum molecular weight of 70,000, but Showed enzyme activity only with the components of molecular weights 1#0.000 and 270.000. It was also noted that the 270,000 molecular weight component did not require the addition of FMN for maximal activity whereas the 1#0.000 molecular weight component did. These findings suggest that the enzyme exists as a tetramer when flavin is firmly bound and as a dimer when the flavin is loosely bound or becomes free. Tolbert and Cohan (96) found glycolate oxidase activ- ity to be low in etiolated material and to increase upon greening. These observations and those of Kuczmak and Tolbert (51) established that the glycolate oxidase activity in etiolated wheat could not be detected unless the material was homogenized in the presence Of either glycolate or FMN. This substrate-protected or alternate form Of glycolate oxidase was isolated and purified by Baker and Tolbert (5) who found the Spectrum of the enzyme to be different from typical flavo- proteins. Similarities of the Spectrum with and stimulation of the enzymic reaction by ferredoxin led the authors to ~53 1r te at CE 23 Speculate that the enzyme might have ferredoxin or non-heme iron bound to it. MAD-glyoxylate reductase. first isolated and charac- terized by Zelitch and Ochoa (105). should be included among the enzymes Of the glycolate pathway, but the signifi- cance of this activity is uncertain. The suggestion has been made by Zelitch (106) that the combination Of NAD- glyoxylate reductase and glycolate oxidase in the plant may function as an MADE-oxidizing system, thus getting rid Of excess reducing power. Such a system might work in conjunc- tion with the chloroplast NADP-glyoxylate reductase described by Zelitch and Gotto (112) to reoxidize the pyridine nucleotides Of the chloroplast during periods of over- reduction. The acidic pH Optimum (6.2) Of the NAB-glyoxy- late reductase activity in comparison to that Of glycolate oxidase (pHopt = 8.?) make the possibilities of the two activities acting as an MADE-oxidizing system somewhat remote. A more probable function Of this enzyme may be found in its D-glycerate dehydrogenase activity. Zelitch found the enzymic rate to be four times greater with hydroxypyruvate as substrate than with glyoxylate. Holzer and Rolldorf (#3) and Laudahn (55) have concluded that the same enzyme is reSponSible for both activities and have preferred the name NAD-D-glycerate dehydrogenase for the enzyme in view Of the lower Km for hydroxypyruvate (ca. lo'YM vs. 10'2M) and the larger VMax (# times greater with 2# hydroxypyruvate). The enzymatic reaction involved in the conversion of glyoxylate to glycine has been elucidated by Kisaki and TOlbert (#7). An aminotransferase activity utilizing gly- oxylate as the amino acceptor and glutamate and alanine as the amino donors was shown to be located Specifically in leaf peroxisomes. The high degree of substrate Specificity and sharp pH Optimum at pH 7.3 led these authors tO con- clude that a single enzyme was reSponsible for the conver- sion of glyoxylate to glycine. The next step in the glycolate pathway involves the conversion Of two molecules of glycine to one molecule each Of serine, COZ, and NH3. Because Kisaki and TOlbert (#7) could find no evidence of glycine decarboxylation or con- version tO serine in the peroxisomes, it was concluded that these steps occur elsewhere in the cell. The site of this conversion appears to be the mitochondria (w. J. Bruin and N. E. TOlbert, unpublished results). . The conversion of L-serine to hydroxypyruvate is accomplished by means of an aminotransferase activity. Sallach (1#) has described an L-alanine:hydroxypyruvate aminotransferase activity as being present in a number Of plant tissues and has implicated this activity in the gluconeogenic flux of the glycolate pathway. This amino- transferase activity was found to have a distribution similar to that Of the D-glycerate dehydrogenase (99). 25 Sallach was able to demonstrate the formation Of D-glycerate and L-alanine from L-serine and pyruvate through the use of the isolated enzymes D-glycerate dehydrogenase and alanine: hydroxypyruvate aminotransferase. In this case the amino- transferase activity was reversed, i,g,. serine and pyruvate were used as substrates. The last reaction unique to the glycolate pathway is the phOSphorylation of D-glycerate to give 3-P-glycerate. Such an enZyme activity has been described recently in higher plant extracts by Cheung. Rosenblum. and Sallach (1#) and Hatch and Slack (38). Both preparations were Specific for D-glycerate and.ATP and required Mg ions for activity. The enzymes of the glycolate pathway as described above act as a system for the conversion of 2 molecules of glycolic acid to 1 molecule of the sugar precursor 3-P- glyceric acid. In thisconversion one carbon is lost as C02. The system also requires the net input Of amino groups in the form Of L-glutamate, the net input of reduc- ‘ing power in the form Of NADH, and one ATP is required for the conversion of Dbglycerate to 3-P-glycerate. Peroxisomes Peroxisomes were first recognized as distinct cel- lular organelles through the work of de Duve and coworkers (2#). During isolation of lysosomes from rat liver, it was .—.- ...——— foune 51ml tase PM 0x1: some cat aci Bee cat to in fr ca 11.“. 81 d2 tc 26 found that the enzymes catalase and urate oxidase showed similar distributions to the lysosomal marker acid phOSpha- tase upon differential centrifugation. Application Of iso- pycnic centrifugation established that catalase and urate oxidase were present in a particle distinct from the lyso- somes. Further characterization Of these particles indi- cated that several flavoprotein oxidases such as D-amino acid oxidase and d-hydroxy acid oxidase were also preSent. Because of the presence of these HZOZ-aproducing oxidases and catalase, these particles were given the name peroxisomes to indicate their role in H202 metabolism. The association Of flavoprotein oxidases and catalase in peroxisomes has been found to hold for liver and kidney from a number Of sources, two protozoans (23), germinating castor bean endOSperm (reviewed in the preceding section under the term glyoxysomes), and leaf tissue (97). The glyoxysomes have been found to contain the complete B—oxi- dation system and the complete glyoxylate cycle in addition to the oxidases and catalase. Muller, Hogg and de Duve (67) have demonstrated the presence Of isocitrate lyase and malate synthetase as well as NADP-isocitrate dehydrogenase in the Tetrahymena peroxisomes but were unable to find the other enzymes Of the glyoxylate cycle. Leaf peroxisomes have been found to contain the enzymes glycolate oxidase, NAD-glyoxylate reductase, catalase (97). glutamatezglyoxylate aminotransferase (#7), and NAD- 27 malate dehydrogenase (103). A summary of the enzymes known to date to be present in peroxisomes and glyoxysomes is given in the Results and Discussion Section. Morphologically the peroxisomes from the various sources and the glyoxysomes appear Similar. They are all bound by a single membrane, Show a dense granular matrix or stroma, and band in sucrose gradients at an equilibrium density of i.2#_1.25 g/cc. Electron micrographs indicate the peroxisomes tO be roughly Spherical in shape with a diameter Of 0.5-1.0 u. Ribosomes have been conSpicuously absent in all preparations tO date. In the case of leaf tissue, a close association of the peroxisomes with the chlorOplastS has been noted by Frederick and Newcomb (29). Because of the presence of the peroxisomes in those animal tissues which are active in gluconeogenesis, de Duve (2#) has Speculated that the peroxisomal function may be related to gluconeogenesis. NO direct correlation has been shown to date, however, with the possible exception that the a-hydroxy acid oxidase Of the liver peroxisomes can oxidize lactate to pyruvate, thus initiating gluconeogenesis from lactate. A.definite gluconeogenic function has been shown for the glyoxysomes which can convert long-chain fatty acyl COA esters to succinate through B-oxidation and the glyoxylate cycle (18). The succinate thus formed is then available for conversion to carbohydrates elsewhere in the cell. The leaf peroxisomes have also been shown to contain carried to the s 28 contain some Of the enzymes of the glycolate pathway which carried out conversion Of the two carbon compound glycolate to the sugar precursor 3-P-glycerate. MATERIALS AND METHODS Plants Spinach (Spinacia oleracea L.) leaves were the prime material used in this study. The Spinach was purchased locally or grown in a growth chamber on a 16-hour day, 8- hour night regime. Other plant material was grown in a greenhouse with double-strength Hoagland's medium applied once a week. Genus, Species and variety, where known, are listed with the results. Preparation Of Fractions by Differential Centrifugation In typical spinach preparations leaf tissue was washed, deribbed and cut into small strips. Subsequent Operations were carried out at 0-#°, either in a cold room 01' in ice buckets. The tissue was homogenized in a Waring blendor at maximum Speed for 10 see with one volume by "eight of grinding medium (0.5 M sucrose. 0.02 M potassium 8>']-y‘33irlglycine. pH 7.5). The resulting slurry was squeezed thr°11lgh 8 layers Of cheesecloth, and the pH (approximately 7) Was readjusted to 7.5 with KOH. The sap was then centri- fuged at 100 g for 20 min, and the resulting pellet was 1‘esufi'rpended in the grinding medium. This was designated the "Whole ChlorOplast" fraction. The sap was further 29 30 centrifuged at 6000 g for 20 minutes. This pellet after resuSpenSion was designated the "Broken Chloroplast" frac- tion. A "Mitochondrial" fraction was prepared by centrifu- gation Of the sap at 37000 g for 20 min and resuSpenSion Of the pellet in the grinding medium. The supernatant fluid after the last centrifugation was designated the "Superna- tant" fraction. Preparations were made with other plants using substantially the same procedure, although in some cases the volume of the grinding medium and the length of blendor time were increased to facilitate homogenization. Sucrose Density Gradient Centrifugation In typical eXperiments a discontinuous sucrose den- sity gradient was prepared in the cold by pipetting succes- sively #eml of 2.5 M, 8 ml Of 2.3 M, 10 ml Of 1.8 M. 15 ml of 1.5 M, and 13.5 ml Of 1.3 M sucrose into a cellulose nitrate tube designed for the Spinco SW 25.2 swinging-bucket rotor. All sucrose solutions were made up at room tempera- ture in 0.02 M potassium glycylglycine, pH 7.5. For studies Of the intracellular location of enzymes, a # ml portion Of the "Peroxisome and Broken Chloroplast" fraction was layered on top of the gradient; and the sample was centrifuged in a Spinco Model L centrifuge at 25,000 rev/min for 3 hours at #°. Fractions were then collected from the bottom Of the tube after puncturing and were numbered in the order of collection. Fraction contents and volumes are given in the 31 Results and Discussion Section. Assay Methods Enzyme assays were run on a Gilford automatic record- ing Spectrophotometer at 25° except where noted. A unit Of activity is defined as that amount Of enzyme catalyzing the disappearance Of 1 umole Of substrate per min at 25°. Malate Dehydrogenase (_:Malate:NAD Oxidoreductase. EC 1.1.1.22) Malate dehydrogenase was assayed Spectrophotometrically by following the oxidation Of NADH at 3#0 mu (103). The assay mixture contained 0.67 ml of 0.1 M HEPES (pH 7.#), 0.03 ml of 0.5% Triton X-100. 0.0# ml Of 0.01 M oxalacetic acid (neutralized tO pH 7.# with KOH), 0.02 ml Of 2.8 x 10‘3M NADH, and enzyme plus water to give a total volume Of 1 ml in the cuvette (d = 1 cm). The reaction was initiated by the addition of oxalacetate, after measurement Of the rate of endogenous oxidation of NADH. The recorder chart Speed was set at 1 in/min. and readings on each cuvette were for a duration of 1.5 seconds. Full scale (250 mm) on the recorder was 0.5A. Under these conditions 1 mm/min = 0.#82 anle/min at saturating oxalacetate concentrations. Glycolate Oxidase (Glycolate:02;Oxidoreductase, EC_1.1. 3.1) Glycolate oxidase was assayed anaerobically by follow- 32 ing the rate Of DCPIP reduction at 600 mu (9?). The assay mixture in a Thunberg tube contained 2 ml Of 0.1 M pyro- phosphate (pH 8.5) containing 1.5 x 10' M DCPIP. 0.05 ml of enzyme plus water, and 0.1 ml Of 0.125 M potassium glyco- late in the side arm. The total volume was 2.5 ml. The cuvette was evacuated with a water aSpirator and then flushed with pre—purified nitrogen which had been bubbled successively through Fieser's solution to remove traces of 02 and then saturated lead acetate to remove traces Of H25 formed through the breakdown Of the dithionite in the Fieser's solution. Complete removal Of 02 was found to be essential for Obtaining linear assays due to the presence Of peroxidative reoxidation Of the DCPIP (97). The reac- tion was initiated by tipping the substrate from the side arm into the main compartment. The recorder chart Speed was 0.2 in/min. and full scale was set at 2.5 A (250 mm). Under these conditions 1 mm/5 min = 0.770 nmole/min at saturating substrate and DCPIP concentrations. Cytochrome c Oxidase (Cytochrome 0:02 Oxidoreductase, EC 1.9.3.1) Cytochrome oxidase was used as a mitochondrial marker and was assayed by following the oxidation Of reduced cyto- chrome c at 550 mu (97). Enzyme (0.5-10 pl) was placed in the bottom corner Of a microcuvette (vol = 0.5 ml) with a syringe and 5 ul of #% digitonen added. After mixing and a no la, en: use as. CW 11: 56' re 33 1 min incubation period, 200 ul Of 0.1 M phOSphate buffer (pH 7) was added. The reaction was then initiated by the addition of 50 pl Of 1.5 mM cytochrome c reduced with dithionite. Care must be taken that the cytochrome c is not over-reduced by the addition of excess dithionite since lag periods in the assay would result. A sample without enzyme must be also used in order to measure the rate Of nonenZymatic oxidation Of reduced cytochrome c. For this assay a recorder chart Speed of 1 in/min was used, each cuvette being read for a duration of 1.5 sec. Full scale (250 mm) on the recorder was 1.0 A. Under these conditions, 1 mm/min a #8.8 picomole/min. ASpartate.Aminotransferase (ASpartate:2-OxoglutarateeAminotransferase, EC 2.6.1.1) ASpartate aminotransferase activity was measured by linking oxalacetate formation with exogenous malate dehydro- genase, thus giving NADH Oxidation followed at 3#0 mu. The reaction mixture contained 0.5 ml Of 0.1 M TES (pH 7.#), 0.03 ml or 0.5% Triton x—ioo, 0.05 ml of stabilized malate dehydrogenase (1,000 units), 0.30 ml of enzyme plus water, 0.03 ml Of 0.1 M d-ketoglutarate (pH 7). 0.02 ml Of 5 x 10'3M pyridoxal phosphate (pH 7), and 0.02 ml of 3.8 x io'3h NADH. The reaction was initiated by the addition of 0.05 ml Of 0.# M LpaSpartate (pH 7). A recorder chart Speed Of 0.2 in/min and full scale Of 0.5 A were employed. Under these conditions 1 mm/5 min = 6#.# picomole/min. 3# Isocitrate Dehydrogenase, NAD and NADP (threO-Ds-IsocitratezNAD Oxidoreductase (EC 1.1.1.#1) and threO-DS-Isocitrate:NADP Oxidoreductase (EC 1.1.1.#2)) The assay for the isocitrate dehydrogenases was modi- fied from that employed by Leighton gt_g;. (57). The forma- tion Of reduced pyridine nucleotide is followed Spectrophoto- metrically at 3#0 mu. The assay mixture for the NADP-iso- citrate dehydrogenase contained 0.67 ml of 0.1 M TES (pH 7.#), 0.03 ml of 0.5% Triton-X-lOO, 0.03 ml of 0.1 M MgClz. 0.0# m1 Of 2.5 x 10'3M NADP+, and 0.13 ml of enzyme plus water. The reaction was initiated by the addition of 0.1 ml Of 0.2 M DL-isocitrate, tri-sodium salt, Ella-free. For the assay Of- the NAD-isocitrate dehydrogenase, NAD+ was used in place Of NADP+. and 1 mM citrate was included in the assay mixture (25). The recorder chart Speed was 0.2 in/min, and full scale (250 mm) was set at 0.5 A. Under these conditions, 1 mm/10 min = 32.2 picomole/min. Malic Enzyme (L-MalatezNADP Oxidoreductase (Decarboxylating) EC 1.1.1.40) Malic enzyme was measured using a modification of the procedure Of Ochoa (70). The reaction mixture contained 0.67 ml of 0.1 M TES (pH 7.#). 0.01 ml or 0.1 M MnClz, 0.03 ml Of 0.5% Triton X-100, 0.0# ml Of 2.5 x 10'3M NADP+, and 0.23 ml of enzyme plus water. The reaction was initiated by the addition Of 0.02 ml of 1 M DL-malate (pH 7.#). Full scale was set at 0.5 A with a chart Speed of 0.2 in/min. 35 Under these conditions 1 mm/10 min = 32.2 picomole/min. PEP Carboxylase (EC #,1.1,§1) PEP Carboxylase was measured using a modification Of the method of Slack and Hatch (87). The reaction mixture contained 0.3 ml of 0.1 M TES (pH 7.#), 0.01 ml of 0.5% Triton X-100, 0.03 ml of 0.1 M M3304, 0.01 ml of 0.01 M dithiothreitol (pH 7). 0.05 ml of 10'2M L-glutamate (pH 7), 0.20 ml enzyme plus water, and 0.05 ml Of 0.01 M PEP. The reaction was initiated by the addition of 1 ac (0.2 umole) NaHln'COB contained in a 0.02 ml volume. The assay mixtures were then mixed and incubated at 30° in stOppered test tubes. The reaction was stOpped after either 15 or 20 min by the addition Of 0.5 ml EtOH followed by 0.1 ml Of glacial acetic acid. The tubes were then flushed with unlabeled C02 for 20 min, and 0.1 ml aliquots were removed and counted. Con- trol tubes contained all ingredients listed above with the exception of PEP. A sample without enzyme was also run to determine the rate Of nonenzymatic oxalacetate formation. Radioactivity was determined with a TriCarb scin- tillation counter using 15 ml of counting solution. The counting solution contained 10 g PPO. 0.1 g a-NPO. 160 g naphthalene. 770 ml xylene, 770 ml pedioxane, and #62 ml absolute ethanol. Citrate Sypthetase_(EC #.1.3.Z) Citrate synthetase was measured by a modification Of 36 the method of Srere, Brazil and Gonen (88). The buffer com- ponent (0.5 ml) in the assay was either 0.1 M potassium HEPES (pH 7.#) or 0.# M potassium tricine (pH 8.1). Other components were 0.03 ml Of 0.5% Triton X-100. 0.10 ml Of 10'3m or 5,5'-dithiobiS-(2-nitrobenzoic acid) (DTNB). (pH 8), 0.05 ml of 10'3M acetyl-COA. and 0.27 ml of enzyme plus water. The reaction was initiated by the addition Of 0.025 ml Of 10'3M potassium oxalacetate (pH 8); and the increase in absorbance at #12 mu was measured. Full scale on the recorder was 0.5 A and the chart Speed was 0.2 in/ min. This assay makes use Of the reaction Of DTNB (Ellman's reagent) with the free sulfhydryl group Of COA. released by citrate synthetase action. This assay is much more sensi- tive than the Old methods of using malate dehydrogenase or following disappearance of the thiOl-ester bond at 232 mu and is less subject to troubles caused by high background readings. Under these assay conditions, 1 mm/5 min = 30 picomole/min. Malate§ynthetase (EC #.1.3.2) The assay used for malate synthetase was modified from the assay for citrate synthetase. The basic reaction mixture contained 0.5 ml of 0.1 M potassium HEPES (pH 7.#), 0.03 ml or 0.5% Triton x-100, 0.10 ml or 10‘3M DTNB (pH 7). 0.05 ml or 10'3h acetyl-COA, 0.01 ml or 0.1 M MgClZ. and 0.26 ml of enzyme plus water. The reaction was initiated by the addition Of 0.025 ml Of 0.1 M sodium glyoxylate. 37 The recorder chart Speed was 0.2 in/min, and full scale (250 mm) was 0.25 A. Under these conditions 1 mm/5 min = 15 picomole/min. Isocitrate Lyase (EC #.1.3.1) The assay method for isocitrate lyase was modified from that of Dixon and Kornberg (26). The reaction is followed through formation of the glyoxylate phenylhydra— zone which absorbs at 32# mu. The assay mixture contained 0.5 ml of 0.1 M HEPES (pH 7.4), 0.03 ml or 0.5% Triton x-100. 0.025 ml or 0.1 M M3012. 0.05 ml or u x 10'2 M dithiothreitol, 0.03 ml Of 0.1 M phenylhydrazine°HCl (neutralized to pH 7 just before use), and 0.32 ml enzyme plus water. The reac- tion was initiated by the addition Of 0.05 ml of 0.2 M sodium DL-isocitrate (allg-free). An extinction coefficient of 1.7 x 104 M'1 cm"1 was used for the glyoxylate phenyl- hydrazone at 32# mu. Aconitase (EC #.2.1.3) and [Egmarase (EC #.2.1.2) Aconitase was determined by the method of Anderson (1), and fumarase was determined by substituting fumarate in the same assay system.. The assay utilized absorption Of 2#0 mu light by the double bond in both gigyaconitate and fumarate, and disappearance of the double bond was measured. {The reaction mixture contained 0.#0 ml Of 0.1 M TES (pH 7.5). 0.03 ml of 0.5% Triton x-ioo, 0.240 ml or 0.1 M 38 (NH4)2804. 0.#00 ml of enzyme plus water, and 0.030 ml Of either 10'2 M potassium gigraconitate or 10-2 M potassium fum- arate (both pH 7.5). depending on which enzyme was being measured. The recorder chart Speed was 0.2 in/min, and full scale was 1.0 A. Using an extinction coefficient for gig-aconitate at 2#0 mu Of 3.55 x 103 M'1 cm'l. 1 mm/10 min a 0.135 nmole/min. Formate Dehydrogenase (EC 1.2.1.2) For the assay of formate dehydrogenase, the method Of Quayle (77) was used wherein the formation Of reduced NAD is followed at 3#0 mu. The assay mixture contained 0.67 ml Of 0.1 M potassium phOSphate (pH 7.0), 0.03 ml of 0.5% Triton X-100. 0.10 ml or 10"2 M NAD+, and 0.20 ml of enzyme plus water. The reaction was initiated by the addi- tion of 0.025 ml Of 0.2 M potassium formate (pH 7). The recorder chart Speed was 0.2 in/min, and full scale was 1.0 A. Under these conditions, 1 mm/10 min = 6#.# picomole/ mine SerineéPyruvate Aminotransferase (EC 2.6.1.-il Serine-pyruvate aminotransferase activity in Spinach preparations was assayed by linking hydroxypyruvate forma- tion to D-glycerate dehydrogenase activity, thus giving NADH oxidation which was followed at 3#0 mu in the Gilford SpectrOphotometer. The assay mixture for gradient fractions 39 contained 0.5 m1 of 0.1 M HEPES (pH 7.3). 0.03 ml Of 0.5% Triton X-100. 0.02 ml Of crystalline glyoxylate reductase (D-glycerate dehydrogenase). 0.35 ml of enzyme plus water, 0.02 ml Of 5 mM pyridoxal phOSphate, 0.03 ml of 0.1 M potassium pyruvate, and 0.02 ml of 3.8 mM NADH. The reac- tion was initiated by the addition Of 0.05 ml Of 0.# M DL- Serine (pH 7). NO pyruvate-dependent Oxidation Of NADH was noted in the gradient fractions. This follows the Observa- tion Of Zelitch that pyruvate does not serve as a substrate for plant D-glycerate dehydrogenase. Starch Gel Electrophoresis The procedure of Fine and Costello (2?) was used for the separation of isozymes of malate dehydrogenase by starch gel electrophoresis. For preparation Of the gel. 3.5 ml Of 0.2 M citric acid, 21.5 ml Of 0.2 M NazHPOu and #75 ml Of distilled water were mixed together. This solu- tion was then.slowly added with stirring to 70 g Of hydro- lyzed starch. The resulting suSpension was then heated with stirring until bubbles appeared (ca. 659).. The slurry was then quickly poured into a 2 l vacuum flask (heated in a water bath to the same temperature) and the mixture was boiled under reduced pressure from a water aSpirator for 1 min. The starch was next poured into forms for electro- phoresis, with filter-paper wicks being used for contact with the electrode solutions; and a Slot former was placed in the gel to allow easy sample introduction. The gel was #0 hydrated overnight before use. The tank buffer used contained 30 ml Of 0.2 M citric acid, 2#0 ml of 0.2 M NaZHPOA' and 1230 ml Of distilled water. For the preparation of the enzyme samples, 0.3 ml fractions from the differential and sucrose gradient cen- trifugation of Spinach leaf homogenates were incubated at ice temperature with 0.03 ml of 0.5% Triton X-100 in order to solubilize enZymatic activity. After 10 min, the samples were centrifuged for 15 min at 39,000 g and the resulting pellet was discarded. For the electrophoresis, 0.2 ml fractions were applied to the gel; these were subjected to 27 mA (210 V) for eight hours at #9. For localization Of the malate dehydrogenase activ- ity, the method of Fine and Costello (27) was used. The staining solution contained 35 ml of 0.1 M potassium bicine (pH 8.5). 2.25 ml Of 2 M potassium L-malate. 0.9 ml Of NAD+ (30 mg/ml). 0.18 ml of phenazine methosulfate (5 mg/ml), and 1.5 ml of penitro blue tetrazolium (10 mg/ml). The gel was incubated in the dark for 30 min at 35° with this solu- tion. activity Of the malate dehydrogenase appearing aS a purple-colored Spot. Protein and Chlorophyll Determinations Protein was estimated by the method Of Lowry, using powdered bovine serum albumin as the standard. Chlorophyll was estimated at 652 mu by the method Of Arnon. RESULTS AND DISCUSSION The detection, isolation, and partial characteriza- tion Of leaf peroxisomes has been accomplished as a group effort in our laboratory during the course of research for this thesis. Some Of these results have been published and constitute a portion Of this thesis. Leaf peroxisomes were first isolated from Spinach (97). Later survey work established that peroxisomes can be isolated from a variety of plants (98). In this survey work my major contribution was the study Of the intracellular distribution of NAD- malate dehydrogenase. The NAD-malate dehydrogenase from Spinach leaf peroxisomes was characterized in detail. and this work constitutes a major portion of this thesis (103). The work on NAD-malate dehydrogenase activity is summar- ized in this section. Studies Of other activities are given in full. NAD-Malate Dehydrggenase My data on the distribution and properties Of spinach leaf NADbmalate dehydrogenase have been published (Ref. 103). Consequently these findings will be presented in summary form in this section. The same technique of organelle separation was used to determine particulate distribution for NAD-malate dehyd- #1 #2 regenase and all other enzymes described in later sections. Details are given in the Materials and Methods Section. The basic procedure involved homogenization Of leaf tissue for short times in a Waring blendor. Buffered 0.5 M sucrose was used as the grinding medium. A fraction enriched in peroxisomes and mitochondria was then Obtained by centrifu- gation at loo-6,000 g. This fraction was then layered on a sucrose density gradient ranging from 2.3 to 0.5 M sucrose. ISOpycnic centrifugation allowed a separation Of chloro- plasts, mitochondria, and peroxisomes according to their reSpective densities. Chlorophyll. cytochrome Oxidase, and glycolate oxidase, reSpectively. were used as markers for these organelles. Figures 1 and 2 present the sucrose density gradient distribution Of NAD-malate dehydrogenase activity. Clear peaks Of activity can be seen to be associated with the peroxisomes and mitochondria. The Egg peaks of mitochondrial activity are probably an artifact Of the non-continuous gradient used. The peroxisomal enzyme as assayed by fol- lowing NADH oxidation exhibited a broad pH Optimum from 6.# to 7.#. The ratio Of the rates with the two pyridine nucleotides (%fifi%§ = 0.006) indicated that the activity was Specific for NADH. The kinetic prOperties Of the malate dehydrogenase activities from the gradient are summarized in Table 1. It can readily be seen that the mitochondrial isozyme is more sensitive to oxalacetate inhibition than is the peroxisomal form. 43 Figure 1 Distribution of NAD-Malate Dehydrogenase and Marker Activities After Sucrose Density Gradient Centrifu- gation Of Spinach Broken Chloroplast Fraction. NAD-Malate dehydrogenase, glycolate oxidase, and cytochrome oxidase units eXpressed as umole sub- strate transformed per min per ml at 25°. o——-O. malate dehydrogenase; I---I . glycolate oxidase; "°"e, cytochrome oxidase. ## Units per ml Fraction Number 0.5 I.3 ' e.5 ' L8 '2.3 '25 Sucrose Molority 45 Figure 2 Distribution Of NAD-Malate Dehydrogenase and Marker Activities After Sucrose Density Gradient Centrifu- gation of Spinach Broken Chloroplast Fraction (Cont'd). NAD-Malate dehydrogenase units expressed as umole substrate transformed per min per ml at 25°. Protein and chlorophyll expressed as mg per ml. o———e. malate dehydrogenase; a-o o—A , protein (1/5 scale); I-o-I chlorophyll. ml Units per a. a e.5 \ X‘". Units per ml 23 0.5 °. F ‘ /. \ ' s-. "k. _i . 1 er: eels-Anew 9 8 7 6 5 4 3 2 l Fraction Number 1+7 TABLE 1 Kinetic Properties Of Malate Dehydrogenase Forms Peroxisomal and mitochondrial fractions taken from sucrose density gradients and used without further purifi- cation. The supernatant is from the differential centrifu- gation. Location Km (0AA) 0AA Conc. That Be- Inhibition at gins to Inhibit 2 x 10'3M 0AA Peroxisomal 1.# x 10'5M 2 x 10'4M 2#% Mitochondrial 5.7 x 10-6M 7 x 10'5M 50% Supernatant 1.3 x io-SM 1 x 10'“M 23% #8 The peroxisomal and mitochondrial forms Of NAD- malate dehydrogenase were easily separated by starch gel electrophoresis. This is shown in Fig. 3. A third dis- tinct activity was seen in the supernatant fraction, but this could not be investigated further because Of the large amount of the mitochondrial and peroxisomal forms present in this fraction. NAD-Malate dehydrogenase activity has been reported by several workers (see Ref. 103) to be present in chloro- plasts, but these reports could not be confirmed in the case Of Spinach leaves. NO evidence could be found for NAD-malate dehydrogenase activity in either whole chloro- plasts or in chloroplast fragments. Hatch and Slack (38) have recently reported the presence Of an NADP-malate dehydrogenase activity in maize chloroplasts. No such activity was detected in preliminary experiments with Spinach chloroplast preparations, but a difference between maize and Spinach chloroplasts could easily be possible. In Ref. 103. it was stated that oxidation of L- malate could not be Observed at pH 7.#, probably because of the unfavorable equilibrium of the reaction. Since this statement was made, an aSpartate-c-ketoglutarate aminotransferase has been found in the peroxisomes. By the addition of L-glutamate to the assay mixture. it is now possible to demonstrate L-malate oxidation as measured by following NAD+ reduction. In this case the unfavorable Figure 3 Schematic Representation of Starch-Gel Electro- phoresis of the Various NAD-Malate Dehydrogenases. Separation and staining techniques are described in the Materials and Methods Section. Homaqenate a @ W Peroxisomal W Mitochondrial Supernatant a» e %@ Cathode Origin Anode 51 equilibrium position is Shifted toward malate oxidation through removal of the product, oxalacetate by transamina— tion. Because of a report by Munkres (68) that NAD-malate dehydrogenase and aSpartate aminotransferase activities are apparently associated with the same protein in NeurO8pora, amino acids and pyridoxal phOSphate were added to the standard NAD-malate dehydrogenase assay using leaf peroxi- somes. NO effect was Observed with #emM L-aSpartate, L- glutamate, DL-Serine. or 0.1 mM pyridoxal phOSphate. The fact that L-glutamate had no effect may seem surprising in view Of the fact that aSpartate-a-ketoglutarate aminotrans- ferase activity is present in leaf peroxisomes (discussed in the next section), but Kisaki and Talbert (#7) have shown that this reverse (glutamate-oxalacetate) aminotrans- ferase activity of peroxisomes is very low. One may also conclude from these data that serine-oxalacetate amino- transferase activity in the peroxisomes is low relative to the NAD-malate dehydrogenase activity. Aminotransferase Activities In preliminary experiments on other transaminase activity Of the peroxisomal fractions, it was found that serine plus pyruvate exhibited a relatively high rate of activity. This transaminase was measured by linking hydroxypyruvate formation to the D-glycerate dehydrogenase activity present in these peroxisomal fractions. This can 52 be done with peroxisomal fractions, utiliZing L-serine, L-aSpartate, or glycine as amino donor Since dehydrogenase or reductase activity is present for each Of the corres- ponding ketO acids. In this study it was found that no NADH Oxidation was Observed with peroxisomal fractions in the presence Of either pyruvate or a-ketoglutarate. Alanine and glutamic dehydrogenase activities likewise could not be detected in peroxisomal fractions. The num- ber of possible combinations of amino donors and acceptors which can be studied with such a system is extremely limited (6 combinations exist), but several activities which are believed to be highly Specific were detected. In Table 2 are summarized the data on the six amino- transferase combinations which could be tested. Signifi- cant activity was detected only with the combinations serine + pyruvate and aSpartate + a-ketoglutarate. .Addi- tion Of glyoxylate. oxalacetate, and hydroxypyruvate (all at a final concentration Of 10 mM) to separate control cuvettes established that the reSpective dehydrogenase and reductase activities were not limiting. The two amino- transferase activities appear to be quite Specific with reSpect to substrates. This was also found to be the case with the glutamate-glyoxylate aminotransferase Of the peroxisomes (#7). Using a different assay procedure, Kisaki and Talbert were able to test a number of amino donors and acceptors in reaching this conclusion. 53 TABLE 2 Aminotransferase Activity of Leaf Peroxisomes as Measured by Coupling tO Endogenous Dehydro- genases. The assay mixture contained 0.5 ml of 0.1 M HEPES (pH 7.3). 0.05 ml Of Fraction 3 from a sucrose density gradient, 0.35 ml Of distilled water. 0.03 ml of the potassium salt Of a-keto acid. and 0.02 ml of 3.8 mM NADH. The reaction was initiated by the addition of 0.05 ml Of 0.# M amino acid (pH 7). No rate was Observed in the presence Of a-keto acids alone or amino acids alone. Dehydrogenase or reductase activities were in excess as measured by addition Of appropriate a-keta acids. The rate Of serine-pyruvate aminotransferase activity was set at 100. - T .Amino Acid a-Keto Acid Relative Rate Pyruvate 100 DL-Serine a-Ketoglutarate 3 Pyruvate 0 Glycine a-Ketoglutarate 0 Pyruvate 18 LqASpartate a-Ketoglutarate 179' 5# The lack of reaction using glycine as the amino donor is not surprising when one takes into account the unfavorable amino-group-transfer potential, i.e.. the highly unfavorable free energy change in converting gly- cine tO glyoxylate. Kisaki and Talbert (#7) also found the glutamate-glyoxylate aminotransferase to be "irrever- sible" and have discussed this point in some detail. In order to determine whether the aSpartate amino- transferase activity found in Fraction 3 could be attrib- uted to peroxisomes, the distribution of this activity throughout differential and isopycnic centrifugation was studied (Fig. #). Marker activities for the peroxisomes and mitochondria are included for comparison. PEP Car- boxylase activity is also included since it appears that a component of this activity is located in the chloroplasts as is evidenced by the relatively high rate/ml in both the Whole Chloroplast fraction and Fractions 7 and 8 of the gradient (broken chloroplast fragments). Some Of the activity in Fraction 5 may be attributed to chloroplasts since it has been observed that intact chloroplasts are present along with mitochondria in Fraction 5. The dis- tribution of PEP carboxylase will be discussed further in a later section. The aSpartate aminotransferase activity exhibits a peak coinciding with the cytochrome oxidase peak. thus indicating mitochondria contain aSpartate aminotransferase 55 Figure # Distribution Of ASpartate Aminotransferase and PEP Carboxylase Activities Among Differential and Sucrose Density Gradient Centrifugation Fractions. Abbreviations used: w-CHL, Whole Chloroplast Fraction; B-CHL. Broken Chloroplast Fraction; MIT, Mitochondrial Fraction; SUP, Supernatant Fraction as described in the Materials and Methods Section. D . Percent Of total activity; , Rate/ml. expressed as nmole per min per mg protein. 2 e 4:— C)\ «,0 C.) (\ A (\J 0 Percent of 'L'obal Activity .4:— ox (p o o r) (\J O 56 SUCROSE DENSITY GRADIENT CENTRIFUGATION DIFFERENTIAL CENTRIFUGATION Aaeaa N Heads x adoaav Ha\apam .3233 dance co patches 0 O 0 O 0 0 O. O .# q; 2 1. 0 O 0 O 0 O 0 O . . . . A. 15 2 1 .4 SJ 2 1 AU 0 O O . _ 4 4e — _ d a - — _ _ 1 a Na 0 em W 3N 1 am a? / 6335.... la r a 6 F I|+o 7 :7“ MW Ilql d m” , Wm / /7 OIG 0 0 0 no _ O 0 O 0 0 0 O 0 _ no 0 O O _ .# 1, a2 1 _e .# 1, 2 1 _ .# 15 2 1 .# 15 2 1 _ _ _% Apabapo< Hapoa mo pzaoham _ _ T _ _e _ _ _ am _ _ _ _w AHIHE N HIGHS Nswflaaav HE\apdm _ _e 0 no 0 O _r n. no 0 0 s 0 O _ s O O 0 n. t 0 O 0 0 _a 0 0 _ _ _a 0 0 0 no _0 0 0 0 0 d 0 O as 8 [b u. 2 n .# a) 2 1 .i 8 .# i i x X m 0 0 A e e e t t a a l t 0 r C a V9 D. l s G _ A A _ b _ _ O 0 nu 0 O O 0 Re Re Au b. 9~ no ,0 MW MW 57 activity. A portion of the animal aSpartate aminotransfer- ase activity has been demonstrated by Lardy (53) to be mitochondrial. The relatively high rate/ml Of the Whole Chloroplast Fraction is taken to be an indication Of the presence Of aSpartate aminotransferase activity in chloro- plasts. PEP Carboxylase can be seen to show a similar high relative rate in the Whale Spinach Chloroplast Frac- tion. Rosenberg. Capindale. and Whatley (82) have pre- sented evidence for PEP carboxylase activity in Spinach chloroplasts. The low percentage of both aSpartate amino- transferase and PEP carboxylase activities in the Whole Chloroplast Fraction may be a reflection Of solubilization of these enzymes upon homogenization. Heber (#0) and Mukerji and Ting (65) have reported the presence Of aSpar- tate aminotransferase activity in chloroplast preparations from Vlgigflgggg and Opuntia reSpectively. In comparison of the rate/ml for cytochrome oxidase and aSpartate aminotransferase. more aSpartate aminotrans- ferase activity is found in Fraction 3 than can be accounted for by mitochondrial activity. Kisaki and Talbert (#7) also found a large amount of aSpartate aminotransferase activity coinciding with the peroxisomal fraction. Close examination Of the substrate Specificity Of the glutamate- glyoxylate aminotransferase studied by these authors indi- cates that the aSpartate-a-ketoglutarate activity cannot be attributed to substrate non-Specificity Of the glycine- 58 forming enzyme. Investigations Of the Specific activity of the aSpartate aminotransferase activity as detailed in the Summary and Conclusions Section indicate this activity to be #0 times greater than the glutamate-glyoxylate amino- transferase activity. From all these considerations, it is concluded that peroxisomes, mitochondria, and chloro- plasts all contain aSpartate aminotransferase activity. ASpartate is thought to be an intracellular trans- port form Of oxalacetate (53). Thus it is not surprising to find aSpartate aminotransferase activity in peroxisomes, mitochondria. and chloroplasts since all three organelles apparently contain enzymes involved in oxalacetate metabo- lism. Data for the distribution of serine-pyruvate amino- transferase activity on a sucrose density gradient are presented in Fig. 5. Clear correSpondence Of the amino- transferase activity with that of the peroxisomal marker, NAD-glyoxylate reductase, indicates that the serine-pyruvate aminotransferase activity is peroxisomal. This aminotrans- ferase activity has been previously described by Sallach (1#) and implicated in the Operation of the glycolate path- way, but the intracellular localization was not determined. With reSpect to various plant sources, Willis and Sallach (99) were able to demonstrate that the aminotransferase activity closely paralleled the activity of the D-glycerate dehydrogenase described by Stafford. Magaldi, and Vennes- land (90). That is, plants with high glycerate dehydrogenase 59 F1 e Distribution of Serine-Pyruvate Aminotransferase, NADP-Isocitrate Dehydrogenase. NAD-Isocitrate Dehyd- rogenase, Fumarase, Citrate Synthetase, Aconitase. Malic Enzyme, and Marker Activities After Sucrose Density Gradient Centrifugation Of Spinach Broken Chloroplast Fraction. Cl, Percent of total activity; I, Specific activity, expressed as nmole per min per mg protein at 25°. Data for NAD- and NADP-glyoxylate reduc- tase activities from A. Oscar and N. E. Talbert. Activity Percent of Total 2:205 79: x TEE x 2055 33:04 2:88 mama ”mam mama asp. mama ' q d 4 d + q q d d H q d u q d u - u d J) u m m w m m . a m N r em N 4 m mu m m .m cm. m n .m m m. m F x F e m» .m Mm m w 9 WW 6 h b h h 3 h r b P P p P h bin 12 P p P P wwwm wwmm mm m mwmm wwmm 5384 .28 so Booed 0 llllllllllllllllllllllllllllllllllll 6 3295 79.: x TEE x 2065 33:04 026on m m msmsmmam mmmm swam 3 a . l in l a a .1 a q 4 a _ 4 l Dehydrogenase NAD-Isocitrate _ nan / 5 I , é é / é / Gradient Fraction Number Gradient Fraction Number MOP-Isocitrate galls i 8 kph #P-L # Lborb mwmm mwmm mwmm wwzm asses slop so roots act p12 sex is: aci Pi] dei pet ti. of pr! 81' ex? 1011 61 activity had high aminotransferase activity, and conversely plants with low glycerate dehydrogenase activity had low serine-pyruvate aminotransferase activity. This parallel- ism can now be interpreted in terms of high peroxisomal activity versus low peroxisomal activity. This demonstration in the peroxisome of serine- pyruvate aminotransferase activity coupled to D-glycerate dehydrogenase activity establishes the ability of the peroxisome to convert serine to glycerate. Thus all reac- tions Of the glycolate pathway leading to the conversion of glycolate to glycerate have been demonstrated to be present in the peroxisome with the exception of the conver- sion Of glycine to serine. The serine-pyruvate and aSpartate-a-ketoaminotrans- ferase activities of the peroxisome have been found to exhibit several common properties. Neither of the activ- ities is stimulated by the inclusion of 0.1 mM pyridoxal phOSphate in the assay mixture. This was also found to be true Of the glutamate-glyoxylate aminotransferase (unpub- lished results Of T. Kisaki and N. E. Talbert). Likewise, bath Of the aminotransferase activities studied here were inhibited 10-20% by the addition Of 0.01% Triton x-100 tO the assay. This Observation contrasts strikingly with the Observations that the glycolate oxidase, NAD-glyoxylate reductase, and malate dehydrogenase activities of the peroxisome are stimulated 30% by the addition of the non- ianic detergent Triton X9100. NAD? the 0ft 62 NADP-Isocitrate Dehydrogenase AS was found in the case Of the NAD-malate dehydra- genase, two isozymes with reSpect to particle localization were found. The distribution of the NADP-isocitrate dehyd- rogenase is given in Fig. 5. NAD-glyoxylate reductase has been used here as a peroxisomal marker. It can be clearly seen that the peak Specific activity (Fraction 3) for the NADP-isocitrate dehydrogenase coincides with that of the peroxisomal marker in a sucrose density gradient Of Spinach particulate material. Relative to the Specific activity of the NAD-glyoxylate-reductase, Fractions 5, 6 and 7 can be seen to have a high Specific activity for NADP-isacitrate dehydrogenase. This activity parallels that found for the mitochondrial marker cytochrome oxidase. Thus NADP-iso- citrate dehydrogenase activity is located in both peroxi- somes and mitochondria. The large amount (50%) and high Specific activity of the NADP-isocitrate dehydrogenase found in the supernatant of the gradient (Fraction 9) may be an indication of leak- age Of this activity from the peroxisomes and/or mitochon- dria. Such was not found in the case of NAD-malate dehyd- rogenase. however. Upon fractionation of the original leaf hamogenate. 90% of the NADP—isocitrate dehydrogenase was found in the supernatant fraction from a 39,000 g centrifugation (data not given). In contrast NAD-malate dehydrogenase which is also localized in peroxisomes and 63 and mitochondria Showed only #0% Of its activity in super- natant fractions. It is difficult to conceive of one enzyme (NAD-malate dehydrogenase) being rather tightly bound to cellular organelles while another is easily lost unless the second enzyme (NADP-isocitrate dehydrogenase) is located.gg the membrane(s). NO evidence to support this membrane localization exists tO date. It is thus .inviting to ascribe the supernatant NADP-isocitrate dehyd- rogenase activity to a cytoplasmic or soluble isozyme. Some of the gradient supernatant activity might be attrib- uted to contamination from this cytoplasmic activity. NADP-Isacitrate dehydrogenase has been found in all peroxisomes tested to date. This includes peroxisomes from rat liver, Tetrahymena, and Spinach leaves. The Specific function of this peroxisomal activity, however, is unknown. In the case Of the Spinach leaf peroxisomal NADP-isocitrate dehydrogenase, the relatively low Specific activity may preclude its playing a major role in the.per- oxisomal metabolism. This point will be discussed further in later sections. Muller. Hogg and de Duve (67) found a somewhat Similar distribution for NADP-isocitrate dehydrogenase in Tetrahymena. They found about 50% of the actiVity in the peroxisomes, 5% in the mitochondria, and the rest was found to be soluble. The distribution of activity from spinach leaves agrees in localization, but the quantitative aSpects differ considerably. 6# NAD-Isocitrate Dehydrogenase, Fumarase, Citrate Synthetase. and_Aggnitase As is seen in Figure 5, NAD-isocitrate dehydrogenase, fumarase, citrate synthetase and aconitase activities paral- lel that of the mitochondrial marker cytochrome oxidase. This is true both of the Specific activities and percentage distributions. It is thus concluded that these activities are exclusively mitochondrial with reSpect to organelle distribution. No conclusions as to occurrence of cytoplas- mic isozymes can be drawn from the data. The NAD-isocitrate dehydrogeanse activity was mea- sured in the presence Of 1 mM citrate. This was in keep- ing with the report that the enzyme from Brassica ggpig is stimulated by citrate (25). The enzyme from Brassica was reported to be unaffected by AMP. This is in contrast to reports from animal systems where the enzyme is activated by AMP. NO effort was made in the present study to Opti- mize assay conditions with reSpect to activators. Thus higher Specific activities might be obtained by the addi- tion Of activator(s) other than citrate. Specific activ- ity figures Should be regarded as being minimal. Fumarase was assayed in the presence Of 20 mM ammonium sulfate. Once again no effort was made to Opti- mize activator and assay conditions. These results must also be regarded as being minimal with reSpect to Specific act 1V1 ty 0 65 Citrate synthetase was assayed at pH 7.#, utilizing the disulfide interchange reaction between GOA-SH and Ellman's reagent. Early reports utilizing this assay had reported the disulfide interchange reaction to be base- catalyzed. A.pH of 8.1 was used in these reports. In this study the rate Of reaction in the citrate synthetase assay at pH 7.# was found to be 1.## times that at pH 8.1. If the interchange reaction is base-catalyzed, it must be that the increased enzymic activity at pH 7.# more than compen- sates for the lower rate of disulfide interchange. Similar results must have been found by Rock and Beevers (#1) who used a similar assay for malate synthetase. These authors used a pH Of 7.1 in their assay procedure. In assaying the sucrose density gradients for pos- sible malate synthetase activity, it was found that the mitochondrial citrate synthetase exhibited a low rate when glyoxylate was used as substrate in place of oxalacetate. The rate with saturating glyoxylate was found to be about 5% Of that with oxalacetate. These data are presented in Table 3. NO reaction was Observed with pyruvate as sub- strate. It can be seen that the combination of oxalacetate and glyoxylate resulted in 20% inhibition over the control. The addition Of 10 OM palmityl-COA was found to give 25% inhibition (Table #). .ATP and ADP to a lesser extent were also found to give inhibition. These findings are in agreement with those found by Hathaway and Atkinson (39) 66 TABLE 3 a-KetO.Acid Specificity of Citrate Synthetase Assay conditions as described in Methods and Mater- ials for citrate synthetase except for the substrate used. All a-keto acids used at a final concentration Of 2.5 mM. W a-Keto Acid Rate Relative Rate _ (Arbitrary units) (0AA = 100) Oxalacetate 252 100 Glyoxylate 12 5 Pyruvate 0 0 Oxalacetate + glyoxylate 20# 80 67 TABLE # Effects of Adenine Nucleotides and.Palmityl-COA an "Malate Synthetase" Activity of Citrate Synthetase Assay conditions as described in Materials and Methods. Additions Relative Rate Control 1 ' 100 2 100 + AMP. 1 mM 100 10 mM 81 + ADP. 1 mM 75 10 mM 50 + ATP, 1 mM 56 10 mM #7 +Palmityl-CaA, 10 uM 75 68 in the case Of yeast citrate synthetase. The differential effects produced by the degree of phOSphorylation Of the adenine nucleosides probably reflects control of citrate synthetase activity by the energy charge of the cell as advocated by Atkinson (#). This activity of citrate synthetase with glyoxylate was found to be stimulated by the addition of Mg++, with maximal stimulation (30%) occurring at a concentration of 1 mM Mg++. To my knowledge no reports exist in the litera- ture concerning either utilization Of glyoxylate by citrate synthetase or stimulation Of citrate synthetase by metal ions. The significance of this "malate synthetase" activity of citrate synthetase is unknown. It should be noted that neither fumarase nor citrate synthetase activities were found in the peroxisome fractions. These two activities were checked because Of the presence Of NAD-malate dehydrogenase activity in peroxisomes. With reSpect to TCA enzymes involved in malate or oxalacetate metabolism, the peroxisomal NAD-malate dehydrogenase appears to be isolated. The fact that aconitase activity was not found in the peroxisomes may indicate an isolated role for the NADP- isocitrate dehydrogenase. NO attempt was made to determine if any a-keta acid dehydrogenase activity was present in peroxisomes, but this possibility seems remote in view of the mitochondrial localization in animal tissue. 69 Malic Enzyme The distribution of the malic enzyme is given in Fig. 5. In the particulate fractions the malic enzyme activity is clearly associated with the mitochondria. TO my knowledge a mitochondrial localization has not been reported previously from plant leaf tissue. Several reports have located malic enzyme activity in chlorOplast fractions, but these reports have been limited to cactus (65) and maize (87). In the case Of the bovine adrenal cortex, Simpson and Estabraok (86) have reported the occur- rence Of two forms Of malic enzyme. The mitochondrial farm is thought to function in the direction of malate oxidation. thus providing NADPH for the 11 B-hydroxylatian system. The cytOplasmic form is thought to function in the direc- tion of the reductive carboxylation of pyruvate to regen- erate L-malate. The two enzymes thus may mediate the trans- port of NADPH reducing equivalents from the cytosol into the mitochondrion for use in steroid hydroxylation. Because malic enzyme activity has not been measured in Spinach leaf homogenates, no conclusions can yet be drawn concerning the function Of the mitochondrial malic enzyme activity, but it may be that the NADPH-Shuttle mechanism prOposed by Simpson and Estabraok for bovine adrenal cortex will find more general application. 70 PEP Carboxylase DATA for the distribution of PEP carboxylase activ- ity in a sucrose density gradient are given in Fig. 5. The enzyme activity does not closely parallel any of the marker activities. There does appear to be a chloroplast component "'fifiifitfi" Of this activity as evidenced by the relatively high rates obtained in the Whole Chloroplast Fraction and Fractions 7 and 8 Of the gradient. The significance of the high rate «r of the Mitochondrial Fraction is not clear at this time Since the activity does not follow the distribution Of the cytochrome Oxidase. Consideration Of this rate plus the high rate Of Fraction 5 from the gradient have led to the tentative conclusion that mitochondria also contain PEP carboxylase activity. Rosenberg, Capindale, and Whatley (82) reported the occurrence of PEP carboxylase activity in chloroplast extracts from Spinach leaves but a rigorous test of the intracellular localization of the enzyme was not undertaken. Mazelis and Vennesland (6#) found some PEP carboxylase activity in chloroplast fractions from Spinach leaves, but they found higher activity in the small particulate frac- tion, which presumedly would correSpond to a mitochondrial fraction. PEP Carboxykinase activity was also indicated to be present in this fraction. Because no correlation could be drawn between the PEP carboxylase and glycolate oxidase activity. it was con- 71 cluded that peroxisomes do not contain PEP carboxylase activity. Activity was found in fractions containing mito- chondria and chloroplasts, but, because of the anomalous behavior of the PEP carboxylase activity with reSpect to the marker activites, conclusions as to chloroplast and/or mitochondrial localization can only be regarded as tenta- tive at best. Formate Dehydrogenase Because Of the presence in the leaf peroxisomes of various organic acid dehydrogenases, and because of the implication by Zelitch (108) that formate is one of the products Of the $3,3izg oxidation Of glycolate, an inves- tigation of the intracellular localization Of formate dehydrogenase was made. The distribution Of the activity as shown in Fig. 6 indicates that the particulate formate dehydrogenase activity is mitochondrial. Mazelis (63) had previously‘indicated a particulate localization for the enzyme from leaf tissue, but the exact nature of these particles was not determined. The Specific activity figures for the formate dehydrogenase should be taken as minimal since optimum conditions were not determined for the assay. The assay pH used in this study was the same as that determined to be Optimal for the cabbage leaf enzyme by Mazelis. Formate was determined to be saturat- ing under the conditions used. 72 Figure 6 Distribution Of Formate Dehydrogenase and Marker Activities After Sucrose Density Gradient Cen- trifugation U. Percent of total activity; ’, specific activity expressed as nmole per min per mg prO- tein. Percent Of Total Activity Leo. 30 20 10 #0 30 20 10 #0 30 20 10 73 Gradient Fraction Number 9l817i61514|3lzl1 Glycolate Oxidase k\\\\\ - l—l Formate Dehydrogenase Ea \\\\\\\\\m \\\\\\\\\\\\\\\ ‘ l L O Cytochrome xidase é / I °‘ \\\\\\\L\\K\l:!1es infers a basic difference between glyoxysomes and leaf peroxisomes. Citrate synthetase and aconitase have been shown by 8# TABLE 5 Summary of Intracellular Localization in Spinach Leaves Of Enzymes Studied Symbols: +, activity present; 0. activity not detected; -, activity not searched for; tr, trace activity. Peroxi- Mitochon- Chlora- Enzyme or activity some drion plast NAD-Malate dehydrogenase NADP-Isocitrate dehydrogenase ASpartate aminotransferase Serine-pyruvate aminotrans- ferase Glyoxylate oxidase NAD-Isocitrate dehydrogenase Fumarase Citrate synthetase Aconitase Malate synthetase Isocitrate lyase .Malic enzyme Formate dehydrogenase lNAD-glutamate dehydrogenase NAD-alanine dehydrogenase ITAD-aSpartate dehydrogenase PEP Carboxylase JPEP carboxykinase IiydroxySSpartate dehydratase Cl-Oxidation B-Oxidation ()xalate oxidase Catechol oxidase Phenylalanine ammonia-lyase -++-+ IOOIIII~0II+++IS++++IO +++ I+OIIII+IIIOOIIOOOOIO+OO OOOOOOOOOOOOOOOOOOON‘I‘F * 85 Breidenbach. Kahn, and Beevers (11) to be present in the castor bean endOSperm glyoxysomes. Similarly. malate syn- thetase and isocitrate lyase, which are present in the glyoxysomes, could not be detected in leaf peroxisomes. From these results it is concluded that leaf peroxisomes do not contain a functional glyoxylate cycle. This con- clusion can be inferred from the lack of isocitrate lyase and malate synthetase activities in crude homogenates Of mature leaf tissue (7). Glyoxysomes, on the other hand, contain all the enzyme activities Of the glyoxylate cycle (10, 11). Inability to detect B-Oxidation activity in peroxisomes is taken as another indication Of the differ- ence between leaf peroxisomes and glyoxysomes which have been shown tO contain B-Oxidation activity (18). The reaction Of glyoxylate with acetyl-COA as <3atalyzed by citrate synthetase appears not to have been :reported previously in the case Of the plant enzyme. The <>ccurrence Of such a reaction may explain formation of malate from glyoxylate as proposed by Asada gt 31. (3) in Erpinach leaves. The finding of NADP-isocitrate dehydrogenase activ- ifby’in peroxisomes, mitochondria, and supernatant fractions Suggests a tranSport or Shuttle function for these locational isozymes. Since mitochondria do not readily oxidize exo- genous citrate (78), one or both of the extra-mitochondrial fCIOIIE‘InS may be active in the oxidation of externally-supplied 86 citrate to a-ketoglutarate. This is perhaps more Of a pos- sibility in the case of the supernatant activity, since large amounts of aconitase activity have also been found in supernatant fractions from tobacco (7#). In either case the extra-mitochondrial oxidation of isocitrate would supply the cytoplasm or peroxisome with NADPH which could then be utilized in biosynthetic sequences. Cleland (16) has also suggested that the extra-mitochondrial NADP-iso- citrate dehydrogenase(s) may be carrying out the reductive carboxylation Of a-ketoglutarate. The presence Of malic enzyme in Spinach leaf mito- chondria may also be an indication Of a shuttle mechanism for reducing equivalents. Simpson and Estabraok (86) have suggested the Operation Of a malate-pyruvate shuttle in the tranSport Of NADPH reducing equivalents into the mitochon- dria Of bovine adrenal cortex. The possibility Of similar shuttle mechanisms Operating in plant tissue has not yet “been fully explored. The mitochondrial localization Of Inelic enzyme activity may argue against a Significant con- tzribution Of this activity to the total photosynthetic C02 fixation Of Spinach. The localization in Spinach mitochon- d1?1a differs from that found in maize by Slack and Hatch (8 7). These authors found malic enzyme activity to be associated with the chloroplasts after nonaqueous isola- ticarl. The presence Of formate dehydrogenase and serine 87 transhydroxymethylase (unpublished results of W. J. Bruin and N. E. Talbert) in the mitochondria is suggestive Of an active role of the mitochondrion in C1 metabolism. An interrelation Of C1 metabolism and TCA cycle activity is suggested by the finding of Magar and Homi (60) that folate is an allosteric inhibitor Of NADP-isocitrate dehydrogenase activity. Inability to detect NAD-glutamate dehydrogenase, alanine dehydrogenase, and aSpartate dehydrogenase activ- ities in peroxisomal fractions is taken as an indication that peroxisomes are not able to carry out the reductive amination Of a-keto acids. This implies that amino groups must be supplied from an external source for the aminotrans- ferase activites found in the peroxisomes. A close rela- tionship must therefore exist between the peroxisomes and :mitochondria and/or chloroplasts with respect to amino acid Imetabolism. The close Spatial association of peroxisomes. (chloroplasts, and mitochondria as described by Frederick sand Newcomb (29) is of Special Significance in this reSpect. The absence of significant PEP carboxylase and PEP (Bexrboxykinase activities as well as malic enzyme in peroxi- scnnal fractions may indicate inability Of the peroxisome to c<>rrvert C# compounds to C3 compounds and vice versa. Gluco- ru3<>genesis from C4 acids thus appears unlikely in the peroxi- somes. An unusual pathway for glyoxylate metabolism has 88 been demonstrated in Migrococcus denitrificans by Kornberg (#8). Glyoxylate is first transaminated to glycine. The glycine is then condensed with another glyoxylate to give erythrO-hydroxyaSpartate. Finally the hydroxyaSpartate undergoes a dehydratase reaction to give oxalacetate. Because Of the presence of glyoxylate and glycine in the peroxisome, hydroxyaSpartate dehydratase activity Of per- oxisomal fractions was assayed. No activity was detected. This finding, plus the fact that Kisaki and Talbert (#7) did not find any other amino acids beside glycine in per- oxisomal experiments with 1“Colabeled glycolate and glyoxy- late, indicate that peroxisomes do not metabolize glyoxy- late by the B-hydroxyaSpartate pathway. Glycolate and glycolate oxidase have been implicated in the generation Of H202 needed for a-oxidatian (13), but the failure to find a-Oxidation activity in leaf peroxisomes :may indicate that sources Of H202 other than the peroxisomal glycolate oxidase must be utilized in this process. Such is jxrobably also true Of peroxidase activities Since no peroxi- dase activity was detected in leaf peroxisomes (97). The inability to demonstrate oxalate oxidase in 31>1nach leaf particulate fractions is probably a reflection 01? the inertness of oxalic acid in this plant. Oxalic acid is known to make up about 80% Of the total titratable acid content of spinach leaf (79). Finkle and Arnon (28) have indicated localization of oxalate oxidase in "cytoplasmic 89 particles" from sugar beet leaves. Further investigation Of this localization in sugar beet leaf seems merited, eSpecially with reSpect to the possibility Of a peroxisomal localization. The enzyme phenylalanine ammonia-lyase catalyzes the deamination of phenylalanine to trgggecinnamic acid, the first step in the transformation Of phenylalanine to chloro- genic acid (115). This activity was checked in peroxisomal fractions in an attempt to determine if the peroxisomes were involved in phenolic metabolism. The absence of detect- able activity is taken to be an indication Of the nonparti- cipation Of the peroxisomes in phenolic metabolism. Peroxisomal.ActivitieS Comparison Of the enzymic content of the peroxisomes from various sources and the glyoxysomes reveals that only the presence Of catalase and a flavoprotein oxidase is com- :men to all these microbodies (Table 6). The word "micro- _ body" is used here as a generic term including all cellu- :Lar'organelles containing catalase and one or more H202- praducing oxidases and bounded by a single membrane. This lrlcludes peroxisomes and.glyoxysomes. If Acanthamaeba is BXcluded from this listing (the data from Acanthamaeba has been published in abstract form only) . it is apparent that, more Specifically. only catalase and an a-hydroxy acid Ollixiase are common to all these microbodies. This diver- 311337 with reSpect to other enzyme activities has been 90 I I o + + o emanaspamm evades I I I I + o soapaaHNOIm + o + o I o cmaedwo means I + an o I 0 IA 0 + + + I 0 ID .amaaaxo mace Ozaad + o + I o SaMSOIwSOq o + o + + Rammeawo opaaoohawv adenoipsosm I cmaadwo pace axoaemmis + + + + + + adempao aaooaa moaaax Habdfl, ,aaaaus asap mobaoa mpabapod Seaman Isusaoa Sam pam Insure Hopmao noaaaam (lellll'ei'el. . .mpabapoa come» .9» “Mom peachacm pa: mpabapoa .I Acopoapop pa: apabdpoa .o ”Snowman mpabapoa .+ ”maopahm maaommxomao use macarom mSOHHa> Scam maaomdwoaam mo moapabapod_oamuam o mqmdfi x . w I , , .. s..a:.,V.\Y/ 91 .mpamnobaao apapm aawH£oaz .paaaaoa .m .z.usa Hoeaa> .A .m .soaSSOSasaaoo Hanomaam a .mpdmhabdso eschew .naaaoo .m .9 .aoapaOdszaSOO Haaomaama Amos .wm Emma Ass .mmvxsm .mmv Ase .smv Ame .sav .se .ssv. assess mass oesmpso moosescsom a+ + apahapsamOpaMIdIapapaaamd I + opabsahaIaaHHam a0 + opaHhNOhHwImpaaapSHo I I I I mmahammsanposdas I I + o o + Aamaposoaa apaHmNOmHoIoezv n a amaaowonaasaa mpaHaOmHOIm D-glycerate + L-aSpartate + 2 a-ketoglutarate If a-ketoglutarate undergoes reductive carboxylation by NADP-isocitrate dehydrogenase, a source of NADPH in the peroxisome would-also be required. In this case "2 iso- citrate" would replace "2 a-ketoglutarate" as products. An alternate scheme utilizing an as yet unknown NAD- linked dehydrogenase to generate NADH within the peroxi- some can also be envisioned. Hydroxypyruvate and oxalace- tate might then compete for NADH. In this case the flow of carbon in the.malate-a3partate and isocitrate-glutamate pathways as shown in Figure 7 might be reversed from that in the first scheme. The flow of carbon from glycolate to glycerate would remain unchanged in this alternate scheme, but more NADH would be required as a consequence of the conversion of aspartate to malate. If the substrate of this unknown dehydrogenase is designated as AHZ and the ‘product as A, then the sum of the reactions would be as follows: 105 2 glycolate + Lpaspartate + Isocitrate + NADP+ + AHZ.____9 D-glycerate + L-malate + L-glutamate + NADPH + A One feature of this alternate scheme is that the glycolate- glycerate and malate-aSpartate pathways might conceivably operate independently. The Specific activities of the leaf peroxisomal enzyme activities are given in Table 7.' Using a figure of 250 umole C02 fixed per hr per mg chlorophyll in intact Spinach leaves and assuming that 50% of the carbon fixed is going through glycolate (107), it is possible to calcu- late a rate of 30 nmole glycolate synthesized per min per mg protein in crude homogenates (assuming 30 mg protein per mg chlorophyll - Ref. 103). It has been estimated that the leaf peroxisomes may account for 1.5% of the total leaf protein (98). Using this figure, a rate of 2 umole per min per mg peroxisomal protein can be calculated for glycolate synthesis in the intact leaf. Thus, in order to account for glycolate metabolism without accumulation of intermediates in the peroxisome, all enzyme Specific activ- ities based on mg peroxisomal protein must exceed the critical value of 2 nmole per min per mg protein. From Table 7 it can be seen that all the peroxisomal enzymes except the NADP-isocitrate dehydrogenase and the NADP-gly- oxylate reductase activity have Specific activities which exceed this critical limit. This implies that all the enzyme activities pictured in Figure 7 are active enough 106 N.: H.m mommnowonumsou manhoomaoummdpom odwaomam memm< Ga Hopasz . .onmn smbaw mmdvdxo opdfloomam mo hpabapoe odhaoomm 039 on mbdumaoa Umpdflommhpxo ones can mnoapmacaoam oaomawoaoa mean whoa madmd muses Iaaoaxo Sony some» one: modpabdpom Hosea 909 spam .Uopoopmu was mpmdaaoaoano no waspsonoopaa Scam soapmsaampsoo o: no mappaq .sodpmaoma oaowaxoaoa now psodvmpw mpdmsem omonozm msodsauaoo d waauaaapz psmaaaoawo so 809% semen ommuHHo mama Ioome use .mmdpofidon opdahxomeImdz .omsdmwoammnmu epdeaIde .omcampdo new dawn .oaomdxoaoa on» ad .sdopona ma hhzoq no woman one mmaawam mpabdpom oamaooam moapabapod cahusm Heaomdwonom mo moapa>apo< camaoomm copmadoaeo m mqmda 107 .pnmpaoe .m .2 sum chanm .e .3 co apes cosmdansdmam .mCOadepCoomoo mHmoD wnapmHSQmm pd osadb Umdeondede .m.: mo endamxomaw op mpwbsnaamxonpm: spas money on» no oapma m wQHSSmmm .dpmo ommposuoh opmHmNOhHwImdz Scam Uopddfioamoo .zOApHpand mpenpm Inna o: wQHaSmmm .:0apmhpcmosoo opmameSm mnapmHSpmm we made» popdaoamhpxmn .mmdnowohphsod opmhcomeIQ v ----- TOLBERT ET AL.»——I.F.AF PEROXIsOML‘s 137 tained the bulk of the peroxisomes. The gradient of 5 layers was prepared at 4" by pipetting 4 ml of 2.5 M sucrose (85% w/v), 8 ml 2.3 M sucrose (68% w/v), 10 ml of 1.8 M sucrose (61% w/v). 15 ml of 1.5 M sucrose (51% w/v) and 15 ml of 1.3 M sucrose (44% w/v). These percent values for w/v are based upon g sucrose per 100 ml final volume at room temperature The 2.5 M sucrose had been prepared in 0.02 M glycylglycine and it was diluted with 0.02 M glycylglycine for the other sucrose concentrations. The gradient was centri- fuged for 3 hr at 4" at 25.000 rpm (44.600g to 106.9009) in a swinging bucket rotor SW 25.2 of the Spinco centrifuge. Model L. In this time. the particles had sedimented to a sucrose molarity which had high enough specific gravity to stop the particles. and longer periods of centrifugation could not alter their distribution. In recent literature citations con» cerning the location of glycolate oxidase (14) and malate dehydrogenase (16) in chloroplast prepara— tions, investigators have used non-isopycnic sucrose gradient centrifugation procedures which. when checked by us. did not separate the peroxisomes from the chloroplast fractions. The gradient was divided into aliquots of 3 to 12 ml by draining from the bottom of the centrifuge tube through a needle, and fractions 1 (at the bottom) to 9 (at the top) were collected as indicated in figure 2. This procedure was selected on the basis of an investigation with spinach leaves (21). The 0.5 M sucrose fraction which had been layered on the gradient (fraction 9) retained the soluble enzymes. In the upper portion of the 1.3 M sucrose layer and at the interface region between 1.3 M and 1.5 M sucrose were bands of chloroplasts as observed microscopically and by chlorophyll analysis (fractions 7 and 8). The interface region between the 1.5 and 1.8 M sucrose layers (fraction 53 contained the mitochondria and some whole chloroplasts. At the interface between 1.8 and 2.3 M sucrose (fraction 3) were located the peroxisomes. Although a contin- uous gradient has produced a cleaner separation of peroxisomes completely free of chlorophyll. a (lis- continuous gradient was more convenient and faster for this survey because of the viscosity of 1.8 to 2.5 M sucrose. A 2.3 M sucrose layer was used rather than one of 2.0 M in order to stop the peroxi- somes in a sharper band. No significant amount of enzymatic activity other than peroxisomal activity has been found below the 1.8 M sucrose layer. The percent activity among the sucrose gradient fractions in figure 2 was calculated on the basis of the total activity recovered from the gradient. In general. nearly 100% recovery on the gradient of the activity from the 60009 pellet fraction was achieved, except for cytochrome c oxidase and malate dehydrogenase, for which an increase in total ac» tivity was observed. This increase suggests that the values obtained with the initial homogenates for these 2 enzymes are too low. Clymla/r Oxidase (liC/.I._:.I). Glycolate:0._. oxidoreductase was assayed anaerobically by follow— ing 2.0—(liehlorophenolindophenol reduction (21,28). Additions were made to a Thunberg cuvette (d = 10 mm) in the following order: 2 ml 0.1 M pyro- phosphate, pH 9.5. Containing 1.5 X 10'4 M dye: 0.05 ml S X 10" \1 FMN (final concentration. 1.0 X 10" M ) I 0.05 ml 0.5 % Triton X-100 (001% final) : 001 to 0.2 ml of enzyme, and in the side arm, 0.1 ml of 0.125 M sodium glycolate (final concentra- tion. 5 >4 10 3 Mi). The final volume was 2.5 ml. The cuvette was evacuated and flushed 10 times with N: which had passed through Fieser's solution to remove traces of 0:. Dye reduction at 25° was measured at ($00 nl'u, by an automatic recording Gilford spectrophotometer. A unit of activity was expressed as 1 change per min. which was equivalent to 4.78 nmoles of dye reduced per min. The use of Triton X-100. which probably solu— bilizes the particulate membranes. increased glycolate oxidase, glyoxylate reductase. and malate dehydrogce nase activity in the peroxisomal fraction by 30 to 40 %. A final concentration of 0.01 % Triton X—100 was found to be sufficient, even with the high sucrose concentration from the gradient. Catalase activity was not increased by Triton X—100, although catalase is the only enzyme whose activity is increased in liver peroxisomes by use of Triton X-100 (1). All attempts to run the glycolate oxidase assay aerobically with a high concentration of cyanide to inhibit peroxidase reoxidation of the reduced dye were unsuccessful. as discussed previously (21). In the aerobic assay, the peroxidase in the soluble frac— tions resulted in lower total enzyme units. The peroxisome fraction (No. 31 from the sucrose gradient contained so little peroxidase that the aerobic assay could be used with this fraction. However, aerobic assays gave incorrect overall re— coveries because of low values in fractions with peroxidase activity such as the supernate. Only by the anaerobic assay could a quantitative recovery of the enzyme from the various fractions be obtain-ed. GIym‘yla/e Redudase (EC 1.1.1.26). The assay for this enzyme involved following the oxidation of NADH at 340 111]). (25). Additions were made to a 1—ml Beckman cuvette (d = 10 mm) in the fol- lowing order: 0.4 ml of 0.02 M glycylglycine buffer, pH 7.5; 0.05 ml NADH containing 0.2 mmole: Triton X—100 (final 0.01%): enzyme. water. and substrate to make a final volume of 1.0 ml. After determining the endogenous rate for 1 to 3 min, 0.04 ml 0.125 M sodium glyoxylate (final concen- tration, 5 X 10‘3 M) was added. A unit of activity was expressed as 1 OD change per min. which was equivalent to 161 umoles of NADII oxidized per min. After completing this survey, it was found that glyoxylate reductase, as obtained from the peroxi- some fraction of spinach leaves by Triton X-100 treatment, was not assayedat optimal conditions. The pH optimum was found to be 5.8 as compared to 7.5 with the soluble enzyme or 6.3 to 6.6 for the 138 PLANT PII YSIOLOGY SPINACH [ SUNFLOWER l TOBACCO so _ l Glycolate Oxudose I Jr 80 60 ‘ ‘ r 1- 60 l A 20 - 1 FE - 20 _=a [ E 3 4 Ca Pa 3 .3 1 -1 60 ‘5 80 ~ 1 a 60 - NADH Glyoxylate Reductose - 40 T >. 40 - a 2 ; r12 2 _a 1 cm A?! I 03 T _ "l c g 80 h 1 Catalase ] _ " 40° E F- 60 — ‘ ~— 300 x '5 4° ' E [E 1 l‘é . 2” 200 3 / O "‘ 20 _ ‘ 5- I00 E Q Ca 1 212 15 a c ‘ - 1200 ‘ cf 80 — 1 I g .3 so — lMalate Dehydrogenase E» 300 E ' / 4o — a E {E l 3‘ 400.9 20 - 4 9.: r2 4 E E r, eo — so 1n 0‘ 0 mg Total Chlorophyll 8 6 F113. 1. Cytochrome c Oxidase —- 40 20 ‘mg E_ _ E E & — l 1 5 ~ —- 1500 2 I 1 ° L- _ 1ooo ‘1 | ‘ 0 ~ 4 500 E I . . _ .72 w— 8- MIT su1D ' w- 8— MIT sup 4 w- 8- MIT sup ,9 CHL CHL CHL CHL 1 C L CHL bl11/y1111c;1~~.'1_v>1>f 1111111111“ 1111111 11111111111111] 1:1‘1111'1111g11111111. The leaf h1111111g1‘nat1‘s “(TC separated into ;4 111119 1111111 4111111111”; 11111111- chl 11'1:111l1>1\ 1\\- (1111 :1 11111111., 11111111 containing 11111‘11xis11111cs and broken chloroplxts 11111111. 4 §‘IIIU()1/ 1111111 01111111111111 1111111111111111r11 (\111) 11111 >11;11~1'11:111‘ 1511111. 17111‘ the enzymc assay 111 each trac- 111111.1111- 1111111 11111-:1111~1nts 111 111-111-111 111 1111 111121] 211‘11\11_\ 1-1' 1111- l11111111g1-112111‘ and the hatched bar the specific gravity. ‘\1 the 1111111111 111 1l11 1 111111 1111-1111111 11:11 1'1111'1‘ \1-111~ 1‘hl1'11'1111hyll :11111 1111‘. hatched bar protein Figures continued next page 139 LEAF I’EROX ISOM ES TOLBERT ET A 1.. 680338: v.8 moment—~83 5 329» 3.888 085 Cow 880.28 0.8 83.3 och .32 03 :88 83? 9.3% .8898“ no: 83 3:29 035 :8: 83.3080: 330 05 5:5 .232“ vamwmxo 9 0803095 05 mucouaaa< .0838 how 05 94. van 0:8 395 how co ovw 33 AN 33 cowawnmbcou “cog—3m Emma—ow omega watsv 828on0: 05 mo cotomuw “ma—acacia 530.5 05 Sew .3150“ 3258 9 089.533 mo @30qu . a: A 3 :8 _Nv .80 “N mom «.2 X md mm coma». 2: X 9o numb N an 8 M Q. onshmwsm we 8 2.1. 3 Km .2 XS 8 8me .2 x 3 .2 m 8 2 _ on :80 no on 33 mm coo ”2 X Nam vm oofimofi i: X fiv 3 N— «mv M: A com 39.6mm R mm 3n 2 30 «2 X fiNN am 80de c2 Xmaw 9. mm vmn mm 2 Km :mom vw mm N2: 3 mm“ as XndN vN 08.3w. 2: X cs 9 on N2: 2 _N O8 @223 mmmam mm om hug om Gm; no. X Q3 3 893m. 2: X 5w w to 82 w“ om m E «3:3 3 3 $2 8 mm .2 v3.8 RN 8w.~8 .2 xx: 2 2 w: m... a 82 3.. A .3 AN: 35 am .2: .1. we: .2 x mam 8 83% .2 x 3 mm a. ma 3 a. 82 82309 SW mv o3. _ mo mNN m2 X v.0 mm oom._w_ a: X Wm gm 2 Nwm. «v 3 cm: Sac—tam Nu mm as a 8m .2 x “.2 2. 80.3 a: x 3. mm a. N8 an _N 3m 62% £833.; £80:— Ewmoa mcofloahw 5803 Emma? 3238... £803 Emma? 2269: £808 EMEB mcotumb E303 EMEB 33:2th TwE pots fiu 82:3th 7M5 33 TM 32:03.23 er 3.5 7w ova—3E3 TME Ho? . w 82:3th TME 8.5 Tm 5 0&9 X TEE X 33:5 5 ok» X _ EE X .338: 926me .. 05:533th ummcuwohwmzuv 3332. 5 0% X TEE X 8.9:: own—Emu :H mm. X TEE X 835: cH 05 X 758 X 3.2:: om Sun—om: 32.98.30 3%me own—09:0 BEAN ‘ WHEAT PEA 8653503 am 3.3.5.:qu ozuafiwxm BBB 5208 .19.... ._ TEE _. 3.9:: 53:3 2:99am EZRA on. .20... 1 m mmwmmwmmwmmmmm.» m I+|TIT11:» I . 1 In». 1.», Nag/,1». 1 1 L, _ I1 m m C E $ m m m V; I u m m G C W. e m . N £5.34 .20» .o 28.8. SUGAR CANE CORN .2 “.33. 5.203 ToE x TEE x 3.06: 53.34 23on £205 9.: .20» n . . wwmswmwmm m mm . 1101.0»09»,:010 u m I 1 m1 - I - I 1 R M O ‘ o m . u u w m u m n m m m. In W. o o G C r H M D A m N .1 .. PIG WEED GDP 60- 401- 203 w w m _ w m m m m m :23 .28 s 23.... 40L 201- 283.6 2. .22 ‘ 140i PLANT PH YSIOLOGY SPINACH SUNFLOWER GYOdlen? FFOCNOD Number 1 . l Groalent Fraction Num5er 9181716151413121| 918l716I5l4l3I21 Glycolate Oxidase E ,2 'O E o C O 2‘ IE 8 < E .9 ‘6 E a) 8 G) a. so» 7.: Cytochrome [c Oxidase 40+— ’5 ,4 | 20_ 55:” 2 , l E | \ 2»— O‘ E l =>’ 1 c l '— ‘ Q. 9 x O _ .7 ‘ ’ LC) /? J 4 . .1 ., ,, slelrlslsialslzllj> 7.65432 Grfldlenf Frocnon Number Gradient Fraction Number 05! r3 1 :5 l I [25 \O.5l|3 I l5 1 LB ~l Sucrose Molomy J Sucrose Molom continu TOLBERT ET ALr—LEAF PERUXISOMES 141 TOBACCO ‘ PEA BEAN PIG WEED l Groom n j I act-on Humour 5.09."; r, I-on Humour (rotten! FrocIIon Number ‘l’l .Gmdsnl. Froct Nan I F_’__i..";‘I3L‘13L11| 9 01 Icifila.sia I _LLJ.L.ZA_LL§_L‘_.LJ m Wu: I I . + I 0 <> 000 W‘ 6ch «on Oman 0 . Glycolate Once“ ‘01» “I T ‘ . 4» 003 l’ *0. . rl \ $0. 1’ H ' I-ooz , 7 + 01 . _ . «I oon .. _l mmd‘LnLhr—j Fun _ LL-‘L.LLJ_1_‘_1..L:1-.._Q_ Q l — i.____x::n_l;1=-:s.£:l_... fiOr— E i W GlYOIV'O'O Reductou + o '0 5 .0 F W Glyolykml Wm 10 - l {7.1. 1 ”I cucum- u' v5 5:.- ‘ . E g l i g . t : . “' ' :- I r E l l n a l . _ __.’ I 4'. a ‘ I l ,1 . : 7 . c I l ' - . ‘- O ‘ ' . _ I i l ' -o- d . O : .. 3 5 . j ' t j I ‘ + , jg 3 . 2 ~ 1 E 1: I A .. . .» U H fl 44 7‘ r“ >1: 744 ‘ 4 J,’ . . '1'. I- ! l .+.G 6%.: El: '4 9“.+’7' F §+£""Fh U II V 0 V Tl I h ',C a”- . 3 ° ‘91 '9‘ 1‘9"”! -7 ...CI'90-9,"J 77,9ng Number fl Gregg: Frochm ho: . Gradual! Fraction Numb:3 (. . 35 to" I) IS I. 33 I15 *‘T‘—T I ‘Sgdou 9.. 4"“ 'v _i Sucrose Motom 05 I! I3 T I. l u 7:] CT I! 1 I5 T It I fag ._ .,._- __,_ L . _ ___ -1- .J l _ Sucrose Moon ! Sucrose Mona, _J WHEAT SWISS CHARO CORIN- ' so GAR CANE r 3'3"”; E50"? ‘- Pr" , F - —_ wool lent ‘ o-f v Nu'nbe- __ '1 , 7 I've-"3N “out” farts.“ ‘ ' 1.1+" "y”r '. "[1" W y,LL.9.L_I'.I_1I14,‘ “3+; ‘7‘1 LL94. LJS L:‘s .,.?.L _4 ‘w _u p "m 4,1 ,4 .!.'I‘ III [9103‘ '.‘.' 1‘ ‘ I . 7 L i . . r .. GlycoIato 0'2”" ‘+ . D ’ P ' Glycolate Ondmo v.‘ , a I l ‘E + DD ‘ I ‘ + . _H‘ 4:, - l i 5 A av." at I125 SLaI L 0 6 a ‘ 2:! Li {W , ‘ 4. o 4 + 2 . r ‘ ‘lu. - C 7 I... r: 7 . - ’1' “Rh—1’1 4——}-‘~4 ‘1 ' fl -_._ L. -;r.‘ #3 :9 3::— u‘—-. sq _. ? L " :H_ j ‘- ‘r Li r rh— ‘ .‘j 7;- -—\-’—‘ i-vrlbj-g - 5,; L— F‘I ” 7'0 5 O. - ’ ‘ - NAD" Glyoxylate Reductou #- coo ‘E ~- I- g c .5, - ' + n“ u ’c 4- ‘ a g ‘ 7 5A an.“ m -o . 54:0. ‘ _ 3 NADH Glyoxylate Reductase H ‘ _ 5 2’1» I"— J...‘ l l O )O‘ .2 E: I ' a '0‘ . I ‘ h I .. e01 V + E . . 2 ' ,J': ' 3 1. «Ln.-- 52—3: -L- L.,)...” L xii—[:17 filmy-2.-.“... ._._‘._ _ " 5 - 7- -- - —w -- 9 " 4: l » 0000 |c : + m. '5 Z ‘4“ l- l E '3 - I E U o A C - on I“ ‘4 my - Comma “"7 - v: ‘ Catalase . ,, _ a ._ «xx g 7: " - g f, a A]: _. o E III '" "l ,. " ‘ LF‘L 1r WE ' ‘ -I , l , 9 7, L. ,LJ -..--l,-r‘ L- Li 3.": -‘ ._ . .In-L. Liz! :24 H I.— _ ,,, _ ,1 E - . 5m ‘rm .. ._ Sung" ea __ . i=1 H ...‘ ‘1 l I .—_, ,,; I t T o 3 . h c e 40 " C_ ‘ ‘ : 8 63;“ 3 t . - _, A” E I . so §> 3 ' l +-~ .:.L_ W Malate Dehydrogenase 2 “ u Macon D'hyd'm’m” ‘ : l1 I :' f I l l + 20 g V t_ I i l i i 5 ’ L C- . . . . O 1'1 L.“ fi‘ *‘ ‘I , L I : , . n .x. C .7 _ ' . a ._ U) ' . l | I I I' . I": L—-— 1* ; '0 I-., ,_ -- - i ’ :I I”. ‘ .=-‘ fl -1..- -3... .4 .21 -1-.- I {3 .‘-_- - L .L— -1 4-12.. _..r.2.:..a _ . -I H .1 - .IJ.1 ._ -_ ‘.‘I ‘._I - I . I -,,I _ l '_’x,: l "_ ‘ 7» A - . a so» ‘ r‘ ‘ Cytochrome c Ondase .- Cytochrome c 01:60:. + “ ‘ 7' I‘ i s f—L’b ., (- ‘ * Hi‘ .23 r" j i1: 3 “*I « .L. 5 - I“. ._ 3‘: _ m I - 3 . '43 I“ r~ a ,- ,. .In: - ~I _ -- i’ 11,, U J4 ’1 h’rCHlA—IZI cs— LL--17J j~-—= 4, - ._ , . - J 1 J’ ‘ .‘W 2:. $—-'-\ .‘ J [I] 5.-.»; - i _,.I_--,l J Li} 5. L—Cl- W I E r: g 2 .— Ti 3 {'1 t c E :5, ~ ”_j 1‘ + I: E . I". ,— Chievoohl: .. II! kw. \ l : D ‘ + . ' F‘: ‘1 0 §'”,II :11? +‘% 8 ‘2 =' I? H? g I g 3‘1"; g l L ‘1 T 2 a s i 'l l 1* J! l i i *7 ‘. E U l—;»iI——.-—+—‘ .' nib—£1! M ‘1 ' g ‘ ll; 4‘4 _fir‘ £2:- T—T—__a \ L1 . . :3 t “HIT—.fi {’7? i 1 - l :o-b—{m m- 5‘..— 3' room! Fro-(t flying-g, _ ' _ abjodwnl P10C'I’V‘I m l ., .- J’Jv f.“- if J" .‘1 'ViuTiwt - ‘ 1' I; 413' "31} I :n " n v fés'T—TS—F—I . I? - 25 ha. FEET I *—“ *4 Di) TES ,1 I: III 3 :5 ‘5? I! ' e” ' I9‘ 7‘53 725' I533“? “0'9":1. __ __._J L._.____. ..S.‘.”_'°" 37‘3"'1_-_. I K" "" "I" ' "1 , L“ 7, haunt.» wound L__’_— FIG. 2. Enzymic assays of sucrose gradient fractions from broken chloroplasts. The fractions are numbered on the horizontal axis as removed from the bottom of the centrifuge tube. The designated sucrose molarity is that used to prepare the gradient, and the scale is proportional to the volume ot each layer of sucrose solution. The total volume of each gradient was about 54 ml and the actual volume of each fraction from the gradient is depicted by its band width. Consequently, the total chlorophyll (open bar) and protein (hatched bar) in each fraction is rep- resented by the width plus height of the bar. With respect to the enzyme assays only the height of the bars are to be considered. The percent of the total activity on the gradient is indicated by the open bars. while the hatched bars Show the specific activity of the enzyme in each fraction. 142 crystalline enzyme (25 ). PLANT PHYSIOLOGY For optimal conditions. a final substrate concentration of 5 X 10‘2 M was indi- cated, which is comparable to previous values of 9.1 X 10'3 M (25). Thus, the specific activities for glyoxylate reductase reported here (except for wheat which was assayed at pH 5.8 with 5 X 10‘2 M substrate) are estimated to be about 40 % of maxi- mum values. Sufficient reevaluation of the data were completed to verify that the reported results did not alter the conclusions concerning the distri- bution of this enzyme amongr the various fractions. 0 flier Assays. These procedures have been de- tailed in a paper on the characterization of peroxi- 5011165 from spinach leaves (21). Catalase (EC 1.11.1.6) was assayed by the disappearance of ll._.0._. as measured spectrophotometrically at 340 mp. A unit of activity was a change of 1 OD in 1 min at 25°, and was equal to 2.76 “moles of H.202. cytochrome In the c oxidase (EC 1.0.3.1) assay. the rate of oxidation of reduced cytochrome was measured at 550 mp. Malate dehydrogenase (EC 1.1.1.37) was measured by the Spectrophotometric rate of NADH oxidation with 4 X 10“ M oxalacetate as substrate. A more detailed characterization of malate dehy- drogenase in leaf peroxisomes will be published else- where. cedure and Protein was determined by the Lowry pro- chlorophyll by its absorption at 652 mp according to Arnon’s procedure. Results Actual}! in Homogenatcs and Fractions From Differential. C cntr-ifugutimz. in table. U, the specific activity of each enzyme in nmoles >< min‘l X g“ wet weight or mg" protein in the different leaf homogenates and the percentage of this activity in the combined particulate fraction-s have been recorded. The distribution of these enzymic activities in the whole homogenate among the 4 cellular fractions obtained by differential centrifugation of the leaf homogenates is plotted in figure 1. The fractions have been designated by the centrifugational proce- dure ( Methodsl after their major component. Thus the 1001.} pellet contained whole chloroplasts (W—Chl). pellet \ ms The bulk of the protein in the 60000 associated with broken chloroplasts (B-Chl), yet this fraction contained in addition most of the peroxisomal activity and a substantial part of the mitochcmdrial cytochrome c oxidase activity. The percent distribution of the different enzymatic activities (open bars), and the specific activity (hatched bars) of each enzyme showed a significant difference among the plants. The amount and specific activity of glycolate oxidase and glyoxylate reductase were of the same order of magnitude. from 1.450 Glycolate oxidase values ranged min” > g”‘ wet weight for \ an to] es sunflower to 0.030 for corn leaves. and glyoxylate reductase from 1.3 for wheat to (1.03" for sugarcane. Due to its high turnover number. the specific activity of catalase was immense. and catz‘ilase activity. like glycolate oxidase activity. varied among the plants from 11,800 ,umoles >< min”1 X g"1 we pea to 500 for corn leaves. Total mal genase also varied among the plants. of cytochrome c oxidase. was more nee among the plants and reflects the ubiquit of mitochondria. Sucrose Density Gradient C cniri} Pcrnxistmtes‘. Figure 2 shows the dis the various enzymes among the 9 fracti from the isopycnic non—linear sucrose den separatimi of the 6000;] or broken chlo tion. The whole chloroplast and mitocl tions from the differential centrifugatii subjected to the sucrose gradient separa they contained less of the enzymes ass peroxisomes. However. from a previm tion (21), the small amount of glycolat peroxismnal activity in the whole chlo mitochondrial fractions had banded : sucrose gradients to that in the broker fraction. As indicated by the chlorophyll analysis (fig 2). most of the chlorop sucrose gradient were found in fractio which consisted of 1.3 M to 1.5 M suc fractions were visible as 3 distinct green the upper part of the 1.3 M sucrose lay at the interface with the 1.5 M sucrose small one at the interface between 1.5 sucrose (fraction 5). The chloroph} particles in fraction 5 appeared to b observed with the light microsc0pe. wl fractions 7 and 8 appeared broken. \Vh chloroplast fraction from the differentia tion was used instead of the broken chic tion. more chlorophyll was present ir (data not shown). Mitochondria were predominantly loc tion 5. as illustrated by the location of c oxidase and 1 of the malate dehydro; (fig 2‘). Peroxisomes were found in fractic sucrose gradient which represented t between 1.8 and 2.3 M sucrose (fig particles in fraction 3 from spinach examined by electron microscopy. tli spheroid, 0.5 to 1.0 pi in diameter. and dense granular matrix or stroma Slll‘l‘( single membrane which was ruptured ii (’21). In this survey, the peroxisomes terized by their location on the sucr and by their enzymic content of glyco glyoxylate reductase. catalase, and part < dehydrogenase activity. The presence each of these enzymes at the top of tin fraction 9, which corresponds to the : particulate region of the gradient. may of the fragility of these particles. Since were not washed. some activity from enzymes of the original homogenate “'t pear in fraction 9 of the gradient. TOLBERT ET AL.——LEAF PEROXIsOMIas 143 To estimate the relative distribution of each zyine between the peroxisomes and the cytoplasm. e sum of the particulate activities obtained by dif- "ential centrifugation can be compared to the tivity in the supernate (fig 1). This estimation es not account for breakage of the particles during inding which is undoubtedly severe. A maximum about 50 % of the glycolate oxidase in homogenate spinach and sunflower leaves was found in the mbined particulate fractions. whole chloroplasts. roxisomes plus broken chloroplasts. and mitochon- ia. Examination of these 3 fractions indicated It the same particle. peroxisomes. moving to the me place on the sucrose gradient. contained this rcolate oxidase activity. In subsequent handling the particles during gradient development. more the enzymatic activity was solubilized and ap- ared in the soluble fraction. No. 9, of the sucrose adient. Movement across boundaries of different crose molarities has a shearing effect upon par- les. How much of the activity in the top soluble 1(‘1101'1 9 of the sucrose gradient is from disruption the particles or from removal of supernatant zymes occluded with the particle cannot be re- lved. This degree of apparent breakage varied tween 20 to 30% for sunflower and spinach and to 00 % for pigweed. corn. and sugarcane. Ex- iination of the spinach peroxisomes by electron croscopy showed that nearly all of them had a when membrane or were more severely ruptured 1). The small amount of peroxisomal activity which is found in fraction 2 of 2.3 M sucrose is an arti— ft of the procedure. Removal of the fractions )m the gradient was based upon visual observations. 1 attempt was made to stop the collection of lCthI‘l 2, after this very light green solution had lined from the delivery tubing. This slight color emed to be due to traces of chlorophyll-containing iterial. Under the best of conditions. fraction 3 .s faintly yellow in color. It was never possible completely separate fractions 2 and 3 after 1 idient run which started with a concentrated mix- 'e of particles. Some contamination of fraction 3 0 fraction 2 resulted in enzyme activity in fraction )f lower specific activity. probably due to the green terial seen in it. Contamination of fraction 3 0 occurred. and the enzymatic specific activities )Ol‘th for it are not maximum. Isopycm’c Linear Gradient. Fractions 2 and 3 m a spinach preparation were combined and rerun a second isopycnic linear gradient between 2.3 i 1.7 M sucrose. The peroxisomal activity banded ween an estimated sucrose molarity of 1.95 to 0. and the peroxisomes were completely devoid chlorophyll and cytochrome oxidase activity which nained at the top of the gradient. No activity 5 found below 2.1 M sucrose. In this experiment. peroxisomes in about 2.0 M sucrose of fraction 3 re diluted only to 1.5 M sucrose before rerunning the linear gradient. During this recentrifugation, about 30 0;; of the glycolate oxidase activity was solubilized and was found at the top of the second gradient. This result gives some measure of the loss of enzymatic activity from the spinach particles. In the original grinding medium. more severe rup- ture of the particles could be expected. Fraction of Total Protein as Peroxisomes. From the data only a limited approximation can be made concerning the total amount of cellular protein which may be in the peroxisomes from Spinach and sun- flower leaves. About 1.5 to 2.0% of the protein of the, 60009 pellet was found in the peroxisomal frac- tion, No. 3. after sucrose density gradient centrifuga- tion. According to the marker enzymes this fraction was reasonably pure, but its actual purity on the basis of protein from other particulate material was not known. This value should be corrected to 3 to 4 9} of the total protein of the 600037 pellet on the basis of the amount of marker enzymic activity which was found in other fractions on the gradient, par- ticularly the solubilized top fraction. About 20% of the total protein of the homogenate was generally in the 60005] or broken chloroplast pellet. Thus 0.6 to 0.3% of the total protein of the homogenate appeared to be in the peroxisomes. This value may be twice as large on the basis of an estimated 50 % rupture of the peroxisomes during grinding. Thus about 1 to 1.5% of the protein of the spinach homogenate may be peroxisomal. Grouping of Plants by Photnrespiration, Enzyme Activity, and Peroxisomes. In the plants surveyed. the activity of the enzymes associated with the per- oxisomes varied over a wide range. In an attempt to categorize the. data. the plants have been grouped with respect to photorespiration (table I). total glycolate oxidase activity (table II), and the relative amount of this enzyme which remained in peroxi- somes during the isolation procedure (figs 1 and 2). Sunflower and spinach leaves, which exhibit photorespiration, contained a high level of glycolate oxidase (1450 and 514 nmoles X min'1 X g‘1 wet tissue or 49 and 21 nmoles X minl' X rng’1 protein) in the crude extract. Likewise, values for the spe- cific activity of glyoxylate reductase, catalase. and malate dehydrogenase activity were high in these plants. In the original extract. 40 to 50% of the activity of these enzymes was found in particulate fractions and 50 to 60 % in the supernate. On the sucrose gradient of the broken chloroplast fraction, a single peroxisome fraction (No. 3) contained most of these enzymatic activities. These plants, then, are characterized by the presence of the most stable peroxisomal fraction. active enzymes for glycolate metabolism. and photoreSpiration. As such, these plants provide a basis for comparison with the other plants. Corn and sugarcane leaves contained only 2 to 5% as much total egColate oxidase. glyoxylate reductase, or catalase as spinach or sunflower leaves. and only traces of these enzymic activity were found in the area on the sucrose gradient where peroxi- 144 PLANT PHYSIOLOGY somes should be located. These 2 plants represent the best example of plants without photorespiration and the least amount of peroxisomal enzymes. It is noteworthy that the total activity of each of the peroxisomal enzymes was similarly low in these plants compared to sunflower and spinach. In figure 1 for the fractions from the crude extract. the specific activity scale for glycolate oxidase is one-seventh that used for plants with photorespiration. The specific activity of glycolate oxidase in the supernate from corn and sugarcane leaf extracts was about 2 nmoles X min" X mg“ protein as compared to values between 40 and 90 for plants with photo— respiration. On a wet weight basis. corn oxidized 29 nmoles glycolate X min"l X g" and sugarcane 48. which is to be compared with values of 500 to 1500 for other plants. These low values are not due to poor homogenation of these fibrous tissues since amounts of protein and chlorophyll in the ex- tracts were comparable with amounts found in other leaves (fig 1). However. it must be emphasized that the activity of glycolate oxidase and catalase from corn and sugarcane leaves was still sufficiently great to be measured easily and reliably. Signifi— cant amounts of these enzymes are present in corn and sugarcane. whereas very large amounts of these enzymes are present in spinach and sunflower. For corn. sugarcane, and pigweed. there were only trace amounts of marker enzymes on the sucrose gradient at the location for peroxisomes Again. note the scale change for specific activity in figure 2 when comparing these plants with sunflower or spinach. The smaller amount of enzyme activity which was first separated with the 60009 pellet or broken chloroplast fraction by differential centrifuga- tion was not stable to repeated centrifugation and after sucrose gradient centrifugation was located at the top of the sucrose gradient. The presence of trace amounts of activity in fraction 3 from all the plants indicate that there are some peroxisomes in these plants. The results are qualified because of the difficulty in grinding corn and sugarcane leaves which might have resulted in greater rupture of the particles. For this reason. preparations were made with old leaves as well as very young leaves, but similar results were obtained. Although the neces— sary longer grinding period could have ruptured most of the peroxisomes. it should not have destroyed such enzymes as glycolate oxidase and catalase and the values for total activity should not be in serious error. Although pigweed, like corn and sugarcane. does not exhibit photorespiration. its conmleinent of glyco- late pathway enzymes was greater. The crude pigweed homogenate oxidized glycolate at the rate of 266 nmoles X min"l X g‘ wet weight. or 7 nmoles X min‘1 X mg‘ protein. This amount of activity was. half that found in spinach leaves. Similar results were obtained with leaves from green house-grown plants that were flowering or from vegetative or florally induced plants grown in growth chambers. It was hoped that pigweed leaves. with- out photorespiration, would be easy material from which to extract enzymes. However. the leaf homogenate from greenhouse leaves was so gelatinous that only part of it could be squeezed through cheesecloth. and from plants in the growth chamber. the homogenates were rich with saponifiers and extremely foamy. The total amount of enzyme ac- tivity on a leaf weight basis is thus minimal and true values are probably similar to those from spinach leaves. From the pigweed broken chloroplast fraction. only traces of peroxisome activity were observed. and of this small amount. most of it was found in the soluble protein fraction of the sucrose gradient. In this respect. the pigweed preparations gave gradi- ent distribution patterns similar to that from corn and sugarcane and differed from those obtained with spinach and sunflower preparations. Tobacco. pea. and wheat leaves, plants with plin- torespiration. contained relatively high levels of glycolate oxidase. glyoxylate reductase, catalase. and malate dehydrogenase activity. However, from these species, more of the enzyme activity was found in the supernate {65—80 %) rather than in the particu— late fractions of the original extracts. Thus. either there were fewer peroxisomes in these leaves or else the yield of peroxisomes by the isolation procedure employed was poor. The activity in the broken chloroplasts was located on the sucrose density gradient in 2 bands. 1 in fraction 3 as for spinach and sunflower peroxisomes and 1 in fraction 5 at the interface of the 1.5 to 1.8 M sucrose layer. Peroxisomes in fraction 5 could be accounted for by more broken particles, by less dense whole particles. or by 2 species of peroxisomes. Further study is needed to delineate between these possibilities or other explanations of the results. Currently. it is assumed that fraction 5 contained broken peroxi- somes to account for its glycolate oxidase activity. Homogenates of bean leaves, a plant which has photorespiration, and Swiss chard leaves contained high levels of glycolate oxidase and glyoxylate re- ductase. However. 80 % or more of these activities were found in the supernate after differential cen- trifugation. Of the amount of activity in the broken chloroplast fraction. only traces were found in the peroxisome fraction 3 as well as in fraction 5 after sucrose gradient centrifugation. and most of the activity was recovered in the top layer which is assumed to be soluble protein. Note that the specific activity scale for these for the plots in figure 2 were magnified 25-fold. Again, these low yields of per- oxisomes could be due to rupture of the particles during grinding. These results seem to be inconsistent with the concept that peroxisomes are characteristic of plants with photorespiration. In fact, bean leaves are particularly active for glycine synthesis. In previous studies. the leaves of the same bean variety incor- porated 50% of the total 1*COg fixed in the first 'romuca'r li'l‘ .\I..~—l.l~2.\F meanxrsoMics 145 10 to 30 sec of photosynthesis into glycine by the glycolate pathway (unpublished). Thus. bmt and Swiss chard may be characterized as having an active glycolate metabolizing system and active enzymes of the glycolate pathway which mainly ap- pear in the soluble fraction after grinding the leaves. The results with the last 2 groups of plants could be reconciled by the assumption that the differences reflect the degree of rupture of the peroxisomes or the inefficiency of isolating the peroxisomes by the single procedure developed for spinach leaves and used for all the plants. All 3 groups of plants with photorespiration contained active enzymes of the glycolate pathway. The major difference was in the distribution of the enzymes between peroxisomes and the supernate. Similar Distribution Pattern for Glycolate Oxi— dase and Glyoxylate Redm‘tase. Glycolate oxidase and glyoxylate reductase in the isolated peroxisomes had about equal total and specific activities and the same distribution on the sucrose gradient. A some- what similar percentage of both enzymes was found in the broken chloroplast fraction from each plant. liach of these activities in the broken chloroplast fraction, when put on a sucrose gradient, was found to distribute similarly between a peroxisome fraction (No. 3 or No. 5) and the soluble or non-particulate fraction (No. 9). In all cases. a substantial part of the activity was in the soluble fraction even though this part of the activity had originally sedimented with the broken chloroplasts. Electron microscopic examination of spinach peroxisomes had indicated that they were nearly all sheared or ruptured. and. thus. partial solubilization of activity is to be ex- pected. The data suggest that the 2 enzymes are associated in the cell in the same organelle: i.e., the peroxisomes. From the data. it is not possible to ascertain whether there are 2 pools of each of these enzymes, 1 soluble and 1 particulate. Distribution of Catalase and Malate Dehydro- genase. Distribution of catalase activity was similar to that for glycolate oxidase and glyoxylate reduc- tase. \Ve wish to emphasize that catalase did not follow the chlorophyll-containing bands on the sucrose gradient. With sunflower. spinach, and tobacco, it was present mainly in peroxisomes and it was nearly absent in chloroplasts. The data sug- gest that for sunflower and spinach leaves. at least. peroxisomes may be the only site of catalase activity. A similar distribution of glycolate oxidase and catalase is consistent with the concept that. during the oxidation of glycolate by peroxisomes. the H._.O._. produced is immediately decomposed by the catalase. For sugarcane, corn, Swiss chard. and bean. from which poor yields of peroxisomes were obtained, a higher percent of the catalase activity was found with the soluble fractions. as was true for glycolate oxidase. From corn and sugarcane leaves. only one-tenth as much total catalase activity was found as in other plants. and this fact was also true for glycolate oxidase. Characterization of malate dehydrogenase of the peroxisomes is being published elsewhere. There are different malate dehydrogenase isozymes, in leaves ( 16). so that the percentage of the total malate dehydrogenase activity found in the peroxisome fraction is affected by the recovery of the peroxi- somes as well as that fraction of the total cell activity attributed to the peroxisomes. The present data clearly indicate that in all the plants, malate dehy- drogenase activity is in both peroxisomes and mito- chondria. Like catalase. the specific activity of malate dehyt'lrogenase in the peroxismnes was high. Discussion I’eroxisomes containing glycolate oxidase. gly- oxylate reductase. catalase, and malate dehydrogenase were isolated in good yields from 5 of the 1f) plant species analyzed. Spinach and sunflower leaves were the best plants so far examined from which to isolate peroxisomes. and about half of these enzymatic activities remained with the peroxisomal particles after grinding in 0:5 M sucrose. Only small to trace amounts of peroxisomes. as indicated by total enzy- matic activity for glycolate oxidation, were isolated from the other 5 species. As discussed later, the low yield of peroxisomes from some of these species may be due to the fragility or loss of protein from these particles under the isolation techniques used. The results establish that peroxisomes are widely distributed in plant leaves. Our data support the concept that plant species with relatively high levels of glycolate pathway enzymes are obligate photorespiring plants. with the exception of pigweed. Of the plant species which have been stated to exhibit photorespiration (refer- ences in table I). all contained a relatively high but variable level (500—1500 nmoles X min‘1 X g“ wet weight) of glycolate oxidase. Swiss chard might be included in this group. but no published literature was found with respect to its photorespiration. The data from corn and sugarcane also supports this view as they have but 2 to 5 % of the glycolate oxidase and glyoxylate reductase activity of plants that exhibit photorespiration. Both of these species do not photorespire. Pigweed is an exception as it does not exhibit photorespiration (24). yet its leaves had 25 to 50 % of the level (266 nmoles min‘l X g" wet weight) of glycolate oxidase as had photore- spiring plants such as Spinach. bean, and wheat. Even this specific activity value for pigweed is too low because of difficulty in filtering the leaf homog- enate. Obviously, data from pigweed on the absence of photorespiration yet abundance of enzymes asso- ciated with glycolate metabolism necessitates con- tinuing the search for a better understanding of photorespiration. As a corollary, it can be. proposed that in eit'n all the glycolate pathway enzymes are functionally organized and exclusively contained in the peroxi- somes. This proposition is based on the supposition 146 PLANT PHYSIOLOGY that the stability of the peroxisomes is a function of the plant species and the isolation techniques. The isolation procedure used was developed for spinach and used without change for all other species. Elec- tron micrograph observations have indicated that the membranes of the isolated peroxisomes or the peroxi- somes themselves were nearly all broken. Thus, the peroxisome yields, as measured by enzymatic activity. are at best very minimal. It is easy to visualize that the observed relative distribution for glycolate oxi- dase between the supernate and the peroxisomes may not represent the actual in viva distribution. Thus. the results may be considered quantitative for the amount of total enzyme present in each plant. but only indicative of the presence of peroxisomes. The grouping of plants by peroxisome stability used in the results should not be considered as any type of botanical classification. The results empha- size the great range of total activity for glycolate oxidation and variability in the amount and stability of the peroxisomes. If more plants were surveyed, other groups may become evident. Before any clas- sification can be done on the basis of peroxisomes, it would be necessary to work out isolation proce- dures for maximum yield of active peroxisomes from each plant. The magnitude of this task is suggested by the. fact that procedures to isolate active whole chloroplasts from many different plants is still not available. It is interesting to note that spinach provides a good yield of both chloroplasts and per- oxisomes. At best, our present results indicated the diversity that exists among plants for glycolate oxi- dation. In no case were peroxisomes entirely absent. and it is possible that the enzymes of the glycolate pathway are present in peroxisomes in all plants. Plants such as bean and Swiss chard. which had much total glycolate oxidase, yielded no more peroxi— somes than corn or sugarcane. For bean and Swiss chard, particular effort should be made to improve the isolation procedure for the peroxisomes. The function of the peroxisomes and its relation- ship to photosynthetic efficiency is of interest. These data prove that photosynthesis and photorespiration. as measured by glycolate oxidation, occur in distinct and separate entities in the cell. The data obtained with those plant species that have photorespiration indicates a great diversity in the level of glycolate pathway enzymes (peroxisomes). This enzymatic data indicates that there should be a wide variation in the magnitude of photorespiration among plants. Such has been the case for compensation point analyses. However. any attempt to correlate the compensation point as CO._. production with the amount of glycolate oxidase activity in different leaves would also have to consider the efficiency of CO2 fixation. Although corn and sugarcane leaves do not ex- hibit photorespiration. the presence of 2 to 5 % as much glycolate oxidase. as in sunflower or spinach. is still a substantial amount of activity which could catalyze a rapid flow of carbon through the glycolate pathway. Homogenates of sugarcane leaves oxidized glycolate at the rate of 48 nmoles >< min“1 X g"1 wet weight of tissue or 3 nmoles X min‘1 X mg‘1 protein. Previous results cited in the introduction for glycolate-“C metabolism by com leaves can be accommodated by this level of enzyme activity. The complete absence of photorespiration in corn and sugarcane and the failure to release “CO._. during glycolate-“C metabolism in the light cannot be due to the absence of these enzymes. The absence of photorespiration in these leaves may be accounted for by a combination of a lower rate of glycolate metabolism and the highly efficient CO: fixation process catalyzed by P-enolpyruvate carboxylase (7. R. 17 ). \Ve would interpret our data to indicate that peroxisomes and chloroplasts are separate entities. It seems unlikely that the peroxisomes are in whole chloroplasts and break out of the chloroplasts during centrifugation as do starch grains. In all the plants tested, the ratio of amount of chlorophyll or protein to amount of glycolate oxidase or glyoxylate reduc- tase in the “whole" chloroplast fraction was small compared to the ratio in the “broken" chloroplasts. If the peroxisomes were inside the chloroplasts. this ratio ought to be largest for the whole chloroplasts. However. a large part of the starch grains from inside the chloroplasts are pelleted by themselves. even by centrifugation at a low gravitation field to remove whole chloroplasts. Thus, the more dense bodies readily escape from chloroplasts during iso- lation. and the peroxisomes become much denser than the chloroplasts in a sucrose solution. In previous discussions about the enzymes of the glycolate pathway (15,18), catalase usually was not included because of the assumption that the universal presence of catalase would easily dispense with H303 produced by glycolate oxidase. In this survey. a similar distribution pattern between peroxisomes and supernate for all the plants was found for glycolate. oxidase. glyoxylate reductase, and catalase. This data emphasizes that these 3 enzymes appear to be closely associated in the same organelle. The possr bility of several catalases in the cell is not excluded. but little catalase activity was found with the mito- chondria or chloroplasts from sunflower or spinach leaves. Soluble cytoplasmic catalase may also not exist because, in plants with the most stable peroxr somes. catalase activity in the supernate of the original leaf homogenate was no greater on a percent basis than glycolate oxidase. Literature Cited 1, m; Dun-2. C. AND P. BAUDHUIN. 1966. Peroxi- somes (microbodies and related particles). Pb)" siol. Rev. 46: 323—57. 2. DOWNTON, W. J. 5. AND E. B. TREGUNNA. 1963» Photorespiration and glyoxylate metabolism: A re-examination and correlation of some previous studies. Plant Physiol. 43: 923—29, TOLB ER '1‘ ET A I. .-—-I.F. .\ I" EL-SHARKAWY, M. A., R. S. Looms, AND W. A. WILLIAMS. 1967. Apparent reassimilation Of res- piratory carbon dioxide by different species. Phy- siol. Plantarum 20: 171—86. FORRESTER, M. L., G. KRO’I‘KOV, AND C. D. NELSON. 1966. Effect Of oxygen in photosynthesis, photO~ respiration, and respiration in detached leaves. I. Soybean. Plant Physiol. 41: 422—27. FORRESTER, M. L., G. KROTKOV, AND C. D. NELSON. 1966. Effect of oxygen on photosynthesis, photo- respiration and respiration in detached leaves. II. Corn and other monocotyledons. Plant Physiol. 41: 428—31. GOLDSWORTHY. A. 1966. Experiments on the origin Of CO2 released by tobacco leaf segments in the light. Phytochemistry 5: 1013—19. HATCH, M. D. AND C. R. SLACK. 1966. Photo- synthesis by sugar cane leaves. A new carboxy- lation reaction and the pathway Of sugar formation. Biochem. J. 101: 93—111. HATCH, M. D., C. R. SLACK, AND H. S. JOHNSON. 1967. Further studies on a new pathway Of photo- synthetic carbon dioxide fixation in sugar cane and its occurrence in other plant species. Biochem. J. 102: 417—22. JOLurFE, P. A. AND E. B. TREGUNNA. 1968. Ef- fect Of temperature, CO2 concentration, and light intensity on oxygen inhibition Of photosynthesis in wheat leaves. Plant Physiol. 43: 902—06. NOLL, C. R., JR. AND R. H. BURRIS. 1953. Na- ture and distribution Of glycolic acid oxidase in plants. Plant Physiol. 29: 261—65. Moss, D. N. 1966. Respiration Of leaves in light and darkness. Crop Science 6: 351-54. MOSS, D. N. 1967. High activity of the glycolic acid oxidase system in tobacco leaves. Plant Physiol. 42: 1463—64. MOSS, D. N. 1968. Photorespiration and glycolate met;bolism in tobacco leaves. Crop Science 8: 71- 6, PIERPONT, W. S. 1962. Mitochondrial preparations from the leaves Of tobacco. IV. Separation of some components by density gradient centrifugation. Biochem. J. 82: 143—48. RABSON, R, N. E. TOLBERT, AND P. C. KEARNEY. 1962. Formation of serine and glyceric acid by the glycolate pathway. Arch. Biochem. Biophys. 98: 154—63. l6. 17. 18. 19. 23. 24. 25. 26. 27. 1'i«:Rox1s0MI-:s 147 ROCHA, V.. S. K. MURDRJI. AND I, P. TING. 1968. Chloroplast—malic dehydrogenase: A new malic de— hydrogenase isozyme from spinach. Biochem. Biophys. Res. Commun. 6: 890—94. SLACK, C. R. AND M. D. HATCH. 1967. Compara— tive studies in the activity of carboxylases and other enzymes in relation to the new pathway of photosynthetic carbon dioxide fixation in tropical grasses. Biochem. J. 103: 660—65. TOLBERT, N. E 1963. Glycolate pathway. In pho- tosynthetic mechanisms in green plants. NSF— NRC publication 1145. p 648-62. TOLBERT, N. E. AND R. H. BURRis. 1950. Light activation Of the plant enzyme which oxidizes gly- colic acid. J. Biol. Chem. 186: 791—804. TOLBERT, N. E. AND M, S. COHEN. 1953. Activa- tion Of glycolic acid oxidase in plants. J. Biol. Chem. 204: 639—48. TOLBl-ZRT, N. E., A. OESER, T. KISAKI, R. H. HACE- MAN, AND R. K. YAMAzAKI. 1968. Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. Biol. Chem. 243: 5179—84. TREGUNNA, E. B. 1966. Flavin mononucleotide control of glycolic acid oxidase and photorespira- tion in corn leaves. Science 151: 1239—41. TREGUNNA, E. B., G. KRO’I‘KOV, AND C. D. NELSON. 1966. Effect of oxygen on the rate of photores— piration in detached tobacco leaves. Physiol. Plan- tarum 19: 723—33. TREGUNNA, E. B. AND J. DOWNTON. 1967. Carbon dioxide compensation in members of the Antar- anthaceae and some related families. Can. J. Botany 45: 2385—87. ZELITCH, I. 1955. The isolation and action Of crys- talline glyoxylic acid reductase from tobacco leaves. J. Biol. Chem. 216: 553—75. ZELITCH, I. 1958. The role Of glycolic acid oxi- dase in the respiration of leaves. J. Biol. Chem. 233: 1299-1303. ZELITCH, I. 1966. Increased rate of net photo- synthetic carbon dioxide uptake caused by the inhi- bition Of glycolate oxidase. Plant Physiol. 41: 1623—31. ZELITCH, I. AND S. OCHOA. 1953. Oxidation and reduction Of glycolic and glyoxylic acids in plants. I. Glycolic acid oxidase. J. Biol. Chem. 201: 707— 18. '5- Tm; J()1'R.\'.\L or lliotoouxu. ("nuns-Tar Vol. 213, No. 19, Issue of October 10, pp. 517!) 5184, 1008 Printed in U.S.A. Peroxisomes from Spinach Leaves Containing Enzymes Related to Glycolate Metabolism* (Received for publication, February 20, 1908) N. E. TOLBERT,I A. ()Esiza, T. KisAKi, R. H. HAGEMAN, AND R. K. YAMAZAKI From the Departim'nt of Biochemistry, .llz'chz'gan State. ('nircrsity, East Lansing, .lliclu'gan 48823 SUM MARY Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from spinach leaves. After grinding leaves in 0.5 M sucrose, the peroxisomes were removed with the broken chlorOplast fraction by difierential centrifugation. During sucrose density gradient centrifuga- tion, the peroxisomes banded in about 1.9 M sucrose and were separated from mitochondria and chlorOplasts. The particles, 0.5 to 1.0 a in diameter, contained a dense granular stroma surrounded by a single membrane. The leaf peroxisomes contained glycolate oxidase, DPNH- glyoxylate reductase, and catalase. Up to 55 % of the activity for these enzymes in spinach leaves have been found in the particulate fractions after the initial centrifugation. The leaf peroxisomes are probably the site of oxygen uptake during photorespiration. No catalase activity was present in chloroplasts after removal of the peroxisomes by density gradient centrifugation. P-Glycolate phosphatase, TPNH- glyoxylate reductase, D-amino acid oxidase, urate oxidase, and peroxidase were not present in leaf peroxisomes. (.llycolate oxidase was first isolated from clarified extracts of tobacco leaves (1, 2). Subsequent investigatitms (3, 4) sup— ported the concept that the oxidase, as well as DPNH-glyoxylate reductase (5) were soluble cytoplasmic enzymes. (‘hloroplast (6, 7) and mitochondrial fractions (8, 9) have been reported to mntain only a small portion of the total glycolate oxidase ac- tivity. Upon examination by sucrose gradient centrifugation, Pierpoint (10) and, most recently, 'l‘hompson and Whittingham (11) have concluded that there was no glycolate oxidase in the particulate fractions. A reinvestigation of the intracellular localization of glycolate oxidase in leaves was prompted by our own observations of large amounts of glycolate oxidase in broken chloroplast. prepz‘irations. " This research was supported in part by (il'alll (:13 4151 from the National Science Foundation. This is journal article 4307 of the Michigan Agricultural Experiment Station. Parts of this work were abstracted in the Fed. Proc.. 27, SH (1908), I To whom all inquiries should be addressed. De I)uve and Baudhuin (12) have designated the li‘llt'l‘t)l)()(ll0S from liver and kidney, which contain a-hydroxy acid oxidase and catalase, as peroxisomes. The metabolic function of per— oxisomes is unknown. Particles of unknown composition have been reported in plants and designated as cytosomes (13), but there are no reports of particles from leaves with enzymic activity characteristic of peroxisomes. Particles containing enzymes of the glyoxylate cycle have been isolated from germinating 'aster bean cotyledons (14) and from Tetralzg/me'na (15). A similarity between these microbodies and peroxisomes is suggested by the fact that microbodies from Tetrahymena contain enzymes which are also characteristic of liver and kidney [wroxisomes (16). 1'2 X I’l‘LRIM l-ZN TA L PROCEDURE Preparation of Fractions——Spinach (Spinacia. oleracea L.) leaves were purchased locally. Leaves have been stored at 4° for up to 1 month before use. The chilled leaves were washed and deribbed, and then 40 g of tissue were chopped into small segments before grinding at maximum speed for 10 sec in a Waring Blendor with 80 ml of grinding medium. All work was done at about 4°. The standard grinding medium was 0.5 M sucrose in 0.02 M glycylglycine, pH 7.5. The homogenate was hand squeezed through six layers of cheeseclmh, and the pH of the sap immediately readjusted to 7.5. Particles from 70 ml of this sap were then precipitated by differential centrifugation at. 0° for 20 min at each step. The first pellet (120 X 9) con- tained mostly whole chloroplasts, starch grains, and large cell fragments. The second pellet (3,000 X g) was designated as “broken chloroplasts,” although it also contained some mito- chondria and a large part of the peroxismnes. The third pellet- (35,000 X 9), designated as “nnitochondria" contained the re- maining microbodies and broken chloroplasts. The remaining solution was designated “supernatant.” Each pellet was re- suspended in grinding medium by stirring, and the final volume of about 4 ml for the first and second pellet and about ‘2 ml for the third pellet was recorded. Sucrose Density (gradient Ccnlrzl/‘ugation—A ll(_)ll(‘()llllllll()llS sucrose density gradient. of five layers was preparet‘l at. 4° by pipetting 4 ml of 2.5 M sucrose (85.5%), 10 ml of 2.0 M sucrose (68.-1(1), 10 ml of 1.8 M sucrose (til.6‘},7), 10 ml of 1.5 M sucrose (431.3“), and '20 ml of 1.3 M sucrose (44.5%). All sucrose fractions contained 0.02 M glycylglyciue at pH 7.3. After 179 5180 TABLE I Sucroae density gradient centrifugation Experimentally, fractions were numbered as removed from the bottom of the gradient. Fraction Sucrose Volume Type of particles in band M ml 9 0.5 4.3 Supernatant 8 1.3 8.6 Broken chlt.)roplasts 7 1.371 .5 11.9 Broken chloroplasts 6 1.5 5.4 None 5 1.5—1.8 3.8 Whole chloroplasts; mitochondria 4 1.8 6.4 None 3 1 .8-2. 0 4.0 Peroxisomes 2 2.0 7.6 None 1 2.5 3.6 None layering 2.0 to 2.5 ml of the appropriate pellet fraction on top of the gradient, the samples were centrifuged for 3 hours at 4° at 25,000 rpm (44,700 X g to 106,900 X g) in a swinging bucket rotor SW 25.2 of the Spinco centrifuge, model L. Samples of 3 to 12 ml were removed from the bottom of the centrifuge tube through a needle. The approximate leiation of the various particles after development of the gradient. are illustrated in Table 1. Electron .1]icrosc0py—-'I‘he peak fraction containing the per- oxisomes was withdrawn from the gradient and fixed for 1 hour in 30’: glutaraldehyde. The sucrose concentration was then lowered to l M and the particles were pelleted. The sediment was rinsed in 0.5 M sucrose buffered at pH 7.5 with 10 mM N-2— hydroxyethylpil)erazine—N’-2-ethanesulfonic acid, and then postfixed for 2 hours with 1% Os(.).. All fixation steps were carried out in the cold. The pellet was then dehydrated in a gradei senes- of ethanol and embedded in Epon. Sections were stained with uranyl acetate and lead citrate. Glycolate Oxidase—(llycolatc-O, oxidoreductase. (EC 1.1.3.1) was assayed anaerobically by 2,6—dichloroindophenol reduction (4). Additions were made to a 3-ml Thunberg Beckman cuvette (10 mm in diameter) in the following order: 2 ml of 0.3 M pyro- phosphate, pH 8.3, containing 1.5 X 10“| M dichloroindo})henol; 0.05 ml of 0.1 M KCN in 0.01 M NH40H (final concentration of KCN, 2 X 10"" M), at times 0.1 ml of 2 X 10‘3 M FMN (final concentration, 0.8 X 10'4 M); water so that the final volume with enzyme would be 2.5 ml; in the side arm, 0.] ml of 0.125 M sodium glycolate (filial concentration, 5 X 10'“ M). Between 0.05 to 0.2 ml of enzyme was also placed in the cuvette before it. was evacuated and flushed three times with N3 which had passed through Fieser's solution to remove traces of ()2. Dye reduction at 25‘ was measured at 000 my. by an automatic recording Gilford spectrophotometer. A unit of activity was cxpressm’l as 1 0.1,). change per min, which was equivalent to 4.78 nmoles of reduced dye. l’yropliospliate butter with dichlortiindophcnol should be prepared fresh weekly to avoid an initial lag in the assay. Since the soluble glycolate oxidase is a FMN protein. l".\l.\v "was used in the assays. Aerobic assays were unreliable since 1‘ 0 ll“; L’cncratcd by the glycolate oxidase could be used by t‘ltlt"‘iniu.l‘3!t1 pct‘oxidascs to oxidize any reduced dichloro- indoplw'wl which was generated. (‘onipeting reactions then occu'wd' 't'trt) production. dichloroindt:phcnol reduction, and permit! ... it‘."l"ll’tll ot' the rctlitctd :lvc. Inhibition of petitxh . dase ! I l g“ i'ont't'tilt‘atiot s of l\( ‘N, as originallj.‘ i'ccotizn‘crnletl Peroxisomes in Lea vcs Vol. 243, No. 19 (4) for an aerobic assay, did not give maximum rates. Per— oxidases were so active in sap and supernatant that no aerobic reduction of dichloroindophenol could be detected if KCN were omitted. Consequently, anaerobic assays were the only reli- able method, and KCN was left in the assay to ensure against peroxidase activity in case of incomplete anacrobiosis. Cytochrome c Oxidase—From 5 to 25 ul of enzyme was pipetted into a bottom comer of a 0.3-ml spectrophotometer cuvette (diameter, 10 mm) and 5 pl of 4.0% digitonin were added, mixed, and allowed to stand for 1 min. Then 200 pl of 0.1 M phosphate buffer (pH 7.0) and 50 pl of 1.5 mm cytochrome c reduced with dithionite were added successively and mixed (17). Readings of optical density at 550 mg were obtained with the Gilford re- cording spectrophotometer. The first order rate constant for the disappearance of reduced cytochrome c was calculated ac- cording to the method of Smith (18). Other Enzymes—Glyoxylate reductases were assayed by the rate of oxidation of either DPNH or TPNH in the presence of 0.005 M substrate (19, 20). Catalase was assayed by the dis- appearance of H202 as measured spectrophotometrically at 240 my (21). A unit of activity was a change of 1 0.1). in 1 min, and based on the extinction coefficient was equal to 2.76 nmoles of 11202. Peroxidase was assayed by the method of Gregory which was based upon the length of time to oxidize a standard amount of ascorbic acid (22). The assay for P-glycolate phos- phatase has been described (23). Protein and Chlorophyll—Protein was determined by the. Lowry procedure. Although the green color in aliquots from the chloroplast fractions interfered, the recovery of protein in the various fractions indicated that the method gave a valid estimate of protein content. Chlorophyll was determined by its absorption at 652 mp. (24). Aliquots from 1 pl to 1 ml were diluted to 5 ml with water and acetone to make a final concen— tration of 809;, acetone. They were allowed to stand in the dark at 0° with occasional stirring for several hours to com— pletely solubilize the chlorophyll. In samples with high sucrose concentration, the acetone extracted so much of the water that the sucrose and chlorophyll formed a second phase. Since sucrose is more soluble in cold acetone, it was necessary to let these samples stand overnight at — 18° in order to obtain efi'ective chlorophyll extraction. Samples were centrifuged or filtered before reading the extinction at room temperatures. RESULTS Distribution of Glycolate Oxidase among Particles—Spinach leaves were ground in the sucrose grinding medium and separated by centrifugation into whole chloroplasts, broken chloroplasts, mitochondria, and supernatant fractions as described under “Experimental Procedure.” The distribution of certain en— zymes among these fractions is shown in Table II. In this type of experiment, the broken chloroplast. fraction contained 29 to 34"; of the total oxidase activity and had the highest specific activity. About 40 to 55‘}. of all the oxidase, activity was in the total particle fractions. Most of our investigations have been done with the broken chloroplast fraction, because glycolate oxidase from spinach leaves was greatest in this fraction on the basis of amount and specific activity. Removal of the whole chloroplast fraction was beneficial for reducing the load put on the subsequent sucrose gradient. The broken chloroplast and mitochondrial fractions were >lllijet‘1ctl separately to sucrose density gradient centrifugation Issue of October 10, 1968 Talbert, Oeser, K isaki, Hageman, and Yamazaki 5181 TABLE II Distribution of enzymes among fractions oblained by Ilifl'erenlial cenlrt'fugation Data, from a typical experiment, are designated as Experiment A. The percentage of distribution from a second experiment, B, is shown to illustrate the maximum activitv found In the pellet fractions Specific activin is expressed as micromoles per min per mg of protein. Glycolate oxidase ‘ Catalase DPNH-glyoxylate reductase name" 19:81 Distribution 1 l l Distribution p o em Spctific ' ' Specific Distri- activity Expcrr Experi- activity Experi- Experi— atlivily bution m nt A ment B ment A ment B mg humus/mi; ‘7} ‘I'r amulet/min ‘1 'er $127515! 5 9} “'hole chloroplasts. . H. 360 5.5 0.015 5.5 5.6 34,500 96 5.9 6.6 7.7 0.023 6.8 Broken chloroplasts. . .. 618 28.9 0.047 28.9 34.5 184,800 299 31.3 30.7 17.6 0.033 15.3 blitochondria ........ 208 5.2 0.025 5. 2 15.0 29,700 143 5.0 15.5 2.6 0.022 2. 2 Supernatant , .. ...... l 2,730 60.5 0.022 60.4 44.9 341,000 125 57.8 47.3 86.4 0.026 75.7 TABLE III Distribution of enzymes upon sucrose density gradienl cenlrl'fugalion of broken chloroplast fraclion Fractions were numbered in the order in which they were drained from the bottom of the centrifuge tube. For approximate sucrose molarity see Fig. 1. bottom and 9 is the top supernatant. Fraction 1 was in the The broken chloroplasts were from Experiment A of Table 11. Specific activity is expressed as micromoles per min per mg of protein. Glycolate oxidase Catalase DPNH-glyoxylate reductase Fraction Total protein Specific activity Specific activity Specific activity "18 umoltr/min 9; ”mole: "nII'u C}. “malts/min ”1 9 247.2 3.39 0.014 23.9 27,200 110 24.4 3.7-1 0.015 21.0 8 109.0 0.77 0.007 5.4 5,310 49 4.8 0.83 0.008 4.6 7 35.8 0.47 0.013 3 3 3,680 102 3.3 1.05 0.029 5.9 0 14.5 0.31 0.021 2.2 1,570 107 1.4 0.43 0.029 2.3 5 22.5 0.78 0.035 5.5 43 184 3.7 0.81 0.036 4.7 4 9.6 1.80 0.190 12.7 11,450 1,190 10.3 2.48 0.259 13.9 3 5.5 5.07 0.920 35.8 37,400 6,783 33.6 6.57 1.191 36.8 2 , 4 1 1.53 0.370 10.8 19,300 4,689 17.3 1.96 0.478 11.0 1 i 2 0 0.07 0.030 0.5 130 68 1.2 0 0 0 (Table III and Fig. 1). The glycolate oxidase activity was found in a band (No. 3) sedimenting in about 1.9 M sucrose. Activity, also present at the top of the gradient (No. 9), attributed to soluble protein from the supernatant and from broken particles. The. specific activity of the oxidase in the particles was about 92~fold greater than the specific activity in the top fraction. The oxidase activity in the 1.9 M sucrose band was distinc tl_\ separated rom both the chloroplast bands, as indicated bv chloroph\ll analv ses, and from the mitothonrhial fraction, as indicated by cytochrome c oxidase :Icti\it_\ (Fig. l). The particulate fraction containing the plant glycolate oxidase has been designated as peroxisomes alter the terminology of De Duve and Baudhuin (12). These plant wroxisomes scdimcntcd similarly in the sucrose gradient and contained similar types of enzymes as those found in peroxisomes from liver or kidncy. The a—hyt'lroxv acid oxidase activity of the, particles has been designated glycolate oxidase because it was Inorc active with glycolatc than lactate or r -hy(lrt)xybIItyrate. With sutlicicnt substrate for maximum activity. the relative rates of glycolate, lactate, and a-hydroxybutyrate oxidation at pll 8.3 by thc peroxisomes Were about 10030530. Since this activity ratio with the three substrates was the same in c:tclI fraction from thc sucrose gradient, it is assumed that their oxidat ion \\'.‘t.\ catalyzml by the same enzyme. This ratio of activity is similar to that “113‘ reported earlier for the soluble spinach glycolate oxidase (25), but the oxidase from tobacco leaves is much more specific for glycolate than for a-hydroxybutyrate (1). Peroxisomes from rat liver have a similar specificity for glycolate, the shortest carbon chain of the a-hydroxy acid series, while ix‘roxisomcs from rat kidney contained an a-hydroxy acid oxidase which oxidizes the longer chain substrates (ashydroxylnItyratc) faster than glycolate (12). The reason for these substrate difiercnccs is not apparent. The prosthetic group of glycolate oxidase, as prepared from soluble extracts of spinach leaves, is 1°th (3, 4). Addition of excess (10" M) FMN gencrally increases the activity of glycolate oxidase in crude extracts or a fraction precipitated by ammonium sultatc. Similarly, the oxidation of glycolate by iwroxisomcs generally was stimulated as much as 50‘ ,7 by added FMN. ('atnlustt‘atalasc activity had nearly the identical distri— bution among’ the particulate fractions as (lid glycolate oxidase (Table II and Fig. 1). About 40 to .30“; of the catalase activity was in thc particulate fractions. ['pon subsequent sucrose density gradicnt centrifugation of the broken chloroplast or about 70"; of the activity in these fruc- Thc presence of mitochondria traction, tions \vas prcscnt in thc twroxisomc particles. glycolate oxidasc and catalase together in leaf peroxisomes is consistent with data on liver peroxisomes and with the concept 5182 40— Glycolote Oxidase 20~ 40-0-5' t4 ' l5 ‘ l6 ' l.8 r 2.0 ‘2.5‘ Catalase [—— 20 - 4o— DPNH Glyoxylate Reductase 20 > | .t I I I T I l i '2 05 M I5 |.6 l8 2.0 2,5 g— 0 60r- il‘lce tile ‘nafk (If the peroxisomes was removed initially from the sap 3:, ll!" broken chloroplast fraction, previous reports on Peroxisomes in Leaves Vol. 243, No. 19 chloroplast 'atalase could be due to peroxisome particles in the chloroplast preparations. If so, the unknown role for catalase in the photosynthetic apparatus need not be of concern. While isolating plant mitochondria by sucrose density gradient. cen— trifugation, I’lesnicar, Bonner, and Storey observed, but did not comment on, catalase activity at a location on the gradient, which would be characteristic of peroxisomes (see Table III of Reference 26). DPNH-Glyoxylate Reductase—This enzyme was located in the peroxisomes similarly to glycolate oxidase and catalase (Tables II, III, and Fig. 1). 0n the basis of total or specific activity in the peroxisomes (Table III), glyoxylate reductase activity 'as the same order of magnitude as glycolate oxidase. Glyoxylate reductase has not been reported to be a constituent of liver peroxisomes (12), and Zelitch, when isolating this enzyme from plants, believed that it was a cytoplasmic component (5). Enzymes Not Detected in Peroxismnes—The leaf peroxisomes were tested for other enzymes associated with the glycolate pathway (27), or with liver mroxisomes (l2). n-Amino acid oxidase and urate oxidase, which have been found in liver peroxisomes, were absent in leaf peroxisomes. P-Glycolate phosphatase and TPNH-glyoxylate reductase were absent. in the peroxisomes, but some activity for each was associattxl with the whole chloroplasts. The activity of P-glycolate phosphatase was found on the sucrose gradient in the same area as cytochrome c oxidase. This area contained the mitochondria as well as a small band of whole chloroplasts. The broken chloroplast bands containing the bulk of the chlorophyll did not contain P—glycolate phosphatase. Peroxidases are extremely active and abundant in leaves. Both chloroplast and mitochondria fractions contained some peroxidase activity; however, the bulk of this activity was found in the supernatant. When the particulate fractions were further separated on sucrose gradient, no peroxidase activity by the ascorbate assay was. detected in the peroxisome fraction. The catalase activity of the peroxisomes did not show peroxidase activity by the assay employed. Recently, Plesnicar et nl. (26) with the use of sucrose gradient centrifugation, also con- cluded that peroxidase from mung bean hypocotyls was mostly soluble, although some activity was apparently located in microbodies. Perorismne Morphology—An electron micrograph of spinach l¥tlf peroxisomes is shown in Fig. 2. The particles are charac- terized as containing a dense granular stroma surrounded by a single membrane. In some cases, a denser area was visible within the particles. The shape of the peroxisomes varied. but most often they appeared spherical and about 0.5 to 1.0 M in diameter. The fact that the membranes of the particles were often broken is attributed to shearing forces as they passed through the discontinuous sucrose gradient. Thus, it was not surprising that a portion of the enzymic activity attributed to the peroxisomes was found at. the top of the gradients. The pl't“|‘):‘tl‘:tti()tt also contained chromatin material, broken chloro- plasts, and other particles, presumably mitochont‘lria. The amount of chlorophyll and cytochrome c oxidase (Fig. l) paral- lels these visual observations. Grinding .llcdi-mn and Particle Stability—T he grinding mcdnmi of 0.5 M sucrose in 0.02 M glycylglycine at pH 7.5 has been changed. but so far no variation has altered the primary cell» clusions or improved significantly the yield of ix‘roxisonzcs. Without sucrose to prevent osmotic shock, much more of the n.— Issue of October 10, 1968 glycolate oxidase activity was in the supernatant fraction, sug gesting that the particles were labile. This also provides an explanation for previous literature citations that glycolate oxidase was a soluble enzyme. Substitution of NaCl or inannitol in the grinding medium for sucrose resulted in poorer recovery of peroxisomes, and shifted their location on the sucrose gradient to 1.8 M sucrose as if they were somewhat lighter in weight. Substitution of (‘arbowax 4000 for sucrose or addition of poly- vinylpyrrolidone with sucrose was not beneficial. The final concentration of sucrose in the leaf homogenate after grinding was lowered by the dilution from the leaf sap, and an unknown amount of peroxisome destruction may have Occurred. Best results were obtained when the particles were removed as rapidly as possible by centrifugation and further separated on the sucrose gradient. It the leaf homogenate or the resuspended broken chloroplasts stood at 0° for several hours before the sucrose gradient centrifugation, :1 large part of the enzyme activity was found in the supematant or top fraction. Stability of the particles was estimated by resuspending them in media with different concentrations of sucrose. and then removing them again at different time periods (Fig. 3). If resuspended in buffer without sucrose, 60‘} of the activity was immediately found in the supernatant, and it is assumed that the osmotic shock ruptured the particles. When resuspended in 0.5 M sucrose, the peroxisomes appear stable for 2 hours and in 1.0 M sucr0se, most of the glycolate oxidase activity remained with the microbody pellet for about 8 hours. Enzyme activity lost from the pellet fraction was found in the supernatant or suspending medium. We do not understand why a constant level of about 409'; of the glycolate oxidase activity remained in the microbody fraction even after 21 hours in buffer without sucrose. Mitochondria Fraction—The designation, mitochondria. or broken chloroplasts fraction, was arbitrary, as the initial cenv trifuual technique did not clearly separate these fractions. In the examples shown in Table II, 5 to 15% of the total enzymic FIG. 2. Electron micrograph of a peroxisome—rich fraction. X 26, Talbert, Oeser, K isalri, Hageman, and Yamazaki 5183 !———“3\ 70— 60— 50- . - ‘. 401A e 30- % Glycolate Oxidase in Particles on i l i I l 2 4 8 IS 20 Hours after Preparation of Particles Fm. 3. Stability of peroxisomes in grinding media of different sucrose concentration. Equal aliquots of the chloroplast fraction were sedimented by centrifugation from sap prepared in the standard grinding medium of 0.5 M sucrose and buffer. The broken chloroplast fractions were resuspended and stored in difierent grinding media which contained 0, 0.5, and 1.0 M sucrose and buffer. At designated times, aliquots of these suspensions were recentrifuged at 39,000 X g for 20 min before measuring glycolate oxidase activity in the pellet or peroxisomes and in the soluble supernatant grinding media. For assay purposes, the particles were resuspended in the corresponding grinding medium in which they had been stored. Both the supernatant and the resuspended microbodies were assayed for glycolate oxidase with added FMX and KCN. There was little decrease in total activity during the experiment; decrease in activity in the microbody fraction was balanced by a corresponding increase in activity of the supernatant. activity associated with the peroxisome was initially separated with the mitochondria fraction. The mitochondria fraction also contained broken chloroplasts (about 20% of the total chloro- phyll). The fraction designated as broken chloroplast also contained cytochrome c oxidase activity, which is attributed to mitochondria. Thus, the initial separation of the particles into fractions, designated as whole chloroplasts, broken chloroplasts, and mitochondria, as has generally been (lone, is arbitrary and for convenience. When using sucrose gradient centrifugation, thc initial particle separation would not be necessary except to prevent overhauling of the gradient. When the mitochondria fraction was sedimented in the sucrose gradient, the peroxisomes also handed in 1.9 M sucrose as they did from the broken chloroplast fraction. There was no glyco— late oxidase, catalase, or DPNH-glyoxylate reductase in the 1.5 M sucrose region of the gradient where mitochondria were located, as indicated by cytochrome c oxidase activity. Previous re- ports: have indicated that a particulate fraction typical of plant mitochomlria contained part of the leaf glycolate oxidase ac- tivity (8, 28). However, the investigators were unable to couple glycolate oxidation to phosiihorylation. The presence of peroxisomes in these mitochondria preparations undoubtedly accounted for the glycolate oxidase activity. msCI‘sslox The significance and function of peroxisomes in liver and kidney are unknown, but have been carefully considered by De Dave and liaudhuin (12). One possibility has been that. these microbodies function as a site to dispose of cell excesses by the 5184 combined action of oxidases and catalase. This description 111 1'1eroxisomes from spinach leaves establishes a wider distribution for these microbodies. A current survey indicated that per— oxisomes are present in many different plants, although spinach has so far yielded the highest percentage of the glycolate oxidase activity in peroxisomes.1 In considering the function of leaf peroxisomes, two facets of the photosynthetic system provide clues which could not. be studied with the liver and kidney. Glycolate is a major and universal product of (‘02 photosynthesis (for review see. Refer— ence 27). In the light, many leaves have an enhanced rate of respiration, which is called photorespiration and is attributed to glycolate oxidation (29—32). Thus, in contrast. to the liver and kidney, in the leaf a major substrate for the peroxisomes is known. Further, in the leaf, photorespiration may be attributable to the peroxisomes, and photorespiration can be differentiated in viva from mitochondria res[.1iration. The present state of knowledge does not assign a clear function to the glycolate pathway and the peroxisomes. A working hypothesis is that during (‘02 photo- synthesis, P-glycolate is synthesized by the chloroplasts and excreted as glycolate (27) by the aid of the specific P-glycolate phosphatase (25). Excess glycolate would be oxidized by the peroxisomes and account for photorespiration. When glycolateJ‘C has been fed to leaves, it is rapidly c1111- verted into sugars via glycine, serine, and glyceric acid, i.e. via the glycolate pathway (33). The glycolate-”C was not con- verted into C02 and refixed photosyntheti 'ally. If all the initial oxidation of added glycolate-“C were occurring in the peroxi- somes, then the peroxisome function is not to destroy the glyco- late by complete oxidation to C02. H202 production from glycolate oxidase. has been considered as a potential source of H202 for peroxidase metabolism. However, the absence of ix’roxidases in the peroxisomes suggest that gly- colate oxidase and the peroxisomes do not function for synthesis or metabolism via peroxidases. The presence of 7000-fold more catalase activity in the peroxisomes than glycolate oxidase ac- tivity indicates that the H202 produced there is not used. Zelitch has emphasized that the glycolate system could serve as a terminal oxidase for any pyridine nucleotide-linked dehy- drogenase (5). Glyoxylate reductase would 'atalyze the oxida- tion of DPNH and the resynthesis of glycolate, which, in turn, would be reoxidized by oxygen as 'atalyze1‘l by glycolate oxidase. The packaging of the DPNH-specific glyoxylate reductase in the peroxisomes, along with glycolate oxidase and catalase, is certainly convincing evidence that such a terminal oxidase sys- tem could function to dispose 11f excess DPNH. Oxidation of excess DPNH, then, is a possible function for peroxisomes. In this paper, glycolate oxidase, glyoxylate reductase, and catalase are designatml as constituents 111 the peroxisomes. Pre- these enzymes have been considered to be associated This error vionsly, with the soluble fraction of the leaf homogenate. seems to have been caused by the fragility of the peroxisomes. Investigators isolating enzymes from leaves did not grind in 11.5 .11 sncro e (ttt'l even if they did so, when isolating chloroplasts, the 111‘111‘;is11111‘*s\\1'1‘(‘ stable 1111 only :1 1e \\ hours and we re broke 11 if tlw ""'l"|>!l.\ \11-1e diluted l~~ 5. “ 3 1s been shown that .11)‘; of the total glyc 11l:1te oxidase i1 l1‘:1\'1-s can be isolated in particulate fractions. We do uhethcr the other half of the oxidase activity in the '11 represe111s :1 soluble glycolate oxidase or whether it 15y use of t1ch11i1ptes desc1il1e1l vZebu-1111:1111. I’eroxison1cs in Leaves Vol. 243, No. 1‘.) arose from fragmentation of the labile peroxismnes during their isolation. Pierpoint (10) and Thompson and “'hittingham (11), with the use of sucrose density gradient centrifugation to separate parti- cles, concluded that glycolate oxidase activity was only in the soluble fraction from tobacco leaves. Pierpoint’s data indicate that there was activity which moved into the sucrose gradient, but it was ignored. Thompson and VVhittingham probably did not detect the peroxisomes because they ground the leaves in too dilute a solution of NaC‘l or sucrose and used only the whole chloroplast fraction. 011 the basis of the present work, glyco- late oxidase can be found 111 peroxisome particles when a simple isolation procedure 1s followed. .11cknowledgments——Apt1reciation is exp1essed to G. Longo, (,‘. Longo, and M. Jost of the Michigan State 1"Diversity-Atoniic Energy Commission Plant Research Laboratory (ABC (‘ontract AT (ll-1)-1338) for the preparation and interpretation 111 the electron micrographs. $201-$- bib—1 NOCCWKI 1‘2. l3. 15. 16. . SimoN, E. W., Biochem. J., 69, 67 (1958). . SMITH, Z., 11Ielh11d.fl1'ochem. . ZELI'I‘CH, 1., J. Biol. Chem., . ZELI'I‘CH, 1., AND (lorro, A. M., Biochem. J., 84, 541 (1111321 . LUCK, 11. 29. .M’ an. 31). in [Q .. .. «Vs/v . 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