x'l."l|l Uiha.b I..""‘:T::&.‘:‘:Y RE an: :; umte This is to certify that the dissertation entitled LOCALIZATIONOF ENZYMES 0F PURINE DEGRADATION IN PLANTS AND ANIMALS presented by JOANNA FRANC ES HAN KS has been accepted towards fulfillment of the requirements for flD—demmEM 72.5% Major professor Date MW 19/78; MS U is an Afl'lrnnm've Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES —;—_ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. LOCALIZATION OF ENZYMES OF PURINE DEGRADATION IN PLANTS AND ANIMALS By Joanna Frances Hanks A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1982 ABSTRACT LOCALIZATION OF ENZYMES OF PURINE DEGRADATION IN PLANTS AND ANIMALS by Joanna Frances Hanks Compartmentation plays an essential roLe in purine degradation in both animals and leguminous plants such as soybean (Glycine max L. Merr.). Nitrogen fixed by symbiotic bacteria within the root nodules of these plants is transported to the green shoot of the plant in the form of allantoin and allantoic acid. These compounds are intermediates in purine degradation. Two enzymes required for purine degradation, uricase and catalase, were localized within peroxisomes isolated from soybean nodules by iso- pycnic sucrose density gradient centrifugation. Xanthine dehydrogenase was found in the soluble fraction and allantoinase activity was associ- ated with the endoplasmic reticulum. The purine synthesis pathway has been localized in the plastid fraction from soybean nodules. The metabolic conversion of symbiotically fixed nitrogen into allantoin and allantoic acid is not only compartmented within several intracellular organelles, but is also divided between two different cell types. Uninfected cells from nodules were separated from cells infected with bacteria on a sucrose step-gradient. The peroxisomal enzymes, uricase and catalase, were associated only with the uninfected cell fraction from soybean nodules. Allantoinase also had a greater specific activity in the uninfected cell fraction, as did several enzymes whose products are required for purine synthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphog1uconate dehydro- genase, and glucose-6-phosphate dehydrogenase. Although nitrogen fixa- tion occurs only in the infected cells of the soybean nodule, these data indicate that at least the final reactions occur in the peroxisomes and endoplasmic reticulum of the uninfected cells. Further degradation of allantoin and allantoic acid must take place in soybean leaves where nitrogen is needed for growth of the shoot. Allantoinase from soybean leaves and germinating seedlings was localized in the endoplasmic reticulum. No allantoicase activity was found in soybean leaves. Allantoin was degraded by seedlings in viva. Purine degradation was also investigated in fish liver. Allantoin- ase activity was present only in the soluble fraction, while uricase and allantoicase were localized in the peroxisomal fraction. TO MY PARENTS ACKNOWLEDGEMENTS I would like to express my gratitude to Dr. N}E. Tblbert for his financial support, professional guidance, and patience. I also wish to thank the members of my committee, Drs. L.L. Bieber, S. Ferguson-Miller, S.D. Aust, and J.M. Tiedje for many helpful discussions of research and manuscripts. I received financial support in the form of a Graduate Professional Opportunity Program Fellowship from the National Institute of Health, as well as from a National Science Foundation Grant to Dr. Tolbert. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES O O O O O O O O O O O 0 INTRODUCTION 0 O O O O O O O O O O O O Purine Catabolism Ureide Metabolism . . . . . . . . . Statement of the Problem . . . . . . CHAPTER I. PEROXISOMES FROM SOYBEAN NODULES Localization of Enzymes of Ureide Perioxisomes and Microsomes of Additional Enzyme Activities in Nodule Peroxisomes Biosynthesis in Nodules II. UREIDE METABOLISM IN INFECTED AND UNINFECTED NODULE Introduction . . . . . . . . . . Materials and Methods . . . . . . Results and Discussion . . . . . smary O O O O O O O O I O O O 0 III. METABOLISM OF UREIDES IN SOYBEAN LEAVES AND ”8 thOds O O O O O O 0 O O O O O O l. Sorbitol Gradients of Leaf Organelles . 2. Sucrose Gradients of Leaf Organelles 3. Sucrose Gradients of Seedling Organelles 4. Enzyme Assays . . . . . . . . ll 13 15 17 21 27 29 3O 31 33 45 46 47 47 47 48 48 Results and Discussion . . . . . . . . . . . . 1. Isolation of Organelles . . . . . . . . . . 2. Localization of Soybean Leaf Allantoinase . 3. Soybean Allantoicase . . . . . . . . . . . 4. Radioactive Assay for Allantoicase . . . . IV. INTRACELLULAR LOCALIZATION OF PURINE DEGRADATION IN FISH LIVER . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . APPENDIX . . . . . . . . . . . . . . . . . . . . . . Subcellular Organization of Ureide Biogenesis from Glycolytic Intermediates and Ammonium in Nitrogen- fixing Soybean Nodules . . . . . . . . . . . . . . BIBLIOGRAPHY O O O O O O O O O O O O 0 O O O O O O I 49 49 51 57 65 69 70 71 73 89 91 92 99 TABLE 10. ll. 12. 13. 14. LIST OF TABLES Separation of Enzymatic Activities in Fractions Containing Peroxisomes and Bacteria . . . . . . . . Additional Enzyme Activities in Nodule Peroxisomes Total Enzyme Units and Specific Activity from a Representative Step-Gradient . . . . . . . . . . . Specific Activity of Enzymes Involved in Ureide Formation O O O O O O O O O O O O C O O O O O O O 0 Specific Activity of Enzymes Involved in Purine Synthesis and Energy Metabolism . . . . . . . . . . . . . . . Comparison of Soybean and Spinach Leaf Extracts for Catalase and Peroxisomal Isolation . . . . . . . . Allantoicase Activity in Soybean Seedlings . . . . Activity of Enzymes Involved in Purine Degradation in Fish Liver . . . . . . . . . . . . . . . . . . . Tests for the Localization of Allantoinase in the Soluble Fraction by Centrifugation . . . . . . . . Non-linearity of Allantoicase Enzyme Assay . . . . Stimulation of Allantoicase by Magnesium Chloride . Effect of Buffer and pH on Allantoicase Activity . Distribution of Enzyme Activities Between Soluble and Peak Mitochondrial and Proplastid Fractions from Soybean Nodules . . . . . . . . . . . . . . . Recovery of Enzymes in the Proplastid Fraction of Soybean Nodules from Three Experiments . . . . . . to 24 28 36 39 40 SO 68 74 83 85 86 87 95 96 FIGURE 10. ll. 12. 13. Purine Degradation Distribution of Organelle Marker Enzyme Activities and LIST OF FIGURES Specific Activities on a Sucrose Gradient . . . . . . Distribution of Organelle Marker Enzyme Activities and Specific Activities from a Nodule Homogenate Schematic Representation of the Proposed Intracellular Location of the Enzymes of Purine Degradation . . . . Protoplasts of Infected and Uninfected Soybean Nodule Cells . . . Isozymes of Aspartate Aminotransferase Separation of Soybean Leaf Organelles on a Sorbitol Density Gradient . . . . . Separation of Soybean Leaf Organelles on a Sucrose Density Gradient . Separation of Soybean Seedling Organelles on a Sucrose Density Gradient . Isolation of Fish Liver Peroxisomes on a Sucrose Density Gradient . Distribution of Marker Enzymes for Mitochondria, Proplastids, and Bacteroids of Soybean Nodules Distribution of Soybean Nodule Enzymes Responsible for O the Synthesis of Amino Acids and Amides . . . . . . . . Gel Electrophoresis of Soluble and Proplastid Fractions from Soybean Nodules v 14 23 25 26 34 41 53 55 58 76 94 94 94 14. 15. 10 Distribution of Soybean Nodule Enzymes of the Glycine-C1 Pathway . . . . . . . . . . . . . . . . . . . . 95 Proposed Model for the Subcellular Distribution of the Enzymes of Ammonium Assimilation and Ureide Biogenesis in Soybean Nodules . . . . . . . . . . . . . . . . 97 INTRODUCTION Peroxisomes and glyoxysomes, subcellular organelles enclosed by a single lipid bilayer membrane, usually contain flavin oxidases, cata- lase, and other enzymes participating in catabolic metabolism (1). The term microbody has been previously used to describe all uncharacterized small organelles which contain catalase activity and are found in most eucaryotic cells. The term peroxisome is now recognized to include organelles which are characterized by the presence of catalase and/or flavin oxidase activity, and the term glyoxysome may be used if any part of the glyoxylate cycle is present (2). There are numerous examples of induction of peroxisomal biogenesis, including yeast growth on methanol (3) or alkanes (4), and rats treated with the drug clofibrate (5). There are currently three recognized types of plant peroxisomes: leaf peroxisomes, glyoxysomes, and unspecialized peroxisomes (6). Leaf peroxisomes contain the glycolate pathway in which glycolate, a compo- nent of photorespiration, is converted to glycine (l). The glycerate pathway, also found in leaf peroxisomes, allows the reversible intercon- version of serine and glycerate (1). Plants with higher levels of photorespiration (C3 plants) have greater numbers of leaf peroxisomes than do the C4 plants, in which the level of photorespiration is much lower, and peroxisomes are observed primarily in the bundle sheath cells (7). Glyoxysomes, found in the fat-storing cells of germinating fatty seeds, are responsible for the conversion of stored triglyceride to 04 acids, via the glyoxylate pathway (8). The reactions of the glyoxylate pathway found in glyoxysomes are not duplicated in the mitochondria, but 11 12 represent a separate source of C4 acids for energy and gluconeogene- sis. Glyoxysomes are also the exclusive site of beta-oxidation of fatty acids in seedling tissues (8). The question remains whether glyoxysomes become leaf peroxisomes as the tissue greens, or if leaf peroxisomes represent a new population of organelles. Nonspecialized peroxisomes are found in many non-green plant tis- sues, including roots (9) and potato tubers (10). The nonspecialized peroxisomes contain high levels of catalase and low levels of other peroxisomal enzymes such as glycolate oxidase and uricase (9, 10). The number of nonspecialized peroxisomes per cell is generally low, and no major metabolic role has been characterized, other than the function of catalase in detoxification of hydrogen peroxide. It is possible that specialized peroxisomes arise from nonspecialized precursors during cellular differentiation. Liver peroxisomes contain many of the same enzymes and metabolic pathways present in plant peroxisomes (1). Beta oxidation occurs in both peroxisomes and mitochondria of liver cells, but the two pathways differ in many important aspects (1). The various enzymes and pathways found in peroxisomes from differ- ent tissues or developmental stages of the same organism indicate that peroxisomes may be specialized for carrying out unique vital functions in cellular differentiation or in response to environmental stimuli. As outlined above, peroxisomes play an important role in such processes as photorespiration, conversion of stored fats to sucrose (glyoxylate cycle), beta oxidation, methanol oxidation, and detoxification of hydro- gen peroxide. The enzymatic content or number of peroxisomes per cell may vary widely between tissues, for example, liver versus kidney in __. ._‘_'._ ,_ 13 animals, or mesophyll versus bundle sheath cells in C4 plants (1). The topic of this thesis is the role peroxisomes play in the degradation of purines in both animals and plants, and the importance of this func- tion to. nitrogen metabolism in many economically important nitrogen- fixing legumes. Purine Catabolism The nitrogenous excretory products resulting from purine degrada- tion differ between animal phyla due to the loss through evolution of genes for enzymes catalyzing the latter steps of the pathway (11). The respective intermediates in purine degradation are hypoxanthine, xan- thine, uric acid, allantoin, allantoic acid, and finally, glyoxylate and urea (Figure 1). The final enzyme of purine degradation present in primates, birds, and some reptiles is xanthine oxidase, which oxidizes both hypoxanthine and xanthine to uric acid, the excretory product. Most 'mammals, reptiles, and. mollusks excrete the next intermediate, allantoin, the product of the uricase reaction. Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, which is excreted by some fishes. Allantoic acid is hydrolyzed to urea and glyoxylate by allan- toicase, the final enzyme present in most fish and amphibians. The aquatic invertebrates retain urease activity, and therefore excrete ammonia. Xanthine oxidase was found only in the soluble fraction in rat (12) and fish liver (13), but avian xanthine dehydrogenase was reported to be A peroxisomal (14). Xanthine dehydrogenase is reportedly the native form of xanthine oxidase (15). Uricase has been found in peroxisomes from mammals (16), fish (13), amphibians (l4), and plants (17). Catalase, OH c N / N/ \l/ \\ HYPOXANTHINE I l CH Hc C ,/ H xanthine 2 oxidase ('5 I XANTHINE l \ N/ \ N H 0 xanthine 2 oxidase 0H MR URIC ACID l HOC N ” NR COH /’ n (5:: FN/ \N O H 0 H + 2 2 ;) uricase H202 + C02 :0 HZN ALLANTOIN l 0::c H /\ c::o \N/H\N/ H H n—n Figure l. Purine Degradation allantoinase _____> 14 C0 + 2 NH 0H 2 4 T urease 2 HN—C—NH UREA 2 " 2 O + O H HC——-COOH GLYOXYLATE H20 allantoicase NHZ NH 2 u l ::l coon c::o HN--C-——:NH H ALLANTOIC ACID 15 which degrades the 8202 produced in the uricase reaction, is also found in peroxisomes (1). Allantoinase has been reported in amphibian and fish liver peroxisomes (14, 13), and at low levels in glyoxysomes from castor bean endosperm (17). However, an equal amount of activity was associated with the proplastid fraction from castor bean endosperm, and eighty percent of the original activity was lost on the gradient (17). In another report on castor bean endosperm allantoinase, most of the activity was found in fractions of density 1.21 g/cc or greater, but the activity was very low, and the location of other organelle fractions was not adequately characterized (18). Allantoicase has been localized in fish liver peroxisomes (13). Thus some of the enzymes in the pathway of purine degradation have been reported in peroxisomes from a few tissues. This thesis contains a more extensive investigation of purine degradation and the subcellular distribution of the associated enzymes, especially in the plant tissues. Ureide Metabolism Two intermediates in purine degradation, allantoin and allantoic acid, are found at high levels in certain nitrogenefixing legumes (19, 20), and are collectively termed the ureides. Although the presence of relatively high levels of ureides in some plants was reported in 1930 (21) and several workers had suggested purine catabolism resulted in allantoin accumulation as reviewed by Mbthes (22), the significance of these compounds in relation to nitrogen fixation in the nodules of cer- tain legumes has been recognized only in the last five years. Matsumoto ££_gl. (23) have reported that allantoin accumulated only in nodulated soybean plants and [ISNZ] supplied to nodulated plants was recovered in allantoin and allantoic acid (24). 16 The majority (60-80%) of the nitrogen transported in the xylem sap of both nitrogen-fixing cowpeas (19) and soybeans (20) was in the form of ureides. Most of the species transporting ureides were members of the tropical tribe Phaseoleae, of the family Leguminosae, which also includes tribes of nitrogen-fixing plants transporting amides and amino acids, especially glutamine and asparagine (25). Ureide production was correlated with nitrogen fixation, and decreased dramatically in the presence of added nitrate in the growth medium (26). Low levels of uricase and catalase were associated with the peroxi- somal fraction from roots of bean plants (27).. Much higher levels of xanthine dehydrogenase, uricase and allantoinase were found in cowpea nodules (28). The addition of allopurinol, an inhibitor of xanthine dehydrogenase, resulted in an accumulation of xanthine and a decrease in ureide formation. Labeled glycine was incorporated into allantoin and allantoic acid in nodule slices (28). .A pathway for ureide synthesis through xanthine dehydrogenase was also demonstrated in soybean nodules (29). Ureide metabolism in higher plants has been recently reviewed (30). All of the evidence to date indicates that nitrogen is first assimilated into amino acids which are then incorporated into purines. The purines are degraded to allantoin and allantoic acid, which are exported from the nodule. The pathway of purine synthesis and degrada- tion to allantoic acid appears essentially the same as the pathway pre- viously elucidated in animals. kbrk is currently underway in several other laboratories to purify the enzymes of purine metabolism from nodules and to isolate the intermediates in this pathway. The pathway of ureide degradation in soybean leaves is unknown. As described above, low allantoinase activity has been reported in l7 glyoxysomes from astor bean endosperm (17, 18). Allantoinase activity has been characterized from several other plant tissues (31, 32), including legumes (33, 34). Allantoicase activity was reported from germinating peanuts (35) and leaves of non-nodulated bushbeans (36), and was localized in peroxisomes from the skunk. cabbage appendix (37). Allantoinase from skunk cabbage was not peroxisomal, but in the soluble fraction (37). Allantoin induced an increase in glyoxylate in germina- ting wheat seedlings and labeling studies suggested glycine was a pre- cursor and glyoxylate a hydrolysis product of allantoin (38). Uricase and allantoinase have recently been reported in peroxisomes from mustard cotyledons (39). Much of the literature on this pathway is contradic- tory, probably due to the very low levels of allantoinase and allantoi- case in most plant tissues, and technical difficulties in the assays. These enzymes have not been reported or localized in leaves of nitrogen- fixing plants, in which high levels of activity might be expected. Statement of the Problem Since some of the enzymes of purine degradation had been reported in peroxisomes of chicken and fish liver and castor bean endosperm (14, 13, 17) my initial hypothesis was that the enzymes involved in allantoin and allantoic acid metabolism in nitrogen-fixing plants would also be localized within peroxisomes. The presence of the purine degradation pathway in soybean nodules had not been established at the time this project was undertaken, and nothing was known about ureide degradation in soybean leaves. Therefore my task was twofold: first, to identify the enzymes responsible for formation and degradation of ureides in soybean nodules and leaves, and secondly, to determine if these enzymes 18 were compartmented within peroxisomes. Xanthine dehydrogenase, uricase, and allantoinase activities would be expected in soybean nodules if ureides are produced by the known purine degradation pathway, previously localized in animal peroxisomes (13,14). Allantoinase would catalyze the final reaction taking place in the nodule, since primarily allantoic acid, along with some allantoin, is transported in the xylem sap (20). Allantoinase activity would also be expected in the leaves, since some allantoin is transported. If allantoic acid in soybean leaves is degraded by the same pathway reported in fish peroxisomes (13), glyoxy- late and urea would be the products of the allantoicase reaction. Ammonia could be released by urease activity (11). Reactions producing glyoxylate are always found in peroxisomes, where this unstable molecule may be protected from oxidation by H202 by catalase, and aminotrans- ferases are present to utilize the glyoxylate (1). Since peroxisomes had never been previously isolated or described from nodules, it was necessary to modify existing methodology for the isolation of nodule peroxisomes. Nodule peroxisomes were isolated by isopycnic sucrose density gradient centrifugation steps, as detailed in Chapter I (40). Xanthine dehydrogenase activity was present in the soluble fraction, uricase and catalase were localized in peroxisomes, and allantoinase activity was found in the microsomal fraction, in con- trast to reports from other tissues (13, 17). Marker enzyme data indi- cated that the microsomes containing allantoinase activity originated from the endoplasmic reticulum. During the course of this work, we were in communication with Dr. Eldon Newcomb, whose laboratory has been responsible for observation by l9 electron microscopy of the distribution and ultrastructural complexity of plant peroxisomes and glyoxysomes. Newcomb and Tandon (41) proceeded to examine cytologically the peroxisomal distribution in soybean root nodule cells. They found that enlargement of the peroxisomes and pro- liferation of smooth endoplasmic reticulum occurs only in the uninfected cells of soybean nodules. This data was consistent with our initial work (40) and further suggested that the uninfected cells might partici- pate in ureide synthesis via the uricase and allantoinase reactions. In order to investigate this hypothesis, I separated infected and uninfected cell protoplasts on a sucrose step—gradient with a very brief centrifugation, as detailed in Chapter II. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected proto- plasts. The peroxisomal enzymes uricase and catalase were primarily associated with the uninfected cell fraction. Allantoinase, previously localized in the endOplasmic reticulum, also had a much greater specific activity in the uninfected cell fraction. Several of the enzymes in- volved in purine synthesis were present at higher levels in the uninfec- ted cell fraction. These data suggest that at least the latter reac- tions of ureide formation may take place primarily in the uninfected cells of the soybean nodule. I have also investigated localization of ureide degradation in soy- bean leaf cells (Chapter III). Leaf allantoinase was localized within microsomes originating from the endoplasmic reticulum, in agreement with localization in the nodule. No significant allantoicase activity has been found in the leaves, although many different assays were tried. Soyhean leaves have also been reported to be very low in urease activity (42). 20 Germinating seedlings also degrade purines, presumably utilizing ureides stored in the cotyledon (Chapter III). While only traces of uricase were found in seedling peroxisomes, high levels of allantoinase occured in the microsomal fraction. [14C]allantoin was consumed in an in 11.53 assay, indicating allantoicase activity, but this enzyme could not be detected £2.2i552' Since localization of soybean allantoinase in the endoplasmic reti— culum disagreed with previous reports that allantoinase was in peroxi- somes from fish liver (13), I investigated the location of enzymes for purine degradation in fish liver (Chapter IV). I found allantoinase only in the soluble fraction, while uricase and allantoicase were perox- isomal. No allantoinase activity was associated with the microsomal fraction. The unstable allantoicase activity in the peroxisomes was increased ten-fold by addition of 10 mM MgC12. While Inany questions remain ‘unanswered, it is the hope of the author that the following chapters will demonstrate the ubiquitous role played by peroxisomes in purine degradation in such diverse tissues as soybean nodules and fish liver and add to our understanding of the importance of compartmentation in metabolic regulation in all living cells. CHAPTER I PEROXISOMES FROM SOYBEAN NODULES 21 Plant Physiol (Hill ) Oh. oh no 0032 inn-whirl ‘hM,«"INI¢i5.ll5-$INI fill/t) 22 Localization of Enzymes of Ureide Biosynthesis in Peroxisomes and Microsomes of Nodulesl Received for publication October I. I930 and in reused torm January I4. I98! JOANNA F. Hxs'xs. N. E. Toiaiai. AND KAREL R. SCHUBERI Department of Biochemistry. Michigan State University. East Lansing. Michigan 48824 ABSTRACT The intracellular Mention of enzymes involved la the synthesis of the ureides. allantoin and allantoic acid. was investigated in nodules of Glycine mar L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 aaao-oles per min per gram fresh weight) was totally soluble. whereas approximately IS‘T of the total urlease and catalase activities (I and mo nicroraoles per minute per gram fresh weight. respectively) was In the fraction containing htact peroxisomes. Allantoinase activity (6” nanomoles per minute per gram fresh weight) was associated with the microsomal fraction. which apparently originates from the endoplasmic reticulum. The ureides. allantoin and allantoic acid. are the predominant form of nitrogen transported in the xylem of soybean and cowpea plants growing symbtotically (I0. lb). The synthesis of allantoic acrd presumably occurs via the degradation of purines (I. 24). After differential centrifugation of extracts from cowpea or soy- bean nodules. the enzymes of purine catabolism were found in the soluble fraction (l. 24). These results do not rule out the possibility that these enzymes are located in fragile organelles. such as peroxisomes. This possibility is supported by results after careful fractionation of nodule extracts by dilTerential centrifu- gation (I9). One enzyme in the purine degradation pathway. uricase. is normally found in peroxisomes. along with catalase. which de- grades the H10; produced by uricase. Small amounts of uricase have been reported to be present in glyoxysomes of germinating fatty seeds (23) and in microbodies from potato tubers ( l8). Traces of uricase are also present in peroxisomes from other plant tissues (I2. ll). In all of these reports. uricase was easily solubilized and did not appear to be part of the crystalline core of the peroxisome. Xanthine dehydrogenase is generally found in the cytosolic frac- tion. although reportedly it is present in peroxisomes from avian livers (20). Allantoinase and allantoicase have been reported to be present in peroxisomes from amphibian (20) and fish (l7) livers. Approximately oneohall' of the allantoinase activity in castor bean endosperm was associated with glyoxysomes and the remainder was in the proplastid region. Eighty percent of the total activity was lost on the gradient (23). In another report on castor bean 'Research supported in part by grants from the National Science Foundation to N. Ii. T (PCM 78 l589l) and United States Department of Agriculture grant (590l-04l0-9ooz4tt-0) to K R. S, J. F. H was supported by a Graduate Professional Opportunity Program fellowship from the National Institutes of Health. Published as journal article 9647 of the Michigan Agricultural Experiment Station. A preliminary report has been published (9). 65 endosperm allantoinase. most of the actrvrty was found in fractions of density l.2l g/cc or greater. In this case. the allantoinase activity. which was reported only as .4 units. appeared to be extremely low and the location of other organelles was not ade- quately characterized (22). At least part of the pathway of purine catabolism has been considered to be associated with animal peroxisomes and possibly with plant microbodies. This proyect was initiated to examine the organelle distribution ol‘the enzymes associated with allantoin and allantoic acid formation in nodules. which form these compounds from recently fixed nitrogen for transport to the leaves and pods. MATERIALS AND METHODS Seeds of Glycine max L. Merr. cv. Amsoy 7| were inoculated with Rhizobtum japonirum strain 3I|b l IO obtained lrom D. Weber. United States Department of Agriculture. Bt‘llSHllC. MD. Plants were grown in a growth chamber in perlite and watered daily with nitrogensfree nutrient solution. Nodules (l2 g) were harvested from 30-day-old plants and very gently broken in a mortar containing IO ml medium (8) containing 0.4 M sucrose. 0 l M Tricine (pH 7.8). I0 mu DTT. l0 mM KCl. l mu Mg(‘l-,.. and ID mM EDTA. plus 50 mg fatty acrdofree BSA and I00 mg soluble PV P. The entire extract was squeezed through six layers of cheese- cloth and applied to a step gradient of 3 ml 2.3 M sucrose. 5 ml I.9 M sucrose. l0 ml l.8 M sucrose. IO ml L75 M sucrose. l0 ml I.7 M sucrose. 6 ml If) it sucrose. and 6 ml l.3 ht sucrose. To obtain a better separation of microsomes. a gradient of 3 ml 2.3 M sucrose. 5 ml l9 M sucrose. 7 ml |.8 M sucrose. 7 ml I75 M sucrose. 7 ml I? M sucrose. 7 ml 1.5 M sucrose. 6 ml l3 M sucrose. 4 ml It) M sucrose. and 4 ml 0.83 M sucrose. was used. All sucrose solutions were prepared in 0.1 u Tricine (pH 7.8). Gradients were centrifuged in a Beckman SW 25.2 swinging bucket rotor at 4 C in a Beckman L-2 ultracentrifuge. The speed was slowly accelerated by holding for IS min each at 5.000. I0.000. |S.000. and 20.000 rpm. and then run for 5 h at 25.000 rpm (l06.900g). Fractions from 2 to 5 ml were collected from the top of the gradient using an ISCO model fits Density Gradient F ractionator. C atalase activity was determined by the decrease in A at 240 nm (l4). Uricase activity was determined by the decrease in .4 at 293 nm in a l-ml assay mixture containing 0.l mhl uric and in U.l M 2-N-cyclohexylaminoethanesulfonate (Sigma) buffer at pH l0 (I9). Nodular uricase was inhibited by borate buffer and Triton X-IOO. NADH-Cyt e reductase activity was measured by the NADH-dependent increase in A at 550 nm (4). using an extinction coefficient of 2| mu" cm ' (l5). Cyt c oxidase activity was determined by the decrease in A of reduced C yt r at 550 nm (2|). after the extract alone was incubated with 20 pl I? digttonin for l min before addition of buffer and substrate. Triosephosphate isomerase activity was determined by coupling to a-glycerophos- phate dehydrogenase (EC H.995) (2). Potassium-stimulated 23 66 HANKS. TOLBI:RT. AND SCHUBERT Plant Physiol Vol. bit. I98| f' '3 Yrtoaa Phoaptioia 300 : E taomaroaa *‘00 V Y Y Y V v 1 v v v v Y VV—Y T ”o Coiotoaa E - “i '1 I00 1‘ t I t 0 300 'E : l l a r i ‘ ' ' g ‘°" I i «0ng ' no a: g "' 30 I i .50 E '_,‘,r‘ t | '02:: 711-11 '0 . .00 9“ Hydro-youwo 4’ i ”° TC so» «so zoo .E. T 80?- 420 E ”0 o n .- E ,.‘ ‘3 C so - tOt- : g‘ 4'0 g .5 N n Cytochrome c E T c .;¢;T‘q2 1‘ 2 Radoetaaa .50 3 E . it * s | E T .‘ i ' 8 i E ': Q 00 : I l g 40»- r‘l 150 g e 5 1 Tc ’° C 1‘?‘ ’-.‘ :J ' 1’0 E l : ' ' I I ' I . J' ‘ _ ._ _ - J Li to i ; o c e: +¢4 : +c4‘.¢e f I ‘uw'm'n'. ... roam : .0 " : " .0 coi- : Oamorooaooa : g": i l ' I I so i- ' : twine 90c m» 50L- : i : 4 so I l I 201-: , 30 .— -J l- '3 'i 30 HO" i : : t0»- : {-1 i l ‘ d. IO r- _J - '_1 . l0 L4 - _A—.L.._J._L ‘ ° 2 4 a a retains"? a 2 4 3 O to re MIG Fraction Fraction hi. I Distribution of organelle marker enzyme activities (--—) and specific ICIIHIICS (- - -) on a sucrose gradient atter centritugation for is h of a nodule homogenate which contained BSA in the grinding media and was applied directly to the gradient. Units are nmol min ‘ nil ' except catalase which is tn umol min ' ml ' Peroxisomes banded at a density of I25 g/cc. ATPase was assayed by measuring the Pi released (I I). lDPase" activity was measured 48 h after extraction of tissue by determin- ing Pi released from IDP (4). The xanthine dehydrogenase assay mixture contained 5 umol NAD. extract. and OJ M Tricine (pH 8.4). in a final volume of I ml. The endogenous rate at 340 nm was measured and the reaction was initiated with the addition of 0.25 umol xanthine. Hydroxy- butyrate dehydrogenase activity was measured after incubation of extract with 50 pl NH Triton X-IOO for 2 min. and then addition of 5 umol NAD and DJ M Tricine (pH 7.8) to a volume of l.0 ml. The increase in A at 340 nm was measured. The reaction was initiated by the addition of 50 umol fl-oi.-hydroxybutyrate. Allantoinase activity was measured by formation of the product. allantoic acid. Allantoic acid was determined by boiling in dilute acid and measuring the diphenylformazan derivative of the gly— oxylate produced (26). A substrate concentration of I5 mu allan- toin in 20 mar Tricine (pH 7.8) was used in the assay performed on gradient fractions. It was necessary to use a concentration ‘ Abbreviations: lDPase. inosine diphosphatase slightly lower than the K... for allantoin (approximately l9 mH. data not shown). because of the low solubility of allantorn and the high background caused by nonenzymic breakdown of substrate. A 30 mM allantoin reagent was used to estimate total enzyme activity in the homogenate. The assays were stopped after 20 min with I ml 0.l5 M HC I. Sucrose and other components of the enzyme sample reacted with phenylhydrazine in this assay to give an orange color. which in some cases obscured the diphenylfor- mazan product at 520 nm. This artifact could be minimized by boiling the samples and then briefly chilling in ice before adding phenylhydrazine. The use of ultrapure density gradient grade sucrose (Mann Research Laboratories) also decreased the inter- ference with the assay. Controls were run for nonenzymic break. down of substrate. as well as for the presence of product at zero time in the extract. Protein was determined by a modified Lowry procedure (3). Phosphate was determined by the method of Chen er al. (5). RESULTS AND DISCUSSION C atalase and uricase. enzymes found in roxisomes of animal and plant tissues (I2. l7). were present at igh levels in soybean Pldfll Pltysltil Vol 65. I‘ihl Iahle I Separation u/ [riginnitir -l. llllllt'\ in Int. (tum ( uttlammi: Perri tisrimi'i and Bin Irrta (iradieiit fractions from I 24 I 27 g! cc suuose. containing a mixture of peroxisomes and bacteria. were combined. diluted with two parts butter vortexed l min. and centrifuged l h at M51003 Solubilized peroxisomal enzymes appeared in the supernatant. while the pellet contained the bacteria After Breakage and Initial ( oni- Recentrilugatioii btned ’ — ’ l‘rac- Super- Pellet [Inns natant ('atalase nmol ‘min So 53 0| nmol,‘min-mg protein I00 280 it 25 Uncase nmol/min 30 32 t) ()5 nmol/min-mg protein 89 lo4 0 I2 llydroxybutyrate dehydrogenase nmol/min l4 0 8 l nmol/mtn-mg protein to 0 I5 nodules. Nodules contained approximately 2000 nmol min ' g fresh weight ' catalase activity and I umol min ' g fresh weight urtcase activity. This level of uricase is at least l0 times that detected in other plant tissues (l2. l3. IS. 23) including potato tubers and glyoxysomes. and is comparable to that found in rat liver on a protein basis. In initial attempts at isolating peroxisomes all of the catalase and uricase was found in the soluble fraction at the top of the gradient or in the bacteroid fraction. Various methods of chopping the tissue. the use of different grinding media and gradients. and low speed precentrifugation were tried in order to isolate a significant peroxisomal fraction. Separation of peroxisomes from the large numbers of mature bactemtds and vegetative cells presented a mayor problem. because both peroxi- somes and bacteria have nearly the same density. To isolate a peroxisomal fraction very gentle breakage of the nodules was necessary. and only young nodules were used. It was necessary to increase the centrifugation time to o h and broaden the sucrose gradient in the denser region in order to separate peroxisomes from vegetative cells (hg I). Nodule peroxisomes appeared to be extremely fragile. as judged from the large amounts of soluble catalase and urtcase with even very gentle homogenization. Tan- don and Newcomb (manuscript in preparation) are also reporting. based on observations by electron microscopy. that the peroxi- somes in nodules appear broken and degenerated. particularly in older tissue. and sometimes do not have a bounding membrane. Results of enzyme assays after fractionation of organelles on a sucrose density gradient are presented in Figure I. In the upper fractions. which represent soluble enzymes. some sedimentation occured into fraction 2. Xanthine dehydrogenase activity appeared only in the soluble portion of the gradient. A large fraction ofthe catalase and urtcase activuies. presumably from broken peroxi- somes. was also at the top of the gradient. Intact peroxisomes. localized by catalase and urtcase activities. appeared at a sucrose density of I25 g/cc and contained approximately l5’I of the total catalase and uricase activity. Allantoinase activity coincided with the microsomal marker. ('yt c reductase (Figs. l and 2). No activity of glucose-b-phosphatase. another microsomal marker. could be detected. Two peaks of bacterial origin were observed using the marker enzyme. hydroxybutyrate dehydrogenase. The band of lower den- sity was presumed to be mature bacteroids. and the band of higher density. which overlapped the peroxisomal band. to be vegetative cells (6). Other marker enzymes used were Cyt c oxidase for mitochondria and triosephosphate isomerase for proplastids (Fig. 24 MN Allth Of tNlYMLS OI" URLII)E SYNTHESIS 07 l). Specific activities of the marker enzymes are shown as a dashed line in Figure l Specific activities for soluble enzymes in the upper three or four fractions were low because of the addition of BSA to the grinding media. and as a result. the specific activity peaks of the microsomal marker enzyme. ( yt t reductase. and allantoinase were shifted toward highei densities of sucrose than the plotted peaks for total activity The specific actiyities of catalase in the peroxisomes was loft nmol min ‘ mg protein ' and for urtcase it was 89 nmol min ‘ mg protein ‘, In order to assay hydroxybutyrate dehydrogenase. it was nec- essary to break the bacteroids by sonication or treatment with Triton X400. No activity could be measured prior to such treat- ment Catalase and urtcase activity could be measured without sonication or treatment with detergent. and actually decreased if sonicated (data not shown) This suggested that these enzyme activities in fractions of density I24 to I 27 g/cc were derived from two different organelles. To test the hypothesis that hydroxybutyrate dehydrogenase activity in the peroxisomal band was due to contaminating bac- teria. fractions at approximately l.24 to II? g/cc sucrose were combined. diluted with two parts buffer. and vortexed to break the fragile peroxisomes. The sample was then centrifuged I h at “5.0003 in a Beckman TY 65 rotor. and the pellet and soluble fraction were assayed separately. The pellet was washed with 0 5 ml buffer. and resuspended in ID ml. Catalase and urtcase actiy ity were in the soluble fraction (Table I). while the hydroxybutyrate dehydrogenase activrty remained tn the pelleted bacteroids. The spethic activity of catalase and urtcase increased greatly tn the soluble fraction. due to the removal of the large amount of bacterial protein. To examine the organelle location of allantoinase further. the sucrose density gradient was broadened slightly to give better separation in the area ofthe microsomes (fig 2) DTT and soluble PVP. which were found to inhibit allantoinase. were omitted in this gradient. Allantomase cosedtmented with ('yt c reductase. a microsomal marker. As before. specific dClIslIICS in the upper fractions were low. due to BSA in the grinding media. Very few units of allantornase were in the top fractions. and this suggests that the enzyme may be membrane-bound as it was not solubilized upon breakage of the ER. The activity of IDPase. a marker enzyme for Golgi apparatus in plants (4). was mainly in the soluble fraction. The potassium-stimulated ATPase. a marker enzyme for the plasma membrane in plants ( I I). was also in the soluble fraction. Thus. it appeared that the allantornase activity in the microsomes did not originate from either (iolgi or plasma membranes. Whereas allantoinase and (‘yt ( reductase cosedt- ment. it is probable that allantoinase is located in the LR The possibility still exists that the microsomes to which allantoinase is bound arise from the broken fragments ofperoxisomal membranes or from some other source. CONCLI‘SIONS Based on the distribution of the enzymes of purine catabolism, it is suggested that the intermediates in the ureide pathway are metabolized in several locations in the cell (It); 3) Nitrogenase is contained within the bacteroids. which release .itiimonia to the host cell. We have pictured glutamate synthase and glutamine synthetase in the proplastids. inasmuch as these enzyme activities have been localized in leafchloroplasts (25) Amino acids are then used in the synthesis ofpurtnes ( l ). which are degraded to xanthine (25) in the cytosol. Xanthine appears to be conserted to uric acid in the cytosol by xanthine dehydrogenase Uric acid is oxidized to allantoin in the peroxisomes by urtcase. producing H;O,.. which is degraded by catalase. also located in the peroxisomes. Allantoin then appears in the xylem sap (lb) or is hydrolyzed to allantoic acid by allantoinase in the ER. and allantoic acid secreted into bll HANKS. TOLBI;RT. AND SCHUBERT Plant Physiol. Vol 68. I98l 2001 t"‘- I DPose ‘l 200 - -- - .m .12 Lao I“; Allantoinase I50» ‘8 I I I ~ no a 70 : .001» Pi 0.000 1. . E I l'" ’° '°° E sol—r“ L-1 I” 2 so 1 7; i 4» _‘—C1 rt. .0 E ISO _ 8 : : ¢+£ f c f c fi 2 E g o . io ..... I” 5 fl K'-Stimutotod ,5 2 °‘ rm: ' * " +' 8 ATPoso l = -. I I Cytochromoc g c t — : I Roduetoso 1‘0 l 50” r-J I — I _ i ff 'c: l f '0 '5 ’ “ E E E J 2 1 «Is :2 o . o E I E {MU 7!; : 4F 0 OJrvo a o o o o o—-¢ 0* 9‘-9' l I — COIOIOS. g .00 .-. Cytochrome c | ._ .50.. Oxidose >2000 E #70 g 7°°f '7 l 't' . l (500 5 I004 :4, 50 5 3001 E : E race in l 30 ’9 r-a 1 50‘ ' I :00 g E I g ' "’°° :1 ll» I0 :.1 |ml rf ' IOO l '04 —°—*-- ! z 4“s"a iziais 2 ‘ 6 °, '0 '2"'° ' Fraction FIOCIIOD Ts — - 8 '_ f I g E Hydroaybutyroto ; '-1 —. 30 ‘I Dehydrogenoso , I L‘ ‘I '5 g E r E :04» L. ."E. 2 n E o I E .‘2 (I IO“ 0 E C 01» —o v o s -o i {a I l t ' I 700 .t<>02 .I H If we r1 4)” r"- I ’0 ‘p-J"' -r-hJ L1 ! 0 I. t r- OT \.1 “6 b ‘ 2‘ *4; Vs: cii¢ Trot Ara'vnnrs Fraction fltr 2 Distribution of organelle marker enzyme ICIIVIIICS (——~ ) and specific activities (— ~ -) from a nodule homogenate which contained BSA in m.‘ 3mm"); media and was applied directly to a broadened sucrose gradient. and centrifuged for o h Units are nmol min ' ml ' except catalase which n m #mol min ' ml '. Note that microsomal enzymes banded at a density of I. I8 g/cc. 26 Plant Physiol Vol 08. Hall LOt'AT ION OI‘ tleMLS OF UREIDE SYNTHESIS 69 MOLLE (Ill 5 (th PS. Ia. TY Triaiasaa. H \b'itiistii I95o Microdeteiininaiion of phosphuv T _____,_ '“”""*" ‘— rus Anal Chem 2! I750 I758 I " ” o. (Him. TM. 5 Hiiiisi. W New man I977 Isolation of bacteria transforming I, bacteria. and bactermds from soybean nodules Plant Physiol NI 77I 774 7 Curtisrtits MI I980 The endoplasmic reticulum In NE Tolben ed The Biochemistry of Plants. Vol I Academic Press Inc NY. pp 3“ ll“ or, If (itaitsant BP. H Buytas I970 Developmental studies on glyousimies troni . castor bean endosperm J ( ell Biol 44) wt lti2 i; _ __ __-, 2., * XYLEM 9 Hssiss JP. KR Si iii-tar. NL Tot tiiiit tvhiit atalase uricase and allantoinase i (1 235%“le p i SAP from soybean nodules Plant Physiol (>5 5 I If y r ‘ 65 I I0 Hiitrtitioi DI LA AlainsJS Psii RM Rustin-ii I9‘r Allantoin and dlldftliils s-u- I -..-.—-._...~ ‘ \ acid in the nitrogen economy of the cow pea Plant Physiol h: 49's 4w- m “m J a” ll Hutu.” TK RT litiwslti I974 Purification ol a plasma "Icflt'ldllt rs and ”my. -- , - o , - ‘ adenosinetriphosphatasefrom plant fouls Methods tfll\I|i-i. )2 WI 8'. I CYTOSQ l ”x cm. I '2 HI AN“ AH£ H Bl I \tls l‘pl lwldlion HI miclitbndies from plant lissue~ Pianl “'6'". we 0; :5 i I Phystol‘h 037 Ml , 4 -O i W0 “I'M ' a“, m “w". I] Ht ysi. AHC‘. H Biiyiiis P973 Localization of enzymes withir. nllwlt-hllulc\ l " u n" m l PEROXISM l ( ell Biol Sit 37v 3m wit: nerd--~ ._ — — .- -- --—/ l4 Lt‘i Is H I965 catalase In HU Bergmeyer ed Methods ol anymatit anatsiis “ 4-" S "" “ " ’ '"' W "" Ld2 Academic Press. NY pp ”'5 894 hi. 3 Schematic representation of the proposed intracellular location '5 M3:21:111123;Maratrrgmgr 21,:“§;'";.'E 3'13”" (“mum “mum“ of the enzymes of purine degradation (it)(iAT. glutamate synthase. (:5. to M‘ (u I, PR. DW (5,. m (we rump," "“1..th ,n m, “it"; H, “Mm“ glutamine synthetase plants Plant Physiol (>4 4)) Atty I7 Nomi iii T. \ Tutsi“ S Ft may sit \ l9’9 Degradation of uric .ILId to urea and . a «1“ c y‘- the xylem. where both ureides are translocated to the aerial parts H R ”Wm“: m ““5“" J 8"“ mm ‘“ ‘ ‘ ' ‘ . . t Is ll Nil Isolation and characterization ol pert-yisoriies lrorri [strain tubers of the plant. In such a model. the ER may be involved in the H..pp¢.s¢,.¢,-,z phym.” Mm 352 (“.5 ”I; release ofthe negatively charged allanIOic acid This hypothesis is I9 si in am KR (.M Disiinci iviio lnzsmes .ii purine hiiiwnlliems and .mh consistent WIIh the suggestion mm me ER may play a royc m clil'iIsm in soybean root nodules role in ureide bioscnthesis Plant Physioln‘ 5 “menu“ of b0"? large and “nannwkuflc’ ‘md m regulation (7) 20 St oil PI LP V'isisiis. IM At II\ I909 I_nzymatic shdldsltllsllc .-i PCls|\|\UHI¢\ The model requires also that uric acid and allantoin must cross “(.mphmmn m, mm hm m, NM. 4,", \\ s...a 5‘. .h. 3“ 3... the single peroxisomal membrane by diffusion. 2| swim I I95! sinciriipriciiomcmc assay of cut-chronic . man- In tit,i..i ed Methods OI Bttxhemwal Analysis. id 2. Vol 2 L hap l3 Intetscience Puhiiah ers. Inc. NY pp 42743.5 H "RAMIREZ (1 [ED 22 St Amittu AI Rl On I970 Localization ol allantoinase in Elss'\\\c'nlc‘ ot germinating castor beans Biochem Biophys Res ( ommun M 29'- 2% I Aisiss L A. R RsiMitiiii. IS Psti I980 Lyidence tor a purine pathway of ureide 23 Timon RR It Bust“ (97) L'ncag and allantoinase in gly.-usoiites Plant synthesis in N,.-l'ixing nodules ol cowpea Z Pllanzenphvsiol 97 249 200 Physiol 47 3“. 3s] 2 Btistmiiaz (i I955 Triosephosphate isomerase trom call muscle Methods 2a Tairiitt [\a 1x. 31...“ [)D Ruin” (vsii Alumni. acid \\nlh¢\|\ in tnzymol I 3873‘“ soybean root nodule cytosol sia xanthine dehydrogenase Plant Phssltll M 3 Bihsttmt is A. D Wtitsstti's l97o Assay of proteins in the presence of interlering )20}. I200 materials Anal Biochem 74) 24l 250 25 Winsoami RM PI Lit. BI MIIII\ (We Distribution or the enzymes of G Biiwi t s DI H K NM NM ( hdllleIlldIIUfl. enzymatic and Iecttn properties of nitrogen assimilation within the pea leaf celi Plant Physiol M II) .‘Ih isolated membranes from Phuieului ourmi Biochiiit Biophys Acta 443 IN) 20 Via.“ s CD. I V \sDuDItH I970 Uillereritial analyses of giyosylate derisa 374 rises Anal Biochem 33 I43 I.“ 27 Additional Enzyme Activities in Nodule Peroxisomes Nodule peroxisomes were also analyzed for activities of enzymes found in leaf peroxisomes or glyoxysomes. A complete glyoxylate cycle is present only in germinating seeds (8). An incomplete cycle, lacking isocitrate lyase, is present in resting seeds (58). No isocitrate lyase activity was found in nodule peroxisomes, and only traces of malate synthetase were present. Glycolate oxidase was not present in nodule peroxisomes. A low level of aspartate aminotransferase was found in the peroxisomal fraction, but the majority of this enzyme was in the plastid and soluble fractions (Table 2). Hydroxypyruvate reductase activity was absent from the peroxisomes, although some soluble activity was present. This activity may be attributed to lactate dehydrogenase, which was also present at about the same level in the soluble fraction. Only traces of activity were found for the glyoxylate:glutamate and g1yoxylate:serine aminotransferases, normally found at high levels in leaf peroxisomes. Thus it appears that nodule peroxisomes do not contain the enzymes found in leaf peroxisomes or glyoxysomes, but are specialized only to perform the uricase and catalase reactions, which are essential for the produc- tion of allantoin in soybean nodules. Table 2 Additional Enzyme Activities in Nodule Peroxisomes Enzyme Isocitrate lyase Malate synthetase Glycolate oxidase Aspartate aminotransferase Hydroxypyruvate reductase Lactate dehydrogenase Glyoxylatezglutamate aminotransferase Glyoxylate:serine aminotransferase Soluble Activity Specific (units) Activity nmol units mi.n"1ml-1 mg protein"1 0 trace 0 890 120 92 12 56 7.4 trace trace 28 Peroxisomal Activity Specific (units) Activity nmol units min-lml-1 mg protein- trace 100 0.7 trace trace 220 1.6 l CHAPTER II UREIDE METABOLISM IN INFECTED AND UNINFECTED NODULE CELLS 29 30 INTRODUCTION Allantoin and allantoic acid, the ureides, are the major nitro- genous compounds transported in the xylem sap of soybeans (20). Label- ing studies have indicated that the high levels of purines synthesized in the nodule are subsequently degraded to ureides (28). The role of both cellular and sub-cellular compartmentation in this process has been implicated in several recent reports. Uricase and catalase were local- ized in peroxisomes, allantoinase in the endoplasmic reticulum, and xanthine dehydrogenase in the cytosol by fractionation of a total nodule tissue extract on sucrose density gradients (40). By means of electron microscopy, a marked enlargement of peroxisomes and proliferation of smooth endoplasmic reticulum during nodule deve10pment was observed to occur only in the uninfected cells of nodules, indicating an important role of these cells in ureide production (41). In further cell frac- tionation studies, several enzymes involved in ammonia assimilation into amino acids and purine synthesis were localized in the plastid (Appendix I, 59). These included.iasparagine synthetase, phosphoribosyl. amido- transferase, phosphoglycerate dehydrogenase, serine hydroxymethylase, methylene tetrahydrofolate dehydrogenase, one isozyme of aspartate aminotransferase, glutamate synthase, and triosephosphate isomerase. Separation of infected and uninfected nodule cells is necessary to determine the distribution of enzymes and organelles involved in ureide production. I have separated protoplasts on a sucrose step-gradient and assayed the uninfected and infected cell fractions for enzymes involved in purine synthesis and degradation. 31 MATERIALS AND METHODS Seeds of Glycine max were inoculated with Rhizobium laponicum strain 311b 110 obtained from D. Weber, United States Department of Agriculture, Beltsville, MD. Plants were grown in a growth chamber in perlite and watered daily with nitrogen-free nutrient solution. For protoplast isolation, 6 g of nodules from 50 day-old plants were finely sliced with a razor blade in a petri dish containing about 5 ml of l-BS tissue culture medium of Gamborg (60). This medium was removed with a pipet, and the tissue was rinsed four times with fresh l-BS medium to remove broken cell contents. Then 11 ml of l-BS medium and an enzyme solution containing 200 mg Cellulysin (Calbiochem), 100 mg Hemicellulase (Sigma), and 0.2 ml Pectinase (Sigma), 1 g sorbitol, and 9 ml H20 were added. The dish was shaken at 25 rpm for 1.5 to 2.5 h at 25°C. The brei was passed through 100 um nylon mesh. Approximately 16 ml of the protoplast suspension were very gently layered onto the following step gradient, prepared immediately before use, in a 30 ml nitrocellulose tube: 3 ml of 60% sucrose, 7 m1 of 40% sucrose, and 10 ml of BS medium (60). All sucrose solutions were prepared in BB medbmn. The gradient was centrifuged at low speed (about 30 x g) in a clinical swinging bucket centrifuge for 3 to 5 minutes, when two layers of cells at the interfaces could be seen. One ml fractions were collected from the top of the gradient using an ISCO model 185 Density Gradient Fractionator. It was not possible to perform cell counts due to the low yield of puri- fied protoplasts, nor could bacteroids be separated from cellular mem- branes of the infected cell fraction. 32 Uricase, allantoinase and hydroxybutyrate dehydrogenase were assayed as previously reported (Chapter I, 40). Catalase was determined by the decrease in absorbance at 240 nm (53). Iriosephosphate isomerase was determined by coupling to a-glycerophosphate dehydrogenase (43). Aspar- tate aminotransferase was assayed by coupling to malate dehydrogenase (61). Phosphoribosyl amidotransferase was assayed by 5-phospho-or—D- ribose 1-pyrophosphate (PRPP)-dependent deamidation of [14C1gluta- mine (62), which was separated from labeled glutamate by ion exchange (63). Phosphoglycerate dehydrogenase was assayed by phosphohydroxypyru- vate-dependent oxidation of NADH at 340 nm (59). The assay mixture for glucose6-phosphate dehydrogenase contained 2 mM glucose-6-phosphate, 0.2 mM NADP, and 20 mM tricine, pH 7.8. The increase in absorbance at 340 nm was measured. The assay for 6-phosphogluconate dehydrogenase was identical, except for substitution of 2 mM 6-phosphog1uconate for glucose-6phosphate. Lactate dehydrogenase was assayed at 340 nm with 0.15 mM NADH, 2 mM pyruvate or hydroxypyruvate, and 50 mM phosphate buffer, pH 7.5. The assay mixture for malate dehydrogenase contained 0.2 mM NADH, 3.3 mM oxaloacetate, and 20 mM tricine, pH 7.8. Decrease in absorbance at 340 nm was measured. Native polyacrylamide gel elec- trophoresis was performed according to Laemmli (64), omitting sodium dodecyl sulfate. the gels were stained for aspartate aminotransferase activity with Fast Violet 3 salt (Sigma) (65). Protein was determined by a modified Lowry procedure (44). 33 RESULTS AND DISCUSSION Although protoplasts have been previously isolated from leguminous nodules (66, 67), only infected cells were obtained. As illustrated in Figure 5, the uninfected protoplasts we isolated were much smaller than the infected protoplasts and were spherical, while the infected proto- plasts were irregular in shape and had a granular surface. Staining with Calcofluor white indicated no cell wall material remained. The uninfected cells did not come from the cortex, since control experiments using only infected tissue yielded both uninfected and infected proto- plasts, and no protoplasts were obtained from cortex tissue in 3 hours digestion. The uninfected protoplasts proved to be more fragile and were often obtained in lower yield than the infected protoplasts (Table 3). The infected protoplasts had a very high density, probably due to the large number of bacteroids per cell, and rapidly pelleted through 50% sucrose even at low centrifugal force. Attempts to separate the two types of protoplasts by flotation were unsuccessful, and the density of the dextran gradients of Edwards 33 Elf (68) were not high enough to separate the infected protoplasts. The sucrose step-gradient specified above was designed to sediment the uninfected protoplasts at the upper interface, and the infected protoplasts at the lower interface. It was essential to minimize handling of the protoplasts, since the uninfected cells were easily broken by contact with the larger infected cells and the high sucrose concentration necessary for separation. Yields of each protoplast type ranged from 0.5 to 3.0 mg protein, depending on the amount of tissue used and incubation time. Longer periods of incubation with the digestive enzymes yielded fewer uninfected cells but more Figure 5. Protoplasts of infected and uninfected soybean nodule cells. Light micrographs were of a crude protoplast suspension photographed before separation on the step-gradient. Background debris from broken cells was removed in the upper layer of the gradient. Magnification: 25X. 3) infected cell. Size ranged from 100-200 um in diameter. b) uninfected cell. Size ranged from 20 to 50 um in diameter. 34 35 V l .a ..a a (V 1a . . s v... o. r...» .. s. ‘.. 1:7,: rm: '5 my, ‘ ‘ ”(ifs ta 3 .‘g d.- “'3‘.” i ' “ YgL 36 Table 3 Total Enzyme Units and Specific Activity from a Representative Step-Gradient Uninfected Cell Fraction Infected Cell Fraction nmol min"1 Specific nmol min'l Specific Activity Activity Uricase 20 51 4.5 5.5 OHrbutyrate 0.2 0.6 3.5 4.3 dehydrogenase Phosphoglycerate 25 64 20 24 dehydrogenase ' Aspartate 20 51 18 22 aminotransferase Protein (mg/ml) 0.39 0.82 Protoplasts were separated on a sucrose step-gradient as in Materials and Methods. All fractions were 1.0 ml. Specific activity units are nmol min"1 mg protein'l. 37 infected cells, and often resulted in higher levels of cross contamina- tion. Only by using the l-BS and BS media of Gamborg (60) were substan- tial quantities of uninfected protoplasts obtained. More uninfected protoplasts were obtained from nodules of 40-50 day old plants than from nodules of younger plants. Data from a representative separation of infected and uninfected protoplasts is presented in Table 3. Uricase was the marker enzyme for peroxisomes (40) land hydroxybutyrate dehydrogenase was the bacteroid marker enzyme. The lower protoplast fraction at the 60% sucrose inter- face contained most of the hydroxybutyrate dehydrogenase activity, and was therefore designated the infected cell fraction. The upper proto- plast fraction at the 40% sucrose interface was very low in hydroxybuty- rate dehydrogenase activity and was designated the uninfected cell frac- tion. Uricase activity was primarily found in the uninfected cell fraction. Data are also shown for phosphoglycerate dehydrogenase and aspartate aminotransferase, two of the enzymes localized in plastids which are probably involved in purine synthesis (Appendix, 59). The specific activities of these two enzymes were 2 to 3 times higher in the uninfected cell fraction, but about half of the total activity was also present in the uninfected cell fraction, but about half of the total activity was also present in the infected cell fraction. On the basis of total mg protein, the yield of uninfected protoplasts was about half that of infected cell protoplasts. Triton-X-lOO was present only in the assay mixture for hydroxybutyrate dehydrogenase, in order to break the bacteroid membranes (40). Addition of low concentration of detergent (~01%) did not increase any of the other enzyme activities, indicating cellular membranes were probably broken during dilution into the assay ntixture, and were not a barrier to enzyme activity. 38 Average specific activities from 25 different step-gradients are presented in Table 4. Uricase and catalase, the peroxisomal enzymes (40), were predominantly in the uninfected cell fraction. Allantoinase, located in the endoplasmic reticulum (40), was also mostly in the unin- fected cell fraction. These data confirm the hypothesis of Newcomb and Tandon (41) that peroxisomes and the endoplasmic reticulum in the unin- fected cells contain the enzymes which. catalyze the final steps in ureide formation. Average specific activities for several of the metabolic enzymes which are involved in purine biosynthesis are given in Table 5. Since none of these assays contained detergent, it may be assumed that the bacteroids made no contribution to the activities as measured. Phospho- glycerate dehydrogenase is a plastid enzyme, probably involved in syn- thesis of serine, which is required for purine biosynthesis (59). The specific activity of phosphoglycerate dehydrogenase was twice as high in the uninfected cell fraction as in the infected cell fraction. Specific activity of aspartate aminotransferase (Table 5) was 2-3 times higher in the uninfected cell fraction. One isozyme of aspartate aminotransferase in the soybean nodule has been localized in the plastid (59), and aspartate is required for purine synthesis. The presence of different isozymes of aspartate aminotransferase :hi peroxisomes, mitochondria and chloroplasts of leaves has been previously reported (65). Protoplast fractions were subjected to native polyacrylamide gel electrophoreis and stained for aspartate aminotransferase activity (Figure 6). Band 1 is the soluble isozyme, and band 2 is the plastid isozyme (59). The uninfected cell fraction was applied to gel A, 39 Table 4 Specific Activity of Enzymes Involved in Ureide Formation Uninfected Cell Fraction Infected Cell Fraction nmol ing-l mg protein"1 Uricase 65 4.1 Catalase 35 x 103 5.0 x 103 Allantoinase 9.0 3.6 OH-butyrate dehydrogenase 1.5 6.0 Triton X-100 was present only in the assay for hydroxybutyrate dehydrogenase. 40 Table 5 Specific Activity of Enzymes Involved in Purine Synthesis and Energy Metabolism Uninfected Cell Fraction Infected Cell Fraction nmol min'1 mg protein.1 Phosphoglycerate dehydrogenase 58 27 Aspartate Aminotransferase 79 30 6-Phosphogluconate Dehydrogenase 4.4 1.1 Glucose-6-Phosphate Dehydrogenase 3.7 1.1 Triose-P Isomerase 150 140 Malate Dehydrogenase 7.2 3.5 Lactate Dehydrogenase 7.0 5.4 Triton X-100 was not present during the assay. Figure 6. Isozymes of aspartate aminotransferase. Proteins were separated by native polyacrylamide gel electrophoresis and stained for aspartate aminotransferase activity with Fast Violet B. Band 1 is the soluble isozyme; band 2 is the plastid isozyme (59). A - uninfected cell fraction, B - infected cell fraction. 41 |J> Iw Figure 6. Isozymes of Aspartate Aminotransferase ta. 2 | u C . \.\A, J mu . . "'00. a'kbt :1Pe by. 'loa. 3701' u.. ‘I ..:E . fish. “¥A 43 which contained much more activity in Band 1, the soluble isozyme. The infected cell fraction was applied to gel B. Both gels showed essen- tially equal amounts of the plastid isozyme (band 2). The amount of activity of the soluble isozyme in the infected cell fraction (B) was due at least in part to contamination by uninfected cells (indicated by uricase activity). Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydro- genase: are. enzymes of the oxidative pentose phosphate pathway, one product of which is ribulose-S-phosphate, which is converted to ribose- S-phosphate by ribose-phosphate isomerase (69).. Ribose-S-phosphate is one of the substrates of 5-phospho-a-D-ribose l-pyrophosphate (PRPP) synthetase, in the first step leading to purine biosynthesis. There- fore, high levels of glucose-6-phosphate dehydrogenase and 6-phosphoglu- conate dehydrogenase would be expected in cells synthesizing large amounts of purines. The specific activity of both of these enzymes in the uninfected cell fraction was about four times the specific activity in the infected cell fraction (Table 5). Starch is the major source of glucose-G-phosphate in most plant cells (70). I have noted starch gran- uales in the plastids of only the uninfected cells in the electron micrographs of Newcomb and Tandon (41). Triosephosphate isomerase activity is present in both the cytosol and plastid fractions (59). The specific activity of this enzyme was essentially equal in both uninfected and infected cell fractions. The specific activity of malate dehydrogenase in the uninfected cell fraction was about twice that in the infected cell fraction (Table 5). While ‘nodule peroxisomes contained no hydroxypyruvate dehydrogenase, lactate dehydrogenase was present in the cytosol, and utilized .. a j'i'f‘ll'd - I I 'I ,‘i DE #- a l'fi‘K “K .uk-Js i 'Ia : , t I r-AL‘ra _ 44 pyruvate, hydroxypyruvate, or glyoxylate as a substrate (Chapter I). The specific activity of lactate dehydrogenase was only slightly higher in the uninfected cell fraction (Table 5). Results of assays for phosphoribosyl amidotransferase activity were inconclusive. Both uninfected and infected cell fractions contained very low levels of this enzyme activity. While this enzyme has been measured in nodule extracts, the specific activity was low compared with uricase (71), for example, and one would not expect to detect activity in the low yields of pure protoplasts obtained in our experiments. PRPP synthetase was also not detectable. It will probably only be possible to localize these enzymes in protoplasts by labeling studies. Phos— phoribosyl amidotransferase has been localized intracellularly in the plastid (59). 45 SUMMARY Several intracellular compartments of nodule cells participate in purine synthesis and degradation to ureides, including plastids, peroxi- somes, the endoplasmic reticulum, and the cytosol (59, 40). Most of the activity of the peroxisomal enzymes uricase and catalase was associated with the uninfected cell fraction. Allantoinase, which has been local- ized in the endoplasmic reticulum (40), also had a much greater specific activity in the uninfected cell fraction. All of these data support the previous report (41), based on electron micrographs, that peroxisomes are found predominantly in the uninfected cells, where smooth endoplas- mic reticulum also proliferates. Purine synthesis has been localized in the plastids (59), which are observed in both cell types in the soybean nodule (41). Purine synthe- sis might therefore occur primarily in only infected cells, only in uninfected cells, or in both cell types. Several of the enzymes whose products are required for purine synthesis, inlcuding phosphoglycerate dehydrogenase, aspartate: aminotransferase, 6-phosphogluconate dehydro- genase and glucose 6-phosphate dehydrogenase, were present at much higher levels in uninfected cells, and the soluble isozyme of aspartate aminotransferase was predominantly found in the uninfected cell frac- tion. However, the possibility of purine synthesis also occurring in the infected cells cannot be excluded on the basis of enzyme distribu- tion alone. If purine synthesis does occur primarily in the uninfected cells, amino acids are probably transported from the infected cells to the uninfected. But if purine synthesis occurs in the infected cells, a purine intermediate might be transported. These and other questions may be resolved by labeling studies using purified protoplasts. CHAPTER III METABOLISM OF UREIDES IN SOYBEAN LEAVES AND SEEDLINGS 46 47 Allantoin and allantoic acid, the major transport forms of nitrogen in soybean xylem sap, must be hydrolyzed in the leaves in order for the nitrogen to be useful in the growth of the shoot. Our knowledge con- cerning allantoinase, allantoicase, and urease, the enzymes likely to be responsible for the hydrolysis of the ureides in soybean leaves, is fragmentary, as indicated in the introduction. METHODS 1. Sorbitol gradients of leaf organelles. Soybean plants were grown as described in Chapter I. Ninety grams of leaves were homogenized in a waring blendor with 450 ml of 15% sorbi- tol in 0.1 M Tricine (pH 7.8) with 40 g buffer-saturated insoluble poly- vinylpolypyrrolidone. Tb remove debri, the 'homogenate was filtered through four layers of cheesecloth and one layer Miracloth, then centri- fuged at 300 g for 15 minutes. The supernatent was centrifuged at 20,000 g for 30 minutes. The pellet was resuspended in 10 ml of the above medium and layered on a step gradient of 10 ml of 85% (w/v) sorbi- tol, 10 m1 of 70% sorbitol, 10 ml of 60% sorbitol, 10 ml of 50% sorbi- tol, and 10 ml of 30% sorbitol. All sorbitol solutions were prepared in 0.1 M Tricine (pH 7.8). The gradient was slowly accelerated for one hour to 106,900 g and centrifuged for 4 hours at this speed. 2. Sucrose gradients of leaf organelles. Soybean leaves (100 g) were homogenized in a waring blendor with 50 g buffer-saturated insoluble PVP and 475 ml of medium containing 15% sorbitol, 0.1 M tricine (pH 7.8), 10 mM KCl, 1 mM MgC12, and 10 mM EDTA. The homogenate was centrifuged as above. The 20,000 g pellet was applied to the microsomal sucrose gradient given in Chapter I, and centrifuged at 106,900 g as above. 3. Sucrose gradients of seedling organelles Eighty g of shoots from 6 day old seedlings were homogenized in 175 ml of the medium used for sucrose gradients (above) with 38 g buffer- saturated insoluble polyvinylpolypyrralidone. The homogenate was filtered and centrifuged as for the above leaf gradients. The 20,000 g pellet was applied to the microsomal sucrose gradient given in Chapter I, and centrifuged at 106,900 g as above. 4. Enzyme assays Marker enzymes were assayed as in Chapter I. Chlorophyll was deter- mined in 80% acetone at 663 nm. Allantoinase was determined by the diphenylformazan method (72), as in Chapter I. Glyoxylate production by allantoicase was measured by coupling to lactate dehydrogenase (73) or by the diphenylformazan method (72). The substrate was 10 mM allantoic acid in either 25 mM phosphate buffer (pH 7.4) or 20 mM Tricine (pH 7.8). Reactions were incubated 30 minutes at 30°C and stopped with the phenylhydrazine reagent for the diphenylformazan determination. Urea production was measured by ammonia formation coupled to urease (Sigma) added in excess to reactions carried out in Conway microdiffusion dishes (74). The reactions were stopped with saturated K2C03 and ammonia determined (74) with Nessler's reagent (Sigma) after diffusion for 2 hours at 30°C. 48 49 Thin layer chromatography was done on glass cellulose plates con- taining fluorescent indicator in 7 ether:2 formic acidzl water. Ureide groups were stained with p-dimethylaminobenzaldehyde (75). For the radioactive assay for allantoicase, 0.8 ml 2-[14C]uric acid in 0.1 M CHES (pH 9.0, Sigma) was incubated with 2 units uricase (Sigma) for 30 minutes at 25°C. The plant or enzyme sample was then added and the reaction was brought to a volume of 1.0 ml in a small vessel containing 0.4 ml 80% hyamine hydroxide in the center well. The reaction was incubated at 25°C, stopped with 0.2 ml 2 N HCl, and allowed to diffuse for 12 hours before counting the [1°C]C02 absorbed by the hyamine hydroxide. RESULTS AND DISCUSSION 1. Isolation of Organelles Isolation of intact peroxisomes from soybean leaves proved to be a difficult task. Good yields of peroxisomes have been previously obtain- ed only from spinach and sunflower leaves, and only poor yields from other species (76). A comparison of catalase activity, the marker enzyme for peroxisomes, in soybean leaf extracts with the levels of activity previously found in spinach leaves (76) is presented in Table 6. Fewer total units of activity were measured in the soybean homogen- ate, and the specific activity was much lower than that in the spinach homogenate, indicating fewer units were extracted from soybean leaves than from an equivalent amount of spinach leaves. The percentage of the original catalase activity found in the 6000 g pellet, which was applied to the sucrose density gradient, was only 4% for soybean leaves, versus 50 TABLE 6 Comparison of Soybean and Spinach Leaf Extracts For Catalase and Peroxisomal Isolation Catalase Activity Fraction 55thSoybean leaves nggfiSpinach leaves Homogenate, units 41,000 583,000 Total protein, mg 715 2,200 Specific activity 58 265 600 g pellet units 700 - 6000 g pellet units 1,600 175,000 % yield of previous step 4 30 total protein, mg 62 460 specific activity 26 380 Peroxisomal peak, units 800 96,800 Leaves were ground in a waring blendor and the homogenate was filtered and centrifuged at 600 g. The supernatant was centrifuged at 6000 g. This pellet was resuspended and applied to a sucrose density gradient. Spinach data are taken from Tolbert gt El“ (1969) Plant Physiology 44:135-147 (76). Units are umol’min'l. 51 30% for spinach leaves, as if a major part of the peroxisomes were broken during the centrifugation steps. The specific activity of catalase in the soybean leaf extract was less than one-tenth that of the spinach ex- tract at this point. The yield of catalase activity in the peroxisomal fraction of the soybean leaf gradient was less than 1% of the yield on the spinach leaf gradient. Thus fewer peroxisomes were extracted from soybean leaves, and many more peroxisomes broke in the steps prior to the sucrose gradient. While adequate amounts of soybean leaf peroxi- somes could be isolated for localization of the major enzyme components, those enzymes present at low levels might not be detectable. Organelles from cowpea leaf extracts were also separated on sucrose density gradients. Even fewer intact peroxisomes were obtained from COWpea leaves than from soybean. COWpea organelles were also separated on a zonal gradient, but the yield of peroxisomes was very low. Almost all of the catalase activity appeared in the soluble fraction, apparent- ly from broken peroxisomes. Different methods of grinding or chopping the leaves were tried, as well as the addition of 2.5% w/v Ficoll to the medium. None of these methods gave any improvement in the yield of intact peroxisomes. It was necessary to add at least 1 g insoluble polyvinylpolypyrralidine per 10 g of leaves to prevent browning of the extracts due to oxidation of phenolic compounds in the leaves. Much better yields of peroxisomes were obtained from growth chamber-grown plants than from greenhouse plants. 2. Localization of soybean leaf allantoinase Sorbitol density gradients were used in order to avoid the possible interference by sucrose in the diphenylformazan assay for allantoinase 52 (Chapter I). The organelle fractions were broader on the sorbitol gra- dients and much of the activity of the peroxisomal marker enzyme, cata- lase, overlapped the mitochondrial marker activity of cytochrome c oxidase, as seen in fraction 8, Figure 7. Intact peroxisomes of higher purity appeared at a density of 1.25 g/cc in fraction 12, in which the specific activity of catalase was 700 umol min'l mg protein-1. Traces of uricase were also found in the peroxisomal fraction, but the levels were very low, as expected in the leaves. Allantoinase activity co-sedimented with cytochrome c reductase, the microsomal marker enzyme. Allantoinase specific activity in the microsomal fraction was 120 nmol min.l mg protein-1. Total allantoinase activity was 320 nmol min"l gram fresh weight-1, about half the level found in nodules per gram fresh weight (Chapter I). The specific activity of allantoinase in a total leaf extract was 30 nmol min"1 mg protein-1. The Km for allantoin was about 30 mM. The pH optimum of the enzyme reaction was 7.8 to 8.0. Allantoin rapidly hydrolyzed nonenzymatically above pH 8.5. Soybean. leaf organelles were also separated, on sucrose density gradients (Figure 8). Part of the catalase activity was found in frac- tion 7, where the mitochondrial fraction was located, as marked by cyto- chrome c oxidase, but the highest specific activity of catalase, 900 1 1 mg protein' , was found in fraction 12, at a density umol min’ of 1.25 g/cc. This density is typical for a peroxisomal fraction (1). Allantoinase again co-sedimented with the microsomal marker cytochrome c reductase at 1.18 g/cc. Allantoinase maximum specific activity was 70 nmol min"l mg protein’l. Sucrose gave only a slight reaction in the diphenylformazan assay for allantoinase when performed as specified Figure 7. Separation of Soybean Leaf Organelles on a Sorbitol Density Gradient. Organelles were first pelleted by differential centrifugation, resuspen- ded, and applied to the sorbitol gradient. The gradient was fraction- ated from the top (fraction 1 has the lowest density). 53 Catalase Cyto c .239" Oxidose (3%. iMhmamme lflkmqiwfl 50 i E E E 1' so” is O C HMO—H‘O-w—O—Oovmoo : « ——oH—-—v—-o Cytochrome c Uricose Reduclose 30 K) r .i L LrllLli '0 - fraction iJ Figure 7. Separation of Soybean Leaf Organelles on a Sorbitol Density Gradient 54 Figure 8. Separation of Soybean Leaf Organelles on a Sucrose Density Gradient. Organelles were first pelleted by differential centrifugation, resuspen- ded, and applied to the sucrose gradient. The gradient was fractionated from the top (fraction 1 has the lowest density). 55 nmols min.I ml.I Figure 8. Cololo sen— irio‘ as; P‘ .-+-4«r~e e- o 0‘4 O 0 ' jLrAllonloi nose 200" I H1“: tit Cytochrome c Re ductose i L7 l r ___J Cytochrome c Oxidose Cololose SPECIFIC ACTIVITY ‘J I “I . 4 .030 “LI—Hm er] . .v—o-qH-n—v-Q '0 Fraction a 8 nmols gmin" ml”I «9O absorbance / ml I § . é . nmols minI mg protein Separation of Soybean Leaf Organelles on a Sucrose Density Gradient 56 57 in the Methods section. The sucrose reaction was subtracted by running zero time controls. Germinating soybean seedlings also degrade purines, presumably utilizing ureides stored in the cotyledon. While only traces of uricase were found in seedling peroxisomes, high levels of allantoinase occurred in the microsomal fraction (Figure 9). In all of the soybean leaf or seedling gradients, allantoinase was found in the microsomal fraction, probably originating from the endo- plasmic, reticulum. These. results contrast with previous reports of peroxisomal allantoinase (13, 14), but agree with localization of allan- toinase in soybean nodules (Chapter I, 40). 3. Soybean Allantoicase Allantoicase activity may be determined by measuring either glyoxy- late or urea, the products of the reaction. I chose first to look for glyoxylate, since enzymes involved in the metabolism of this compound are always found within peroxisomes (1). Several colorimetric or enzy- matic methods for determination of glyoxylate are available. Noguchi gt 2l° (13) assayed allantoicase by the method of Gregory (73) and localized fish liver allantoicase in peroxisomes. In this assay (73), oxidation of NADH by lactate dehydrogenase is coupled to glyoxylate production by allantoicase (Chapter IV). However, allantoi- case activity could not be detected in peroxisomal fractions or crude extracts of soybean leaves by this assay. Low endogenous rates were present that were not dependent on either allantoin or allantoic acid. Low levels of glyoxylate may be present in most leaf extracts and in the substrates, allantoin and allantoic acid, which decompose slowly to Figure 9. Separation of Organelles From Soybean Seedling on a Sucrose Density Gradient. Organelles were first pelleted by differential centrifugation, resuspended, and applied to the sucrose gradient. The gradient was fractionated from the t0p. Data in Figure 9a, b, and c are all from the same gradient. 58 59 m. , O. ‘ ‘ 1 $85.5 $230 0 mEoEootAo ’ I ‘ HI 8.69.“. a I) T _ rxz~oo+m |UJ /eouoqmsqo '_|LLI |_UILLI stowu + on meCHOEOH—d. rill. i...iiii.-.. . Ira . E stIHiHH. i i U . w i ij H _ r mu . _ _ _. w J If i H as.-. L M . U. u . o r m .. w s f mmocoaomm 0 2.9625 rt. coco. toom . .v on 60 .nm mc:m_c Co. 005 n. . -Io.r well in_-n- i i I. :0... b / 3 3 :nN. n w moi S .Ooo.tw.. U . -.i I w rm. _ _ . . _ 82200 _ 88 . . H E 61 PPPPPP r h if D r P b .om mesmwm c262... c238... . . . .9. . . r .o. ..... AI . o. n _ . - . . . . .I i , F b b t? D r h I 4 1 4 q 1 I 4 1 1 44444 . smoc_x00oEoEoo§oL ii.i.:...iri::tiil ”iiiI.oiIooI.4iIl_ . HIIH M PPPPP H I. § H .10. |_uiaio.td 6w |_uitu S|OLUU mmofinnmm o 9:02.023 7 l_uisi01d but I_uiui S|OUJU J _ i f _ n w . w . S H w w com .4 s. m. 1 , _ l _ HI m _ b . _ d . rt 1 _ 8.. ma 3050,62 .< rt. - on. meOHOQ . W .F c , . it - _ 62 glyoxylate nonenzymatically. The addition of 0.1% Triton X-100 deter- gent and variations in pH from 7.0 to 8.5 did not reveal allantoicase activity in either cowpea or soybean leaves. The addition of 0.8% sodium deoxycholate or 0.2% digitonin did not effect allantoicase activ- ity. The sensitivity of the coupled lactate dehydrogenase assay was tested by addition of glyoxylate. A pulse of 10 nmol of glyoxylate was the minimum amount required for a measurable activity. It is not known whether the expected allantoicase activity would be great enough to per- mit an accumulation of glyoxylate sufficient for detection by this assay. Allantoicase activity might also be measured by coupling to the serine:glyoxylate aminotransferase already present in the peroxisomes. The products would be glycine and hydroxypyruvate, the latter of which could be coupled to hydroxypyruvate reductase monitored in turn by oxi- dation of NADH at 340 nm. This method is attractive because the two coupling enzymes should already be present in the peroxisomes. However, no allantoic acid-dependent activity was found in organelle fractions by this assay. The coupling enzymes were present in the peroxisomal frac- tion at a level sensitive enough to detect a pulse of 20 nmol of glyoxy- late. The most sensitive method for determining glyoxylate is the diphenyl formazan colorimetric method (72). When using this assay for allantoicase, I stopped the reactions with the phenylhydrazine reagent on ice, and proceeded with the color determination. High concentrations of chlorophyll, as in the chloroplasts, interfered slightly with the peak absorbance at 520 nm. Sucrose did not interfere with this assay for allantoicase, since the reactions did not have to be boiled. As 63 described earlier (Chapter I), sucrose is a problem in the allantoinase reaction which must be boiled in order to hydrolyze allantoic acid to glyoxylate. No allantoicase activity could be detected in total leaf extracts or peroxisomal fractions with the diphenylformazan assay for glyoxylate. Both allantoin and allantoic acid were tried as substrates, as well as various detergents and buffers. A spectrophotometric assay for allan- toicase was also attempted, measuring the phenylhydrazone of glyoxylate at 324 nm, but no allantoicase activity was detected. Low rates of activity (30 nmol min.1 ml'l) were occasionally found in the chloroplast fraction, but this activity was not substrate dependent, and was probably due to an artifact caused by the high concentration of chlorophyll. Allantoicase activity did not occur in chloroplasts of seedlings, which were shown to hydrolyze allantoin in viva (see Section 4). The above diphenylformazan assay for soybean allantoicase was per- formed under the same conditions, including the addition of umgnesium chloride, which were successfully used to measure allantoicase from fish liver (Chapter IV). Since this endpoint assay is sensitive enough to detect low levels of activity, the lack of activity in soybean leaves may indicate that the pathway or the enzymes in soybean tissues are different from those in fish liver. Allantoicase activity has been reported in extracts of leaves of nitrate grown bushbeans (36). The assay was unusual in that a small amount of phenylhydrazine was present during the reaction to trap glyoxylate produced. The diphenylformazan determination was performed after the reaction was stopped on ice. I found no allantoic acid-depen- dent activity in soybean leaf or seedling extracts with this assay, although artifactual rates in the absence of substrate were observed. 64 Another possible method of detecting allantoicase would be to mea- sure the urea produced by the reaction. The urea production could be coupled to urease, and the ammonia determined by Nessler's reagent. It was necessary to microdiffuse the ammonia from the reaction chamber before carrying out the color reaction, which is sensitive to inter- ference by many compounds (74). The reaction was stopped with strong base, driving off the ammonia, which was absorbed by acid in an inner compartment of the reaction chamber. Using this procedure, no allantoi- case activity was detected in peroxisomal fractions or total extracts from soybean leaves, with uric acid, allantoin, or allantoic acid as substrates. In fact, the peroxisomal extracts incorporated part of the background ammonia present in the reagents. No urease activity was found in leaf extracts by this method. Alternate pathways for allantoin metabolism exist in micro-organ- isms. Allantoin is fermented by Streptococcus allantoicus through a series of intermediates including allantoic acid, glyoxylurea, and carb- amyloxamic acid, yielding oxamic acid, ammonia, ATP, and NADH as pro- ducts (77). This pathway occurs only in the presence of NAD, MgSOA, and phosphate or arsenate. In the absence of these cofactors, allantoic acid is degraded to glyoxylate and urea. Activity of the enzymes of this pathway could be determined by measurement of oxamate (78) or reduction of NAD at 340 nm. Although detectable levels of oxamate were present in soybean leaf extracts, no allantoic acid-dependent production of oxamate was found, nor was NAD reduced in the presence of allantoic acid and the above cofactors. ‘fl , 1 NJ. on—A e,_ 'I. -t Ina o-._ ‘- 65 4. Radioactive Assay for Allantoicase Ureides were separated by thin layer chromatography or high voltage electrophoresis and stained with p-dimethylaminobenzaldehyde (75), which gives a yellow color for allantoin, allantoic acid, or urea. Uric acid was detected by fluorescence. Soybean leaf extracts contained high levels of allantoic acid and urea when separated by either method. This accumulation may indicate that the allantoicase and urease reactions are limiting steps in the pathway, allowing a build-up in the pools of allantoic acid and urea. A radioactive .111. _v_i!9_ assay was employed for allantoinase and allantoicase. [2-14C]Uric acid was converted to [2-14C]all- antoin using excess uricase purchased from Sigma. The conversion reac- tion, monitored at 293 nm for absorption by uric acid, was essentially complete, since the radioactive allantoin was free of uric acid when analyzed by thin layer chromatography. Any remaining uric acid would not have interfered with the assay, although the specific activity of the allantoin. would have been lower than. expecteds. Soybean pods, leaves, or seedlings were incubated with the [2-140]allantoin in closed vials containing a small center cup filled with 80% hyamine hydroxide. [1°C]C02 from. completely hydrolyzed allantoin was trapped in the base. When extracts were assayed, the reaction was stopped. with. acid, driving off the [1401002. This assay' requires the action of all three enzymes in the pathway; allantoinase, allantoi- case, and urease. Since the [2-14C]uric acid contained a small amount of [1°C]urea, control reactions of substrate 'plus purchased uricase and urease were always run as well as nonenzymatic controls, and any background counts found were subtracted from the enzyme reactions. {Iv ,.sc 1.. 'I'I mm . N... . a - Ill 66 Since several hours were necessary for uptake of substrate in the in ES assay, and the uptake was not quantitative, it is not possible to determine enzyme rates from these data, and many factors such as differ- ences in rate of uptake may have affected the results. However, the data does indicate that a pathway for degradation of allantoin does exist in these plants, although it has not been possible to demonstrate activity of the pathway .i_n $59. With extracts of soybean pods only very low levels of allantoin degradation in the radioactive assay were observed. When samples of these reactions were separated by high voltage electrophoresis, the majority of the label was in urea. In view of the recent report (42) that urease is not present in the shoot, it may be concluded that the final step in the pathway did not occur in these reactions. However, the presence of label in urea indicates possible activity of allantoi- nase and allantoicase. Whole pods also showed very low activity for the whole pathway in 3.5319. (Table 7). Earlier work suggested allantoin was hydrolyzed to glyoxylate in wheat seedlings (38). Wheat seedlings should contain the allantoin pathway, since ureides are stored in many seeds (22), even though mature wheat plants do not contain detectable ureides. Seedlings were also very useful for these experiments because of their small size and high rate of uptake of substrate through the roots. Using the radioactive assay procedure, wheat seedlings degraded 4 to 9% of the labeled allan- toin to [14C]C02 (Table 7). Soybean seedlings had much higher levels of allantoin hydrolysis in 1119, degrading 60% of the allantoin in either light or dark (Table 7). The seedlings probably did not take up all of the [1°C] allantoin au- a D a t‘ I 4" II .‘V aa a li- Ls 67 surrounding the roots. An extract of soybean seedlings degraded only a small fraction of the [1°C]allantoin, in agreement with earlier failures to detect the final steps of this pathway in soybeans in_!i££g (Section 3). All attempts to measure the production of either glyoxylate or urea as a result of allantoicase activity in zitgg have met with little or no success. Results of in 3133 experiments indicate that some path- way for degradation of allantoin does exist in soybean plants, but the identity of the intermediates and products, the level of activity of the latter enzymes in the pathway, and the intracellular location of the pathway remain to be determined. Allantoinase activity was present in all soybean tissues examined, including nodules, leaves, and seedlings, and was always associated with the endoplasmic reticulum. 68 Table 7 Allantoicase Activity in Soybean Seedlings [2-14C]allantoin [14C]C0 supplied released Degradation cpm cpm % Soybean seedlings 24 hours light 174,000 98,600 57 4 hours light 380,000 209,000 55 4 hours dark 380,000 226,000 60 extract 380,000 10,000 2.6 Soybean pods 828,000 6,000 0.7 225,000 7,850 3.5 Wheat seedlings 440,000 16,400 3.7 225,000 20,000 8.9 Allantoicase was assayed in vivo by the radioactive method. Controls were run to determine the amount of [1°C]urea contaminating the [14C]allantoin, as well as any nonenzymatic breakdown, and these low background levels were subtracted. 'In vivo reactions were incubated for 24 hours to allow uptake, degradation, and absorption of [14C]C02, except where otherwise specified. A soybean seedling extract containing 2 mg protein was incubated for 2 hours and stopped with HCl. Specific acitivity of the [2-14C]allantoin was 1000 cpm‘nmol'l. CHAPTER IV INTRACELLULAR LOCALIZATION OF PURINE DEGRADATION IN FISH LIVER 69 .1. A: an! O . .1 A: .i.‘ 70 INTRODUCTION Previous reports disagree as to the intracellular location of some of the enzymes of purine degradation in the lower vertebrates, including fish, amphibians, and birds. Xanthine oxidase was reported only in the soluble fraction from fish liver (13), although no data was shown, while xanthine dehydrogenase from chicken liver and kidney was peroxisomal (14), but could not be detected in frogs (14). Enzyme activities in this latter paper (14) were given as a percent of the total activity or as relative units to the mean activity of all the fractions, with no data on activity or specific activity. Xanthine oxidase and xanthine dehydrogenase appear to be two forms of the same protein (15). Uricase is always in the peroxisomal fraction from all tissues (13,14), and is the final enzyme of the pathway present in chickens (14). Allantoinase was reported to be present in liver peroxisomes from frogs, but was in the soluble fraction in frog kidney (14). It was necessary to include MgC12 1n the grinding media in order to keep allantoinase in the peroxisomal fraction of frog liver (14). In another report (13), uricase and allantoinase appeared to be located in the per- oxisomal matrix and allantoicase in the peroxisomal membrane from fish liver. While uricase and allantoinase were present also in the soluble fraction as a result of peroxisomal breakage, allantoicase was located only in the peroxisomes (13). Allantoicase was reported to be soluble in frog liver and kidney, although no assay or data were given (14). In view of the fact that my results with plant ‘tissues located allantoinase in the endoplasmic reticulum (Chapter III), I have reexamined 71 previous work on this enzyme. The allantoinase assay of Noguchi gt El. (13) was dependent on glyoxylate reduction by excess lactate dehydrogen- ase. When allantoin was the substrate, the activities of both allantoi- nase and allantoicase would be required to produce glyoxylate in this coupled assay. Soluble allantoinase should not have been detected, since allantoicase was present in the peroxisomal fraction only. The authors gave no explanation for the reported allantoinase activity shown in the soluble fraction and did not mention any other assay method. Obviously such discrepancies between the data and the assay cast doubt on the conclusions reached by these authors. Indeed, such a coupled assay, dependent on the presence of both enzymes, is not appropriate for a localization experiment for either enzyme. In my studies on allantoin metabolism, I have used a direct diphenylformazan assay for the separate catalytic products, allantoic acid and glyoxylate (72). Thus allantoin- ase and allantoicase activity can be measured independently. Since questions remained regarding the location of purine degrada- tion in animal cells, and allantoicase activity could not be detected in soybean leaf peroxisomes, I investigated purine degradation in fish liver. I hoped to clarify the localization of allantoinase, which was microsomal in all soybean tissues, and to learn more about optimum con- ditions for assaying allantoicase. Data and experience with the fish allantoicase would be useful in searching for this enzyme in soybean leaves, if indeed the same enzyme were present. MATERIALS AND METHODS Bluegill-green sunfish hybrids were the gift of Jay Gouch of the Fisheries and Wildlife Department. Rainbow trout were provided by Dr. J. tr .r 0* at I .- Ola- an ...... ., . ':Q. :“~ 72 .J. Hoffert from the Physiology Department. Madagascar mouth breeders were purchased from a local pet shop. Grinding media, tissue, and gradient varied between experiments as detailed below. Minced liver was extracted once with a Tektron homogen- izer, filtered through six layers of cheesecloth or Miracloth, and cen- trifuged at 300 g for five minutes. The supernatant was applied to the gradient described below, centrifuged for three hours at 106,900 g, and then fractionated from the top of the gradient. Gradient 1: Six g liver from two bluegill-green sunfish hybrids was homogenized in 10% sucrose in 0.1 M Tricine, (pH 7.5) and applied to a step gradient of 3 ml of 2.3 M sucrose, 5 ml of 1.9 M sucrose, 7 ml of 1.8 M sucrose, 7 m1 of 1.75 M sucrose, 7 ml of 1.7 M sucrose, 7 ml of 1.5 M sucrose, 6 ml of 1.3 M sucrose, 4 ml of 1.0 M sucrose, and 4 ml of 0.83 M sucrose; all prepared in 0.1 M Tricine (pH 7.8). Gradient 2: 1.6 g liver (bluegill-green sunfish) was homogenized in 8.5% sucrose in 1 mM phosphate (pH 7.5) and applied to the above sucrose gradient, prepared in 1 mM phosphate (pH 7.5). Gradient 3: 1.6 g liver from the same fish as gradient 2 was homo- genized in 15% sorbitol, 0.1 M Tricine (pH 7.8), 10 mM KCl, 1 mM MgC12, and 10 mM EDTA (Beever media, 79) and applied to a gradient identical to gradient 2. Gradient 4: 1.1 g liver from two Madagascar mouth breeders was homogenized in 8.5% sucrose in 1 mM phosphate (pH 7.5) and applied to a gradient identical to gradient 2. Gradient 5 and 6: 4.4 g liver from two rainbow trout was homogen- ized in 10 ml Beever media and divided between two gradients as above. (hedient 5 contained 0.1 M tricine (pH 7.8); gradient 6 contained 1 mM phosphate (pH 7.5). 73 Gradients 7 and 8: 2.9 g of rainbow trout liver was homogenized in 0.25 M sucrose, 20 mM Tricine (pH 7.8), 20 mM KCl, 1 mM MgC12, and 20 mM EDTA (medium 7) and divided between two gradients. Gradient 7 was a 48 ml linear gradient of 0.75 M to 2.0 M sucrose on top of a 3 ml pad of 2.3 M sucrose, all prepared in 20 mM Tricine (pH 7.5). Gradient 8 was identical, except for the addition of 1 mM MgC12. Gradients 9, 10 and 11: 3.0 g of rainbow trout liver was homogen- ized in 10 ml medium 7 and divided between three linear gradients of 1.0 M sucrose to 2.1 M sucrose, over a 3 ml 2.3 M sucrose pad. Gradient 9 was prepared in 20 mM Tricine (pH 7.5). Gradient 10 was prepared in 5 mM Tricine (pH 7.5) plus 1 mM MgClz- Gradient 11 was prepared in 20 mM Tricine (pH 7.5) plus 2 mM dithiothreitol. Catalase, uricase, NADH-cytochrome c reductase, cytochrome c omi- dase, xanthine dehydrogenase, and allantoinase were assayed as described in Chapter I (40). Allantoicase activity was measured in the presence of 10 mM allan- toic acid in 25 mM phosphate (pH 7.0) or 20 mM Tricine, (pH 7.5) at 30°C for 10 to 30 minutes. The reaction was stopped by the addition of the phenylhydrazine reagent (0.8 ml) for the diphenylformazan determination of glyoxylate (72). This determination differs from the allantoinase assay, in which the reactions must be boiled to hydrolyze the product allantoic acid to glyoxylate, which is then measured in the diphenylfor— mazan determination. RESULTS AND DISCUSSION Organelles from the liver of three species of fishes were separated on sucrose density gradients by essentially the same methods used in 74 Table 8 Total Activity of Enzymes Involved in Purine Degredation in Fish Liver Soluble Peroxisomal Fraction Allantoinase Uricase Catalase Allantoicase gradient units S.A. units S.A. units S.A. units S.A. 1* 150 - 60 - 700 - 15 - 2 510 92 30 140 220 1120 5 28 3 830 240 31 100 250 920 5 20 4* 400 106 12 45 770 6380 5 2.5 5* 2300 240 250 43 9470 1610 162 28 6 2100 230 74 74 2650 4260 9 7 7 1620 260 29 100 1870 7190 40 80 8* 2090 250 150 32 5470 1200 415 91 9 1150 - 21 - 1160 - 25 - 10 1400 212 34 34 1490 1490 33 33 Peroxisomes were purified on sucrose density gradients as specified in Methods. Approximately 1.5 to 2.0 g liver was used per gradient. Enzyme units are nmol min‘l, except catalase which is in umol -1, and S.A. are units'mg protein-l. Allantoinase data is from the min soluble fraction; no activity was present in the peroxisomal fraction. Uricase, catalase, and allantoicase data are from the peroxisomal frac- tion, except where an asterick (*) indicates the organelles aggregated into a single band. 75 isolation of plant organelles. In several cases, especially in the presence of high concentrations of buffer (0.1 M Tricine) or 1 mM MgC12. all of the organelles aggregated into a single fraction (Table 8, gradients 1, 4, 5), or the mitochondria and peroxisomes failed to separate (gradient 8). Better separation was obtained on gradients con- taining only 1 mM phosphate buffer, and no MgClz (gradients 2, 3, 6). However, more allantoicase activity was found when higher concentrations of buffer or MgC12 were used (gradients 1, 5, 8). In all the gra- clients, including those in which organelles aggregated, allantoinase activity was only in the soluble fraction (Table 8, Figure 10). Enzyme units and specific activities in the soluble or organelle fractions of several of the enzymes involved in purine degradation are given in Table 8. The enzyme activities given are from the peroxisomal fraction, except for allantoinase. Uricase, catalase, and allantoicase activities were highest in the peroxisomal fraction, while allantoinase was found only in the soluble fraction (Figure 10). Levels of uricase and allantoicase varied widely betwen experiments, and were highest in those cases in which the organelles aggregated (gradients 1, 5, 8). Levels of uricase and allaantoicase appeared to fluctuate in parallel, while the level of allantoinase was independent of the other purine oxidizing enzymes. Allantoicase was difficult to localize since the conditions yield- ing the highest enzyme activity and stability caused all the organelles to aggregate into a single fraction. While allantoicase activity was very stable at high buffer concentration (0.1 M Tricine, gradient 1, 5) or in the presence of MgClz (gradient 8, 10), very little activity was found on 1 mM phosphate gradients (gradients 2, 3, 4, 6). Although the Figure 10. Isolation of Fish Liver Peroxisomes on a Sucrose Density Gradient. The 300 g supernatent from a liver homogenate was applied to a sucrose gradient prepared in 20 mM tricine. The gradient was fraction- ated from the top. Figure 10a to 10d data are all from the same gra- dient. 76 77 w Uricose ml"l - a .L A A ' nmols min Cotolose rm 8 umols min‘I ml"l A 8 Fraction Figure IO. Isolation of Fish Liver Peroxisomes on a Sucrose Density Gradient. (J Allantoicase 5 A L A_ A v v f v ml'l IZW ’ Oxidose nmols min I? ir—r—o—no— l 5 Cytochrome c 500» I ”“5 ' +--r—4-~a- l - o ‘a-e-d-e : e v . A.‘ A A A Cytochrome c Reducto se IO 15 Fraction Figure lOb. Uricose 7' £5 £2 $2 404 G. U' E f; 25g Allantoinase E .‘2 O E C IOOv T m» . .E Catalase £2 9. C1 0" E5 .- E mt 5.”. o $— AAAAAA A g ' 5 IO f Y VIS ' Fraction Figure 10c. 79 Allantoicase 80» T .E 33 zo- 9 [3:4 .111 c"mo -' Cytochrome c c» . E Oxrdose T .E E5 2%. 1’2 0 E JUL My..- g c V fij v fifi ' ' ' V v 300 Cytochrome c l Reductose 50‘. L A 4 .4” . 7'1 '1 - ? I0 l5 Fraction Figure 10d. 80 81 same homogenate was applied to gradients 5 and 6, only gradient 5 (0.1 M Tricine) contained a high level of allantoicase in the organelle frac- tion, even though much better separation of organelles was achieved on gradient 6 with 1 mM phosphate. Gradients 7 and 8 are identical, except for the addition of 1 mM MgC12 to gradient 8. While both gradients contained equal amounts of allantoicase in the soluble fraction, gra- dient 8 contained 10 times as much activity on the gradient, in which the organelles unfortunately aggregated. Data from gradient 7 is pre- sented in Figure 10. Gradients 9 and 10 received equal portions of the same homogenate, but differ in their composition, gradient 10 containing less buffer (5 mM Tricine) with the addition of 1 mM MgC12. In this case, both gradients contained about the same level of allantoicase activity and reasonable separation was obtained on gradient 10 (MgClZ). Allantoicase again peaked in the peroxisomal fraction. All the allantoicase data presented were obtained by the direct diphenylformazan assay for glyoxylate (72). All of the peroxisomal fractions were also assayed by the coupled lactate dehydrogenase assay of Noguchi 35 El. (13), but little or no activity was found. This assay is not as sensitive as the diphenylformazan assay, since the Km of lactate dehydrogenase for glyoxylate is quite high (80). Maximum allantoicase activity was found between 10 to 20 mM allantoic acid with the diphenylformazan assay. Since high allantoicase activity had been found when organelles aggregated into a single fraction, samples of all the organelle fractions from gradients 2 and 3 were pooled and assayed to see if some kind of interaction or synergism between different organelles were necessary for allantoicase activity. However, no activity was found in either mixture of organelles. Allantoinase activity appeared only in the soluble fraction from all ten gradients, and was not present in the microsomal fraction, indi- cated by the marker enzyme NADH-cytochrome c reductase. This was surprising, since this enzyme was in the uncrosomal fraction from all soybean tissues, and had been reported in the peroxisomal fraction from fish liver (13). The hypothesis was considered that allantoinase might be contained in very light microsomes which did not sediment into the gradients. To test this, 1.0 ml of the soluble fraction of gradients 2 and 3 was diluted with 2 ml 25 mM phosphate buffer, pH 7.5, and centri- fuged at 145,000 g for 1 hour. The supernatent of both samples appeared biphasic, with an upper cloudy layer and a clear lower layer. These were removed separately, and the pellet was resuspended in 1.0 ml buf- fer. These three fractions from each tube, as well as the original soluble fraction from each gradient were assayed for allantoinase acti- vity (Table 9). Almost all of the allantoinase activity remained in the supernatent fractions, consistent with the probability that this enzyme was not contained in microsomes. The possibility remains that allan- toinase is only loosely bound to fish microsomes and was released under the homogenization conditions for these experiments. It is not con- sidered likely that the soluble allantoinase came from broken peroxi- somes, since no allantoinase activity was found in intact peroxisomes, which were recovered in good yield on several of the gradients (Figure 10). Gradients 5 and 6 were used for characterization of allantoicase. Phsophate buffer (25 mM) was not inhibitory in the assay, and gave values equal to those obtained using 25 mM Tricine. In several instan- ces samples lost all allantoicase activty when diluted 1:10 with l amt 82 83 Table 9 Tests for the Localization of Allantoinase in the Soluble Fraction by Centrifugation gradient 2 Allantoinase units nmol'min"1 soluble fraction from gradient 309 upper supernatent 130 lower supernatent 168 pellet 6.6 X recovery in supernatents 98% gradient 3 soluble fraction from gradient 696 upper supernatent 242 lower supernatent 334 pellet 5.0 Z recovery in supernatents 83% One ml of the soluble fraction from the gradient was diluted with 2 ml of 25 mM phosphate buffer (pH 7.5) and centrifuged at 145,000 g for 1 hour. The supernatent was divided about equally into upper and lower fractions, and the pellet was resuspencded in 1 ml buffer. 84 phosphate buffer and incubated for one hour. Higher rates of activity were found using smaller samples of enzyme, as shown in Table 10. It is not likely that lower rates with increasing enzyme samples were caused by substrate depletion, since 10 mM allantoic acid or 10 nmol of sub- strate was present in the assay, and fewer than 100 nmol of product were produced. The nonlinearity of activity versus enzyme concentration may have been caused. by jproduct inhibition. or limiting amounts of some cofactor. Addition of MgClZ to the reaction caused a dramatic increase in allantoicase activity (Table 11). The gradient fractions containing the highest allantoicase activity were chosen for this experiment, since the soluble fraction contained MgClz from the homogenization media. As mentioned above, greater initial activity was present in the aggregated organelle fraction from gradient 5, which contained 0.1 M tricine, than in the peroxisomal fraction of gradient 6, which contained only 1 mM phosphate. In fact, the allantoicase activity initially present in the peroxisomal fraction of gradient 6 (9 nmol min”1 ml'l) was almost completely gone (0.4 nmol min'l ml’l) when this experi- ment was performed, but activity was restored by the addition of MgClzo Maximum stimulation by MgC12 occured at a concentration of 5 mM or greater, which was included in all further experiments with this enzyme. These results provide a possible explanation for the instabil- ity of this enzyme in dilute buffer, and low activity on 1 mM phosphate gradients. The pH optimum for allantoicase was approximately 7.0 (Table 12). This experiment was complicated by the instability of allantoic acid at lower pH values. The nmol of product formed by substrate incubated 85 Table 10 Non-linearity of Allantoicase Assay Enzyme Aliquot Activity nmol min.1 ml"1 10 ul 102 20 ul 54 30 ul 51 40 ul 33 Aliquots of the soluble fraction of gradient 5 were assayed for 5 minutes by the diphenylformazan assay (72). The enzyme samples con- tained 1 mM MgC12 from the grinding medium, but MgC12 Was not added to the assay. 86 Table 11 Stimulation of Allantoicase by Magnesium Chloride Allantoicase activity MgClz gradient 5 gradient 6 nmol‘min'l‘ml-1 none 114 0.4 0.1 mM 142 2.6 1.0 mM 190 2.6 5.0 mM 311 10.2 10.0 mM 270 9.7 10.0 mM 289 10.6 10 ul of the organelle fraction from gradient 5 or 20 ul of the peroxisomal fraction of gradient 6, along with the indicated addition of MgClz to the substrate, were assayed for allantoicase activity. 87 Table 12 Effect of Buffer and pH on Allantoicase Activity 'Euffgr ‘25 Nonenzymatic Total Reaction Reaction Enzyme rate nmol product'20 min.1 nmol min"1 ml-l Acetate 4.7 17.2 12.4 -16 MES 6.2 4.3 16.0 39 Phosphate 6.8 4.2 19.5 51 MOPS 7.2 0.8 17.6 47 TES 7.5 2.5 15.8 45 Tricine 8.2 3.6 13.3 32 CHES 9.3 1.8 5.5 12 Allantoic acid (15 mM) was prepared in the various buffers with 20 mM MgC12, and incubated both with and without 15 ul enzyme for 20 minutes at 30°C. The reaction was terminated by the addition of the phenylhydrazine reagent and nmol glyoxylate determined by the diphenyl- formazan reaction. The net enzyme rate was calculated by the difference between (nmol from the enzyme reaction minus nmol from the nonenzymatic reaction) divided by (20 minutes X 15 ul enzyme). Control reactions indicated the various buffers had little intrinsic effect on the reac- tion rate. :umumcmyn‘ufl Imam .1) Jr“ dlfi ..1-z' '3 .b \' I‘Q“ r4. - 4 u. r ,4. ..; . I.“ “ in: 21", 'bn tr-L a.“ fa; 88 alone or with the addition of enzyme is shown in Table 12, along with the net.tenzyme rate calculated by subtraction of the non-enzymatic reaction. The addition of pyridoxal phosphate had no effect on the allantoi- case reaction. Dithiothreitol had a slightly inhibitory effect on the enzyme reaction. Xanthine dehydrogenase or oxidase were not detectable in any of the fish liver extracts or gradients. This enzyme is known to be unstable, but sometimes may be stabilized by dithiothreitol (81). However, no activity was found in any of the fractions from a gradient containing 2 mM dithiothreitol, or in a sample of the homogenate to which dithio- threitol. was added. These assays were performed immediately after fractionating the gradient, but the activity was apparently rapidly lost or there was very low activity in the species of fish used for these experiments. The assays were done at both 293 nm and 340 nm for xanthine oxidase and dehydrogenase, respectively, and were quite sensi- tive when. checked. with purchased xanthine oxidase. Since previous workers (13) provide no actual data for this enzyme, little can be con- cluded at this point regarding the localization of xanthine dehydrogen- ase. Xanthine dehydrogenase was in the soluble fraction in soybean nodules (40). , .51 V...- out ‘.. u.. r .q, a q., 3 i "1’ 4. 1 . 1‘ V3. 89 SUMMARY Purine degradation serves very different functions in plants and animals. Animals degrade purines to a form which is convenient for excretion, since animals have no further use for these compounds. In the plant world, purine degradation products are used by many legumes as an efficient form in which to transport nitrogen from the nodule for utilization in other portions of the plant. As different as these pur- poses are, many similarities exist between the pathways of purine degra- dation in plants and animals. The most important of these is the com- partmentation of at least part of the pathway in peroxisomes. The uri- case reaction always takes place within peroxisomes, where catalase is also present t1) destroy the ‘hydrogen peroxide produced by ‘uricase. Allantoicase has been localized in peroxisomes of animals such as fish, which excrete urea. This enzyme is not present in nodule peroxisomes, since the pathway ends with the allantoinase reaction in soybean nodules. Nodule peroxisomes appear to function only in purine degrada- tion, since they do not contain many of the enzymes normally found in glyoxysomes or leaf peroxisomes. The nature of the final reactions of purine degradation in soybean leaves has not been elucidated, but an enzyme such as allantoicase may be involved, and might be expected to be compartmented within leaf peroxisomes if glyoxylate is a product of the reaction. The allantoinase reaction is unusual in that it is localized in different compartments in plants and animals. Allantoinase is associ- ated with the endoplasmic reticulum in soybean nodules, leaves and seed- lings. The enzyme in fish liver is found in the soluble fraction. This 7 .1” TfJ—TTI D 41'". ital-24¢?- ‘i’_" V: 90 result remains an enigma, since the enzymes preceding and following allantoinase, uricase and allantoicase, are localized in peroxisomes, and it is not clear why allantoinase should not: also be localized in peroxisomes. It is not considered likely that allantoinase was released from the peroxisomal fraction during these experiments, since none of the peroxisomal enzymes, uricase, catalase, and allantoicase followed a distribution similar to that of allantoinase. The association of the plant allantoinase with the endoplasmic reticulum may play a role in the excretion of allantoic acid from the nodule cells in which it is pmo- duced. Compartmentation in the soybean nodule is further complicated by separation of nitrogen metabolism between two cell types, the uninfected cells and those infected with bacteria. It is well known that nitrogen is symbiotically fixed into ammonia in the infected cells. The peroxi- somal reactions uricase and catalase, as well as allantoinase from the endoplasmic reticulum, were found only in the uninfected cell fraction. It may be concluded that ureide production is the exclusive function of the uninfected cells, and these cells are also the exclusive location of nodule peroxisomes. The cellular location of the reactions preceding uricase has not been resolved. Purine synthesis has been localized in the plastid (Appendix). Some of the enzymes associated with plastids, as well as several others whose products are required for purine synthe- sis, were present at a greater specific activity in the uninfected cell fraction. However, further work is necessary to establish the hypothe- sis that purine synthesis leading to ureide formation takes place in the uninfected cell fraction, and to determine what metabolite(s) is trans- ported between infected and uninfected nodule cells. [BF-“'95.?- ‘—-i;_."T'_—..‘ “‘ l -.. r. _ t .a ..- it: . a... ..4..J".v.‘a\. 3.434-839!) 3.559L APPENDIX 91 1'l.ttll.tll"v‘.‘ll“ 4‘ ‘I l’lanta t \ptittgtr \ctl..g I‘M." Subcellular organization of ureide biogenesis from glycolytie intermediates and ammonium in nitrogen-fixing soybean nodules Michael .I. Boland'. Joanna F. llatiks'. Paul ”.8. Reynoldsz. Dale (3. BIL‘Hlls". N l Tolbert'. arid Karel R. Schubert' ' llepattment ot lirmhennstty \litliigatt State 1 ntyerstty. last laiistttg \II 48824. and “ Department ol Agronomy l‘IlHt‘lslly ol Miss.»utt. ( olumbia. MUhSZlI l'sA Abstract. Subcellular organelle fractionation of nitro- gen-fixing nodules of soy bean (Clix-me mar (1-.) Merrl tndtcalcs that a number of en/y mes insolyed iii the assimilation of atnmonia into amino acids arid purines are located in the proplastids. These include asparagine synthetase ll:('6.3.l.l). phosphoribosyl amidotransferase (H‘ 2 4.214). phosphoglycerate de- hydrogenase (l-(' l 11%). serine hydroxyniethylasc (H‘ 212 | l. and tnetliylene-tetrahydrofolate dehy- drogenase (12C l.5. l .5) ()f the tvm isoetuytnes of as- parate attiiiititrtttislertise (12C 261]) in the nodule. only one was located in the proplastid fraction. Both glutamate syntliase tl‘(' I4. I . l4) and triosephosphate isomerase (I:(' 5.3.l l) “ere associated at least in part vs tilt the proplastids. ( tlutamine sy nthetase ( H ' 6.3. l .2i and xanthine deliydrogenasetH' I2 I .37) vrere found iii significant quantities only in the soluble fraction. l’hospltoribosy lpy rophospliate sy ntlictase ( 11‘ 2.7.6. I) “as found mostly in the soluble fraction. although small amounts of it were detected in other organelle fractions. These results together \yith recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of antntonium assimilation. amide synthesis atid ure- tde biogenesis tn the nodule. Key words. Ammonium assimilation (.‘Ii-t'me Ni- trogent'txation Proplastid Purine synthesis Root nodule Ureide Introduction It is now well established that a number of legutne species. including soy bean. assimilate most of the am- «lhhrrnulumy 1H,:- tctraliydtololic aud. I‘Rl‘l'm5-pliosplto-z |)-ribose l-pyrtiphosphate l’kl’l’ synthetase ttbm. phosphate py rophospliolttnase tphosphottbosylpyropliospltate synthetase) montum produced by nitrogen fixation into the " ntc- ides". allantoin and allantoic acid tllerrtdge etal. I978; McClure and lsrael I979). The ureides are pro- duced in a pathway involying de noyu purine biosyn- thesis followed by oxrdatton ofthe purine ring (Atkins et al. I980; Schubert th: Boland and belitthert I982. The subcellular location in peroxisomes. endo- plasmic rcttculum. and cytoplasm of the enzymes of purine oxidation has recently been presented tllartks et al. |98l ). Studies on the en/ytnology of purine bto~ synthesis and its ancillary processes in soybean no- dules haye demonstrated the presence of riboseplios- phate py'rophosphoktnase (Phosphortbosylpyro- phosphate synthetase. l’RPP synthetase; l'(‘ 2.7.6 It and phosphoribosyl amidotransferase tl (' 2 4.2.]4). which catalyze tuo of the initial steps of putttie hio- genests (Schubert l98l: Reynolds et al l982ai as well as phosphoglycerate dehydrogenase (H~ l I l 951. se- rine hydroxymethy'lase tl:('2.l 2.l) and methylene FH;ltetraliy‘drofoltcacidldchydrogenasetl:(' l5 LS). which function iii the production of glycine and N5.N'°-methenyl 1H,. precursors for puruie synthe- sis. The leyels of these enzymes are much greater in soy bean nodules than in nodules of lupin. a legume in which the main products of ammonium assimila- tion are amino acid amides rather than ureides (Rey- nolds et al. I982 bl. All these enzymes h.t\t‘ been shou n in recent experiments to increase dramatically in spe- cit'tc activity tn soybean nodules during the onset ot nitrogen fixation atid ureide production tSchuhert l9ttl; Reynolds et al. l982a). The intracellular location of these en/ytnes has been investigated in View of the conipleytty and un- usual structural nature of the nodule cell. and the compartmentation iti different organelles ol the pu- rine-oxidi/ing enzymes (Hanks et al. I98“ t)ur es- periniental approach has been to t'racttonaie nodule organelles on sucrose density gradients and measure levels of the key enzymes in all organelle fractions. will-(N35,.ts'2/tilSS/titr-timt 4o 92 ,. gar—gr”: i—TI - I t 5' '1“?- !- fifth“; 7.? .. ‘ c 40 M .I llolaiid et .il Subcellular organilaliori ol tllc'lylx biogciiests iii socI-eaii nodules including all the cii/ynies lietetolore mentioned and also those inyolycd Ill assimilation ol aiiiriiotiium into amino acids and amides, ' Materials and methods ll "t [\etinc t2 ISUIlq niiriol) H “( laspattatc (I 89 (Iqullllllsill and [I 3"‘( (glutamine (l 5| ltllqiuiiioli were obtained lrotn the RacltoclicintcaH etitre. -\iiieisli.itii. I' Is l‘liospliiiltydriixypyrtiyalc ysas generated lrotn tlic ditiictliylltctal deiiyatryc. which thus ob- tained lroni \igma ( licriiical t o. St I 0111‘. Mo. 155A Tetraliy- dtoltilalc'. 2 PlithPllU-I‘l)-l|htl\c‘ l-p\ttipltcisph.tlc‘ (l’Rl’l’), lttalalc' deliscliogetiasc (porcine heart cytoplasm) r-glycerophosphalc cle hydrogenasc tlype I) and Inil yials ot \4\l)ll. NAI)‘ and NMH' uerc obtained lrotn htgina All other reagents were ol analytical purity lliree separate experiments were carried out. in yyhich the conditions \serc sariecl according to the ayailabiltly' of soybean plants and attempts to stabilize dilleretit err/yines Soybean plants ‘(llltlfll' may (I ) Men cy Anisoy 7” acre groun from seeds (from Michigan I-oiiiidatioti Seed ( orp. Iast lansrng. Mich. l‘S-\) inoculated with Rliimhirmt m/mrrutmt strain lllb Ill) (1' 5 Department of Agriculture. Ileltsyille. Md. LISA) and grossn iii a growth chamber (under cool-yshrte fluorescent and tungsten lamps at an energy llnetice rate of I20 \A m " yctth a I2-Ii phoiopc— Hod and a temperature til 15" (' clay and 20‘ (‘ night. cspc'rinicrtts l and 2) or greenhouse (during the months of August and Serv- tembc-i with additional Iiglii supplied tor a lh-b pltotopciiod lrotn Paired t iro-Iuit and cool-uliite fluorescent lamps at lluence ot 50 \\ In ". and a temperature of .15“ (' day and 25‘ (' night. eyperttiicnt 3) III l’etlrtc (heat-treated yolcanic rock. lliertii -o.Roclt. New lr‘E'C- l'a . l‘\.—\) and watered daily with nitrogen-lice nutrient ""1100" ll rshbc‘clc et al I971). I‘hrce-ueek-old plants were used. listiacts Irutn 1 1 g oI nodules were fractionated on sucrose density gradients .ts described by Ilanlss et al (I9ts'li llie grinding media Cunldlllt'd (14 M sucrose. U l M N-tirsthydrosynietliyliiiiethyl gly- "m‘ l ' “(Niel .rt pll 7 it, I0 niM Kt'l. I0 inM etliylenediatntne te- traaeetic .icrcl (HTfA). I it)!“ Mgt‘ly. 25 pg nil ' pyridoxal phos- Phd't’ and It) mg ml ' latiyoacid-free boytiie serum albumin l)i~ ‘hwmc'k'l “as also included in all grinding media at a concentra- """ ”'43 5 "I M for experiment I. and 20 iii.“ tor subsequent experi- 'I‘¢'“> Reduced gliiiatliione (5 mM) \sas added to the medium lor espeitiiieni 2. to protect phosphoribosyl amidotransferase Ilo- "'"f'm-“k‘ “ere layered on a 48-ml linear gradient of 0 75 20 M sunrise in 20 "in1 Iricirie. pll 7.8. and 25 rig tiil ‘ pyridoxal phos- l‘lh‘” "‘H .i 3411' bottom pad of 2.3 M sucrose Dithiothreitol “3 4‘“) included in all gradients at 2 5 ml“ iii experiment I or 5m“ "I other experiments. and the gradient tn esperiment 2 also ("Married 2 inM reduced glutatlnone A“ thaclietits were centrifuged iii a Ilec‘kmatt SW 25 2 mini 6“ 4“ (‘ tn a I 2 ultracciittituge (Ilec'kman Instruments. l’alo Mm. 1““ - 1'5"” at 25.000 rpm (“10,900 e) Ior 4 5 li. or 5b in the case “‘ c‘l‘k’ltment I. .iltei slim acceleration as described by IlaiiLs “‘4‘ ll‘lb‘li Alter the run. 2- to S-ml tractioiis ssere collected lrotn the top or m,- snydu-n‘ “CI"W "\\(ll‘\. (ilutaniine synthetase (I.(‘ 6.1 I2) was assayed tis- mg the gliiiainyl hydroxaniate synthetic assay according to Far ndeu and Robertson (I980) (ilutainate syniliase (l:(' IAIN) syas as. ”it‘d by measuring ortoglirtarate and glutainine-dependc-nt N ADII d“ill‘l‘i-‘araiict: as described by lloland and Benny ( I977), and aspat . tate aminotransferase (It 2.6.l.li ysiili tIie malate dehydrogenase- "Mcd 3““) 0f Ilergmeycr .iticl Berni (I963) Asparagine synthetase ”1‘5 3 l llwas assayed by the synthesis of I-lo‘fl'lasparagine from labeled aspartate. followed by- separation by paper electrophoresis according to Farnden and Robertson (I980). l’lttispliiitibusyIpyrriplitisplmu syiillic'lasc ysas assayed .15 de- scribed by Schubert (Will). phosphoribosyl atiitdoitatrslerase was assayed using lltt l'kl’l’-depctidcitt deamidation ol l"( |g|utamirie. with subsequent separation ol labeled glutamate by paper electro- phoresis according to Iloliiies et al (I973) hatiihiiie ocliydtoge- nase ysas assayed as described by ltolarid tl‘lh‘ll l’liosphoglycciatc dehydrogenase was assayed by measuring the pliosphohydrosypyruyate-dcpciiderit oxidation oi .NAIHI at .140 nm Reaction mixtures contained 0 I M potassium phosphate bullet. pll 7 5. quM NADII and 25rd of a gradient traction in a yolume or 095 ml. The reaction “as started by the addition ot 50 pl of 20 ll).\1 phosphohydrosypyruyatc- bit-tine liydroxynieili- ylase was assayed by a modilicatioti ol the method of Taylor and “c‘lsslidcll ( I965) l he reaction mixture contained 80 tiiM po~ tasstuni phosphate buffer. pll 7.5. It) pgxml pyridosal phosphate. 0 2"" (us) [t-mereaptoethanol. I nigxml HI, and 25rd ol a gra- dient traction Alter prerncubation for lUmin. the reaction “as started by the addtttoti of serine to 0.8 in M. 20 Mid Iii-"("lsertne per reaction tn a total volutne of 0 4 ml Alter 20 min iii-.ubatioti. deriyatttation and extraction were carried out as described by 'I ay- lor and “cl\>hdc'h (I965). Methylene HI. dehydrogenase was as- sayed by monitoring the increase in absorbance at .140 nm due to formation ot NADI’H and nietlienyl HI, A combined extinc- tron cocllic‘rent of 30.000 M ' cm ' was Used to calculate rates Reaction mixtures contained 50 mM tirs-(hydrmyiiietliylianitito- methane (TrisI-IIL'I. pH 7 5. 2 5 mM dithiothreitol. 400 “M It-l- maldehyde. 400 uM I'H, and 25 rd ot a gradient traction Alter IUniin pretncubation. the reaction was started by the addition ol NADI’ ' to giyc a final concentration of 50 u“ Marker enzymes were assayed as follows lriosepliosphate isomerase (1:65.311). a proplastid niarlcer. was determined by coupling ysith z-glycerophospliate dehydrogenase according to Bet- seiitic-u (I955). Iumarase (LC-l 2 1.2). a marker enzyme lot the mitochondria. ssas assayed in III M potassium phosphate. pll 7 5. by the production oIIumataic. measured at 240 um. iii the presence of 50 mM malate (Biochemica lllltilmdllcm‘ ). and 3-Iiydioitybiityr- are dehydrogenase (l (‘ l I110) iii the bacteroids was measured by ,l-hydrocy'butyrate dependent NAI) ' reduction as described by Ilanlss et al (l98l ). (lr‘l efurmplion-siy. (iel electrophoresis and acliytry staining ol aspartate aminotraiislerase was carried out in tube gels according to Reynolds and l‘al’lldt‘ll (I979). Results and discussion The gradient centrifugation effected separation of titt- tochondria, proplastid and bacteroid fractions as indi- cated by the marker en/y'nies. I‘uniarase. triosephos- phate isomerase and [i-hydroxybutyrate dehydroge- nase. respectively (Fig. I). These organelles were found at densities of I.I8. l.2l and 1.23 g ml ". re- spectively. A value of l.2l g ml " has been reported for proplastids from spinach and pea roots by Millin (I974). The presence of triosephosphate isomerase at the top of the gradient in the soluble fraction is due. at least in part. to proplastid breakage. Enumer- stair/resting dr'i'rrrhiiy‘i‘lt'c ammo acids and nitrides. Glutamine synthetase. the tirsi enzyme re- sponsible for assimilation of ammonia produced by" ' Vol. II. p 7|; Boehringer. Matinheiiii. I R ti 1 3* r1. 1‘ - r:- as. \I l llolatid c'l .il Nitliccllular circanr/atrori ol irrc'rtlt' ltiogcriesrs in soybean nodules 47 " “_ ‘ ‘I ' r i 1 1 -_ I50; -4 b '3 3 TE Mum" amen r n O r i hydra-you're“ '- 3: root ”:19:ng I c i " ‘l > meu I’ ‘ - b - criosor trizirir 2 =0 . * - r- - mnrsrrri arms :5 o t - -a l05 ll “5 l 2 IZS osusrrv (9 mi") Fig. l. Distribution ol inarlcer err/yrires lor iirirocliondrra. proplas- licls .iiicl bacteroids ol soybean nodules lollowing sucrose density gradient ceritrilrigairori lrroscpliosplratc isomerase - U I (proplas- tid riiarkerl t0). lurnarasc tiiiiroc'lrondrral marker) t« i. and [l-lrydiosybutyrate dehydrogenase s 2 5 (bacteroid marker) ta) Results shown are from cum 3 . 2000 ' V v r 7_ "MW. TS .000 _ rm ‘ ' 0 e e - E 400 r m .9an ‘ ,_ 200 ~ 4 t Z 0 v . 4v c A :3 ‘0 ‘ Wm $101M” ‘ U 2 h «it a o \ A ). 5‘ O . e a : U .600 > up." '4 W 000 '- -( o 1 1A l05 It Its l2 I25 ocusrrv (9 mi") “K- 1- Distribution oI soybean nodule eit/yiiics responsible for ”W ‘.\||||tcsrs ol amino acids and ariircles alter sucrose density gra- dient cent rrlugation lrrosepliospliare isomerase and glutamate ‘l'llhslw results .rre ll‘tIIII espt 3. glutamine synthetase and aspar- tate ariirriutranslerasc' results from espl I bacteriods iii the nodule. was found almost entirely In the soluble fraction of the nodule (Fig.2). This result is in agreement with the distribution of this enzyme in nodules of Plrusenlm rulgari's reported by Awonaike et al. (Will) and iii roots of maize. rice. bean. pea. and barley reported by Suzuki et al. (l98l l. Because of the unique role of glutamine synthetase in assimilating ammonium produced by the bacte- roids. a eytOsolic location seems a logical way of Presenting a toxic buildup of ammonium. The location of glutamate synthase in the cell was U i. r. I‘hgl. 3. (ic‘l electrophoresis til soluble to) and (tiiirtlaslitl lit) ll.ic tr-ir. s lroiii so\bc'art nodules. staining lor aspartate aniinorransleiase a.~ iisrry not clear-cut; however. there was a definite peak of enzyme activity associated with the proplastid frac- tion. A proplastid location has been reported for glu- tamate sy nthase in I’lmsmlirs iii/gum nodules (Awou- ailce ct al. I98” and soybean root tissue (Suzuki et al l98l). and our results support this localization. al- though the possibility of an additonal location in the cytosol can not be excluded. Aspartate aminotransferase actisity was round in both soluble and proplastid fractions (Fig. 2). in agreement with the results of Millin (I974). The es- istence of two isoenzyines of aspartate aminotransfer- ase has been reported for lupin nodules by Reynolds and Farnden t I979) and for soybean nodules by Ryan et al. (I972). The faster-moving lupin enzyme III gel electrophoresis is produced in the nodule concurrently with other ammonium assinrilating enzymes during nodule development. and is presumably the primary enzyme responsible for aspartate synthesis III amino- iiia assimilation (Reynolds and I-arnderr I979). Following separation by gradient centrifugation. soluble and proplastid fractions were subjected to electrophoresis on tube gels and stained for aspartate aminotransferase activity (Fig. 3). The proplastids contained only one isoenzynie. while the soluble frac~ tion contained two. the mayor one of which had a lower relative mobility than the proplastid isoeri/yrne The second isocnzy me in the soluble fraction coaiii- 94 stile-1’3???” Qm-sve— - MA \I I Ilhle I. lIistiibutit-ti nl \ll.'\lll\ .M ll\lll\ s licivtuii soluble and peak llllltl‘liutivltldl Jtttl ptn'ilaslul ltJLlItitls lititli unlicxtli IlUtlttlcs‘ Aspaiagtiic I’Rl'l’ s) ttlllv tasc l taclioti l'liuspliotthusyl sviilltclasc .ttiiitltiltdlislclasc‘ \oluhlt U .‘l I l .‘ b .‘Tlllklhlllllsll NI) ll ‘3 NI) l’ioplastids II II It IN 4 ti ‘ ‘ \alues are Itoiii cspciiuieiit No - Itkal titl ' grailtetil liaclion NI) « Itulie detected N” values ale c\ptcssctl iii grated wnh the proplastid isoeiizyme and was probab- ly the result of proplastid breakage. If a comparison Wllh lupins is valid. then the faster-migrating. proplas- tid isoenzyme is specifically involved in aspartate syn- thesis during ammonia assimilation. Scans of the gels indicated that in the soluble fraction. the slower-mov- iiig isoeiizy me accounted for approsiiiiately 70".. of the total activity. Asparagine synthetase. although not involved in ureide synthesis. is an important enzyme in amide- synthesizing legume nodules. and appears to be im- portant in soybean nodules during a developmental phase prior to ureide production (Reynolds etal WXZaI BeeaUsc of the Iovv levels of this enzyme III the soy bean nodule and the difficulties of assay. only the peak fraction front each organelle hand was as- sayed. The results are presented in Table l. The en- zyme was clearly located in the proplastids. The overall disti ibution of this group of enzymes suggests that glutamine is synthesized in the cytosol. vvliile synthesis of glutamate. aspartate atid asparagine occurs in the proplastids. and possibly also in the cytosol. Purine-sinIliuv'iziiig t‘IlIt'Ult'.\. ()nly tvvo enzymes iii- volved directly in purine synthesis have been success- fully assayed in cellsfree nodule extracts. PRPI’ syn- thetase catalyses the reaction immediately prior to the first committed step of the purine synthesis path- “"‘)~ The militifil} of the enzyme activity was in the soluble fraction. although small amounts were asso- ciated mtli mitochondrial. proplastid and bacteroid lraetions (Table I). possibly because of nonspecific binding to these organelles. This occurrence in all organelle fractions is distinct from the ease of proplas- lid enzymes. which vvere not found in significant Quantities in other organelle fractions. The enzyme. however. is highly labile and a large proportion of ”It activity “its lost during the centrifugation. Phosphoribosyl amidotransferase catalyzes the lloilatul ct .il \llh\L‘lllll.ll organization ol l|l\l\l\ biogcneas tlt \rl\l'c'.lll nodules 2000 P _" ‘ ’TT—T“ ' “ rrvasophosphoto momma“ T l000> ( E E 0 , 1L 4* ‘ ‘ no T V ‘ _ l PGA ahflrmhost g 500 r " ‘5 250 i \ 1 3 so , Some Arm-r ‘ g m > “In!“ 4 20 > ‘ E '°’ /\ i 2 ° ‘ ° ' T T u '5 ~2M°Wo f". a l .0th n b x ‘ 5 A. ‘ o i ‘ ‘ I05 II US l2 I25 DENSITY (9 ml" i Hg. 4. Distribution of soy bean nodule ell/)lltvs ol the glycine t , patlmay on a sucrose density gradient Results lot methylene I llJ dehydrogenase vvere taken Ironi espt I. all other activities are from e\pt, 3 Pt: 4 : l-phosphoglyceiate fraction. although the activity in the soluble fraction is probably from broken proplastids, Erin-mes of gli‘eme um/ INt'l/fl’lll’l'rll.‘ synthesis In plants. there are several alternative patlivsays for the synthesis of glycine. Recent studies with nodules (Reynolds et al. I982a. b) indicate that glycine. along with metheiiyl - His. is synthesized from serine which in turn is synthesized from the glycolytic intermediate. phosphoglycerate. by a series of reactions initiated by phosphoglycerate dehydrogenase The gradient profile of this enzyme is presented in I l‘__'. 4. There was a large proportion of activity in the proplastid fraction atid lesser amounts of activity in the soluble fraction. The latter activity was attributed to proplas- tid breakage. A similar distribution in proplastids was found for serine hydrosymethylase and methylene- HI; de- hydrogenase (Fig. 4). Thus. it seems likely that the entire pathway from 3-phosphoglycerate to glycine plus nietbenyI'FH‘ occurs in the proplastids. vvhere the products. glycine and N‘.N“'-nietlienyl Hl.. are conveniently available for a proplastid-based purine biosynthesis. The presence of serine hydroxynicthy- Iase in plastids contrasts with the location ol this enzyme in leaves (Woo I979: Tolbert Will). “here serine hydroxymethylase is iii the mitochondria. Recoveries ul'eitqi'iites m proplastid Iranians. The pro- portions of cnzvmes recovered in proplastid tractions 95 from three different gradients are presented at Table 2. In the calculation of recoveries ot phosphori- bosyl amidotransferase and asparagine synthetase. only the two main fractions of the proplastid band "’5‘ committed step of purine biosynthesis. As vvith as[Imagine synthetase. this enzyme was assayed in $1:Il‘rllimt'llcfractions only. It was clearly located L proplastids (Table I). and also iii the soluble \I l "ill.”ltl ct .il \olut llttlJl t-ieaiii/atioii oi iitcule liitigem-sis in \t'\h\'.lll titululn 49 Iuhk 2 Hunters ol cit/v Ills ~. II) the proplastid ll.|cllv'lls ol sovlieaii Iltklllk“ llt'lll l'llks’ k\l‘\ l Illlc'lil‘ liizviiic l _‘ t Itiiise'lililispltdlt‘ Isulltv‘ldsv ll 3 3 .‘li tiliitaininc synthetase ti | .3 \atitl’illlv‘ dehydrogenase t) l'litispltiiyl\vv'l.tlc' vlv'llytltngc'ttasv‘ (u: 2" 4‘) Heroic ll\vllt)\\lllvlll\ lasc ‘* .tl \lc‘llnlv'ltc' I ll‘ dehydrogenase .‘J -\spaiagiiie svntlictasc' I’ I'liospliotihosvl .iiiiidottaiish iast ‘ ll \spartate .ininiotianslciase .‘l ‘I I’Iastid tsuciizvnic" " 41 I’Rl’l’ sklllllv'ldst‘ ll (illll.illl.tlL‘ svutliasc IV V 4 2‘ ‘ lot methods ol calculation see test " tit aspartate aiiiinotransltiase were taken itito account for the proplastid fraction. and it was assumed that the activities iii the two soluble fractions were the same. although only one was assa.\ed. All other fractions were treated as hav- ing no activity. For all other enzymes. all fractions were taken into account and the four peak fractions of the proplastid band were considered to constitute that band. (lzaeh proplastid fraction contained 2 ml of a Sit-nil gradient.) It is clear that xanthine dehy- drogenase and glutamine synthetase were not located at the proplastids. lhe location of I’Rl’l’ synthetase is uncertain. ()f the enzymes found in the proplastids. there appear to be two classes: those which were probably located exclusively iii the proplastids. for which relatively high and comparable recoveries were obtained in each experiment. and those for which a lower proportion of the total activity was found in the proplastid fraction. The enzymes which are probably found only in the proplastids include those of the glycine-C. path- way. the proplastid isoenzyme of aspartate amino- transferase. asparagine synthetase and phosphoribo- syl atiiidotransferase. To the group with lower reco- veries in the proplastid fraction belong triosephos- phate isomerase. total aspartate aminotransferase and glutamate synthase. Millin ( W74) has reported a simi- lar result for triosephosphate isomerase in pea root llssue. and has suggested the possibility of both solu- ble and proplastid forms of the enzyme. Our results for lhe distribution of total aspartate aminotransfer- iI-‘C activity are consistent with this explanation. An- other possibility is that there might be a second. more ““5"! broken class of proplastids. or an outer mem- brane‘ on the proplastids which is more easily broken. [tissue]; tr'e‘leasii‘iig these enzyme: from the plastids. deh d ' ecvi encc in Table _. phosphoglycerate Y rogenase is a better marker enzyme lor proplas- tids lroni soybean nodules than triosephosphate isomerase. Pro/rout! Him/cl n/ u'l/u/m mgmumlmu u/ mime”! uvsumlulmn m Mir/rem) Ilmlll/t'i lti Hg 5 we present a model for the subcellular distribution of the en- zy mes of ammonium assimilation and ureide liltigcnc- sis based on these results arid the work of Hanks et al. (Will ). In this model. ammonium produced by the bacteroids is incorporated into glutamine in the cytosol surrounding the peribacteioid sac. Further metabolism. at least as tar as a purine ribonucleotide. then occurs in the proplastids The occurrence of the purine biosynthetic pathway in the proplastids is im- plied by the presence of the enzyme catalyzing the first committed step of the pathway. plus ready sources in the proplastid of glutamine. glycine. aspar~ tate. methenyl. Fll4 and presumably ('02. the other precursors for purine biosynthesis. A proplastid loca- tion for this pathway could explain why incorporation of purine precursors into ureides by nodule slices has been low (Atkins et al. I980). Rowe et al. (I978) have reported the isolation of the enzymes of purine synthesis from pigeon liver as a multienzy me complex which includes phosphori- bosyl amidotransferase and all of the enzymes lead- ing to the synthesis of l Ml’. The localization of purine biosynthesis and reactions of glycine and C. metabo- lism in the proplastid presents an alternative mecha- nism for substrate channeling or possible stabilization of a multienzyme complex. There is some question as to the nature of the final product of purine synthe- sis in nodules. or how it is converted to xanthine (Boland l98l: Boland and Schubert 1982); however. the pathway almost certainly leads to a purine mono- phosphate. lieonomy would dictate that hydrolysis of the purine monophospbate should occur in the cytoplasm so that the ribose (or rib0se-S-phosphate) can be re-used for l’RPP synthesis. assuming this oc- curs only in the cytosol. Xanthine dehydrogenase has been shown by Hanks et al. (Hail) to occur in the cytosol of nodule cells. and subsequent oxidation and hydrolysis then occurs in the peroxisomes and endo- plasmic reticulum. ‘ Soybean nodules contain two different types ot cells . larger. bacteroid-containing cells and smaller. uninfected cells. Newcomb and Tandon (Will) have reported that during cell development. the infected cells contain greatly increased numbers of llllltic‘lltilt' dria and proplastids which become crowded around the periphery of the cell. In contrast. in uniiitected cells a large number of peroxisomes are present. to- gether with increased smooth endoplasmic reticulum. Since uricase exists in the perosisomes and allantoin- ase in the microsomal fraction resulting from the en- 96 "he 9."p:C-‘. .‘. ’~ '-.’ 351'? Militan v “‘34:; _ so \l .I liolarid vl .rl Subcellular organization of iirtrde hiogcrrrsrs Ili \-‘\ltc.iti nodules UREIDES / 7r - 7.,” \ ‘- s; . ‘l Namuum] ( i Juan's r Jir 8‘“ 'I 5R ”4‘ work». F a—-——-——- oriomom 1.0“,” H, "‘°""" 5 P ' °'"° (HOOP.- Asmc I ‘ " e .. _ my; REY/CULUM ‘°" .r‘"e»‘.nc ". \ t/ 9.“ FERW/SOW \- 'v"- 'M'". av. Syn'M'i‘H' '1 . —\ prep M v . (Git-reJLYSiS H blulornm. r u'o‘t T P - thcoroto ( I no» mu; b PRPP '— (imam 3:27." Glutomot. ”A“ P more" «brace-u- MI? 0.0qu y' Ionlhme NAD" vwml- Q (MM, 5".“ . .......... P "#0.,py'uvo'. .flIM'u'g I "Mao sauna W‘ " fl. : Isa...- Methylene Ft-t‘ NAD? “ATP , iii-mo momm- nr. .' . ,aw“ e . mm LW : mime;- ' Glycine MolMyl F H. moan Aspartate o—fi- any I I Whom m 1 1 "co" 0‘0 . L I l PURINE BIOSYNT HE SIS Purim ' - - - * w-M T t T T anchoring umol-cc PR ATP gtr_AYP Air aw opzasno Formyt or. c r 7050. J I ’ v u . . MKS. Imposed niodcl lot ”It subcellular distribution of Illv' enzymes of arnriioriruiri assimilation .iiid ureide ITIHL'c'lICsts lll soybean nodules . known enzymes. . reactions yet to be established. oxalacetate. oitoglu. I-ovoglutaratc. (In. glutamine. e/ri. glutamate doplasiiite reticulum (Hanks et al. I‘lts‘l) it has been proposed by Newcomb and Tandon (WM) that at least the two steps of purine breakdown catalyzed by these enzymes are carried out in the uninfected cells. Since a large proportion of the proplastids are in the peripheral portion of the infected cells. it is possible that purine biosynthesis occurs in these cells. l'uture experiments will address the question of the distribution of the enzymes and organelles of ureide biosynthesis between infected and uninfected cells of the nodule. and further clarification of the function til tttidttlc proplastids. ""‘ N‘s-"v“ “M “Iiiiwmt by a gratil to s R s iron. as- t s “Cp'm'l'f'” “' "S”sl'lllllv‘. ‘vlv'ttsc' .iiid l dticatioii .'\tllllllll\ll.tllttll. I utitpetniye Research (iiants (Illice tgiaiit ho. S‘Kil-tl—Ilti-tll-Iiy‘stli M I“ “"‘ ‘l‘l‘l‘t’llfll l" part by a New lealarid Public Service 51ml) Award and is on leave from the Applied lirm‘lieniistrv l)ivr- "ML I" l R l'dllm‘hlon North. New /.c.iland J I- II was stip- "in“! h‘ " “L“IWW I'H'lv‘wiiltatl ()pporttinttv l’rogiam lcllovs- ‘\h'l‘ hunt the National Institutes of Health TIT-h is mutual article . o Hill-I of the Michigan Agricultural l \pctrtiictit Station References Alki , , "‘- ( \. Rainbiid R \l l'ate. .l 5 (lust). lvideuce in. _. I‘Iiiiii~ ' . c pathway Ul tiwuk- “”l'w‘h ttt Ny-lnttig tiodtilcs ol c""Dca / l’tlanzenphysiol 97. 24)) lot) reaction which occurs iii-stlv iii developing nodules (H l Avyonaike ls (l, lea. l‘J. .‘vlillnr III (l‘lhl) ll.e location ol the enzymes of ammonia assnnilairori in root nodules of I'llini u» I!“ Ill/L'iIIM I Plant Sci lctt 23. “W I‘“ Beisetilicrz. (i (I955) lrrosepliosphate isomerase lioin calf muscle Methods l nzyinol I. lit? Ni 5 l'. lsaplaii. N I) . eds '\c.tvlv‘llllv l’ress New \ oik l itllvlull llcrgriieycr. ll l: . Berni. l (WM) (llllldlllolC-vHolit-tcc'lJlL trans— leiase ln Methods of enzymatic analysis lsl l ngl ed PP N37 843. Bcrgiiieyci. ll l .ed .-\cadcitirv l’icss \cvv \oik l tilldnll lloland. M .l . Benny. A (i (l‘l77l l ri/viiies of nitrogen iiiciabohsiii llt leguriie nodules Purification and properties oi \'\l)ll'vlv‘ pendent glutamate sytitliasc fioni lupin nodules I in I “hf chem, 79. 355 3b) Roland M J (NM) NM) \aritliine dehydrogenase Irorii nodules of navy beans partial [)Ullllvdllv‘l) and properties llirslicin Intern 2. 567 57-8 Bolatid. MJ , bchubcrt. KR tl‘lNIi l’riirne biosynthesis .riid c.ll.t- bolisin Ill soybean root nodules Incorporation ol ”t t)_ into \dlllllllk‘. Arch, lhoclieni Biopliys 2|} slim «NI lariideii. K .I l'. Robertson. 1“ (Ivan) Methods for studying v‘ltlylttcs involved iti metabolrsiit tclated to llllly'tlslldss‘ lri Methods ot evaluating biological nitrogen fixation. pp Io‘ Ito. Bergerseii. l J . ed Wiley. ( liicliister lisliheek, b; l‘\;ttls. Il,]_ Brie-(slim. l l tl‘l‘i) \le‘Ulclllc'lll 0| Ittliiigctiasc acltylly OI tltlael legume s\tiihroiit~ ii. sitir rising tlic acetylene reduction assay Agtoii I 05. 439 4” llanks. Ila. lolhert. N l.. Mliuhctt K R ll‘tlbl) localization of enzymes of ureide biosynthesis Iti pcrousotites .iiid iiiiclo- somes ol nodules Plant l’liysiol 08. (i5 6" llei'ridec. l) l- . Atkins. CA” l’atc .l 5 Ramhlhl R \l ((v‘si Allantoin and allantoic acid in the nitrogen v'u'Ih‘m.‘ t" ”W 97 a,” l‘ \l l ltolarid et al \rnitellulai organization of tllv‘lvlc I-rogerresrs lll soybean nodules vovvpea (Irwin lHIL‘Hltlr/ilfil II I “.ilpl l’lalil l’ltystiil 62 am 4% Holmes I \\ .Mclluiiald l '\ 75le old I \l \k'viigairdcti .I II Kelly. W\ ll‘lfill llittiiati glutamine plir-spliotihiisylpyio phosphateatiiidottarisleiase l lliol l lieiii 248 l-IJ I‘tt Mel line I' R Israel. I)“ tl‘l'l‘ll lraiisporl of nitrogen in Ills yyleiii of soy bean plants I'laiit l’liy siol M 4ll th Millni III (I974) llie location ol nitrite reductase and other enzymes related to amino acid biosynthesis in tlie plastids ol rootsand leaves I’laiit I'Iiysrol 54 W) 5.55 \cvvvottilid ll. landori 5 Is (I'lhl) l tiiiilected cells of soyhearr root nodules l lirastrnctiite suggests key role iii ureide prodiry tron Science III ”"4 MW) Reynolds I' ll 5. latiiden Is .I l' (WW) Ilic involvetiicnt of as parlalc anilnotratislcrascs Ili attitiiotiiurii asstriiilaltoli iii loprii nodules l’liyloelicttitstn 18. I025 loll) Reynolds. I’ll S. llolaiid M .l, Blevins, I) (i Randall I) I) Schubert. K R tl‘lhlail rizyiiies of amide and ureide biogenesis at developing soybean nodules I’Iaiit l’hysiol (in press) Reynolds. I’ II S. Blevins. I) (i . Boland M J. Randall. l) I). Schubert. K R (Wit: bi l-iizymes of ammonia assimilation lll legume nodules A comparison between ureide- and aiiirde- trarisporiing plants l’liysrol Plant (in press) Rowe. I’ll. Mcfaitns. h. Madseii. (i. batter. l). Lllrott. ll .‘l (WW) Ur Hutu purine synthesis in avian liver t o purification of the enzymes and Dlv‘f‘v'lllv‘s ot the pathway J bro'r (heat 25} 77II 7’.‘I Ryan I Ilodley l .lotrcll l’l (W73) l'riiilicaiioii and charts- lv‘lt/alliiti ol aspatlali' .ttlttltolt.ittslclasv's Iloiii snleatl Imil nodules and R/IL'H’UIHH III/’iHIlillIN l’liylm'lieinistiy II. ”“- ‘lhl Schubert. R R tl‘lhli l ri/ymes of purine biosynthesis and yalah-il not in (lllt rm Hlil\ l (‘oinparison ol activities with l\_~ tiyatioii .iiid composition ol vvlem cyudatc during nodule development l’lant l’hysiol 68 “IS ll3.‘ Suzuki. A. (iadal. l’. ()aks. A tl‘lryl) Itttrasellular distribution ol enzymes associated with nitrogen assimilation iii roots I’Ianla ISI. 4‘7 «tot laylril. 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