Ill“1Wlilifllflllfilflifltlfilllllfill THE-".516 3 1293 01745 6439 This is to certify that the dissertation entitled ENZYMES OF GLYCOLATE METABOLISM IN CHIAMYDOMONAS REINHARDTII presentedby Nicola Leigh Selph has been accepted towards fulfillment of the requirements for —EhD——degreein131ach_emi_stry 77. aw Major professor Date 0W ’3: /98a MS U i: an Aflirmatiu Action/Equal Opportunity Institution 0-12771 —- 0".- -2 . Ga. ‘7 ‘. o't-J -- "53351? t: " "- lAa . ‘ " r '1” x ‘ ¢“' . . . . 3.. _ c'J‘ . T; u v t ' . .t ‘ no .3 - - .‘L‘ ”at“; ‘53 '33“ .. W Arthw n) 1"».— i—n' —- ENZYMES OF GLYCOLATE METABOLISM IN CHLAMYDOMONAS REINHARDTII By Nicola Leigh Selph A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry l983 Gl30b7g ABSTRACT ENZYMES OF GLYCOLATE METABOLISM IN CHLAMYDOMONAS REINHARDTII W Nicola Leigh Selph Several enzymes of glycolate metabolism (glycolate dehydrogenase, D-lactate dehydrogenase, P-glycolate phosphatase, and NADPHzglyoxylate reductase) were partially purified from the green alga, Chlamydomonas reinhardtii, and some of their properties studied. Glycolate dehydrogenase was purified at least 25-fold, but lost activity upon further purification. It is known to be a mitochondrial enzyme and does not couple directly to 02. It was inhibited by 2- hydroxy-3-butynoic acid, indicating a flavin component, as is the case for glycolate oxidase from higher plant peroxisomes. Glycolate de- hydrogenase was not stimulated by FMN or FAD jn_vitro. It could be coupled to some tetrazolium salts. P-Glycolate phosphatase was purified over lOO-fold, but it lost activity upon further purification. Mg++ was required for activity. An ammonium sulfate fraction had a pH optimum of 8.7. A factor re- quired for this activity was lost upon purification and the pH optimum shifted to 7.5-8.0. Chlamydomonas reinhardtii cells, as well as spinach leaves, con- tained an enzyme which could be detected on native polyacrylamide gels with nitroblue tetrazolium as electron acceptor, and which oxidized Nicola Leigh Selph D-lactate and several other a-hydroxy acids, but not glycolate or L- lactate. The activity with D-lactate was different from glycolate de- hydrogenase or glycolate oxidase, neither of which were strongly reac- tive on the acrylamide gels. In spinach leaves, the D-lactateznitro- blue tetrazolium activity was located in the mitochondria, and in both leaves and algae it may exist as two forms. The algae were found to photosynthetically produce much lactate from 14 C02. The algae contained an active NADPHzglyoxylate reductase, and an even more active NADthydroxypyruvate reductase. The NADPHzglyoxylate reductase was purified 65-fold, and had a pH optimum at 6.2, a Km (glyoxylate) of 0.4 mM, and a Km(NADPH) of 43 pM. It was competi- tively inhibited by aminooxyacetate with a Ki of l mM. It was also inhibited by oxamate, 50 mM bicarbonate, l2.5 mM glycolate, and high concentrations of both its substrates. Algae grown on high C02 had less glycolate dehydrogenase and P- glycolate phosphatase than air grown cells. They had more NADPH: glyoxylate reductase than air-grown algae. Both air-grown and 5% C02- grown algae produced lactate. Both of these algal cultures excreted glycolate, and this excretion was greatly increased when the cells were treated with 1 mM aminooxyacetate. ACKNOWLEDGMENTS I would like to thank my major professor, Dr. N.E. Tolbert, for his aid and attention during the preparation of this dissertation, and for the financial support as a graduate assistant that I received during my many years at Michigan State University. ii Page LIST OF TABLES --------------------------------------------------- vi LIST OF FIGURES -------------------------------------------------- viii LIST OF ABBREVIATIONS -------------------------------------------- x INTRODUCTION ----------------------------------------------------- 1 CHAPTER I: Glycolate Dehydrogenase ------------------------------ 5 LITERATURE REVIEW ------------------------------------------- 5 A. Presence of Glycolate Dehydrogenase in Algae ------ 5 B. Distribution of Glycolate Dehydrogenase ----------- 7 C. Cellular Location of Glycolate Dehydrogenase ------ 8 D. Regulation of Glycolate Dehydrogenase ------------- 9 E. Yellow Chlorella Mutant --------------------------- l0 ~ MATERIALS AND METHODS --------------------------------------- ll A. Algae --------------------------------------------- ll B. Growth of Algae ----------------------------------- ll C. Assay for Glycolate Dehydrogenase ----------------- ll D. Other Methods and Materials ----------------------- 13 RESULTS ----------------------------------------------------- 13 A. Glycolate Dehydrogenase-Crude Extract ------------- 13 B. Various Extraction Methods for Glycolate Dehydro- genase and P-glycolate Phosphatase ---------------- l4 C. Effect of C02 Concentration During Algal Growth upon Activities of Glycolate Dehydrogenase and P-Glycolate Phosphatase --------------------------- 16 D. Yellow Chlorella Mutant --------------------------- 19 E. Purification of Glycolate Dehydrogenase ----------- 22 F. Electron Acceptor for Glycolate Dehydrogenase ----- 25 G. Attempts to Stabilize Glycolate Dehydrogenase After Ammonium Sulfate Precipitation -------------- 27 H. The pH Optimum of Glycolate Dehydrogenase --------- 28 I. Kinetic Properties of Glycolate Dehydrogenase ----- 28 TABLE OF CONTENTS TABLE OF CONTENTS (continued) CHAPTER II: D-Lactate in Green Algae ---------------------------- LITERATURE REVIEW ------------------------------------------- MATERIALS AND METHODS --------------------------------------- RESULTS ----------------------------------------------------- CHAPTER III: A. B C D. E. F G H Gels of Crude Extracts ---------------------------- Ammonium Sulfate Fractions on Polyacrylamide Gels- DEAE-cellulose Chromatography of Ammonium Sulfate Fractions ----------------------------------------- The "D-Lactate Band" in Spinach ------------------- Sucrose Gradient Ultracentrifugation of Spinach Subcellular Organelles ---------------------------- Attempts to Assay the D-Lactate Activity Spectro- photometrically ----------------------------------- Lactate Production by Chlamydomonas reinhardtii--- Conclusions --------------------------------------- Phosphoglycolate Phosphatase in Chlamydomonas rein- hardtii ............................................ LITERATURE REVIEW ------------------------------------------- RESULTS ----------------------------------------------------- A. B. C. m I‘D-Tl CHAPTER IV: Assay 0f P-Glycolate Phosphatase ------------------ Extraction of P-Glycolate Phosphatase from Chlamydomonas Cells ............................... Purification of P-Glycolate Phosphatase from Chlamydomonas reinhardtii ------------------------- Search for Isozymes of P-Glycolate Phosphatase from Chlamydomonas reinhardtii -------------------- pH Profile of Activity of P-Glycolase Phosphatase from Chlamydomonas reinhardtii -------------------- Substrate Specificity of P-Glycolate Phosphatase-- Attempts at Reactivation of P-Glycolate Phospha- tase ---------------------------------------------- Stability of P-Glycolate Phosphatase -------------- NADPHzGlyoxylate and NADHzHydroxypyruvate Reductases from Chlamydomonas reinhardtii ---------------------- LITERATURE REVIEW ------------------------------------------- MATERIALS AND METHODS ----------------------------------- iv Page 32 32 33 34 34 34 36 36 38 39 43 46 48 48 50 50 51 SI 56 57 61 65 65 70 7O 73 TABLE OF CONTENTS (continued) RESULTS ..................................................... A. com CHAPTER V: CONCLUSIONS-- APPENDIX: Presence of Glyoxylate Reductase and Hydroxypyru- vate Reductase in Chlamydomonas reinhardtii ------- Direction of Glyoxylate Reductase Assay ----------- Assay Conditions for NADPH:Glyoxylate Reductase from Chlamydomonas reinhardtii -------------------- Purification of NADPHzGlyoxylate Reductase from Chlamydomonas reinhardtii ------------------------- Kinetic Properties of'NADPH:Glyoxylate Reductase from Chlamydomonas reinhardtii -------------------- Inhibitors of NADPHzGlyoxylate Reductase from Chlamydomonas reinhardtii ------------------------- Page 73 73 75 75 79 82 82 Stimulation of Glycolate Excretion by Aminooxyacetate 88 ---------------------------------------------------- 9l BIBLIOGRAPHY ----------------------------------------------------- 96 Aminooxyacetate Stimulation of Glycolate Formation and Excretion by Chlamydomonas ----------------------- lOO Table 10 ll 12 LIST OF TABLES Page Extraction methods for glycolate dehydrogenase and P- glycolate phosphatase from Chlamydomonas reinhardtii grown with air ----------------------------------------- 15 Disruption methods for glycolate dehydrogenase and P- glycolate phosphatase from Chlamydomonas reinhardtii grown with air ----------------------------------------- 17 Activity of glycolate dehydrogenase and P-glycolate phosphatase in Chlamydomonas reinhardtii grown with air or high CO2 ---------------------------------------- 20 Glycolate oxidation in extracts of Chlorella vulgaris and Chlamydomonas reinhardtii -------------------------- 21 Glycolate dehydrogenase purification from Chlamydomonas reinhardtii grown with air ----------------------------- 23 Substrates that produce an activity stain with NBT in the location of the D-lactate band on the polyacryl- amide gels --------------------------------------------- 35 Effect of NBT on DCPIP assay for glycolate dehydro- genase from Chlamydomonas reinhardtii ------------------ 42 Spectrophotometric methods tested for D-lactate depen- dent activity with spinach mitochondria ---------------- 44 Lactate production by Chlamydomonas reinhardtii grown with air and high CO2 ---------------------------------- 47 Purification of P-glycolate phosphatase from Chlamydo- monas reinhardtii -------------------------------------- 53 Substrate specificity of P-glycolate phosphatase from Chlamydomonas reinhardtii ------------------------------ 62 Effect of some salts on dialyzed P-glycolate phospha- tase from Chlamydomonas reinhardtii -------------------- 66 vi LIST OF TABLES (continued) Table 13 14 l5 l6 17 Page Stability of P-glycolate phosphatase from Chlamydomonas reinhardtii to freeze thawing -------------------------- 69 NADPH:Glyoxylate reductase and NADH:hydroxypyruvate reductase in air and 5% CO2 grown Chlamydomonas reinhardtii -------------------------------------------- 74 Extraction methods for NADPH:glyoxylate reductase from Chlamydomonas reinhardtii ------------------------------ 80 Purification of NADPH:glyoxylate reductase from Chlamydomonas reinhardtii ------------------------------ Bl Effect of some inhibitors on NADPH:glyoxylate reductase at pH 6.2 and 8.1 -------------------------------------- 85 vii Figure 2a 2b 3a 3b 5a 5b 10 LIST OF FIGURES Oxidative photosynthetic carbon cycle in higher plants- Extraction of glycolate dehydrogenase from air-grown Chlamydomonas reinhardtii by different concentrations ofideoxychoTate ---------------------------------------- Time course of deoxycholate extraction from air-grown Chlamydomonas reinhardtii by different concentrations of deoxycholate ---------------------------------------- DEAE-cellulose fractionation of glycolate dehydrogen- ase from Chlamydomonas reinhardtii ..................... Sucrose gradient of glycolate dehydrogenase from Chlamydomonas reinhardtii .............................. The pH optimum of glycolate dehydrogenase from Chlamy- domonas reinhardtii .................................... Rate of glycolate dehydrogenase versus concentration of glycolate ........................................... Dixon plot for inhibition of glycolate dehydrogenase by oxamate ................................................ Inhibition of glycolate dehydrogenase from Chlamydo- monas reinhardtii by hydroxybutynoate ------------------ DEAE-cellulose fractionation of glycolate dehydrogenase from Chlamydomonas reinhardtii and polyacrylamide gels of the peak fractions ---------------------------------- Sucrose density gradient centrifugation of spinach organelles ............................................. Chromatogram of 14C-labelled products from Chlam do- monas reinhardtii after l0 min of photosynthesis with NaHCO3 ................................................. DEAE-cellulose fractionation of P-glycolate phosphatase from Chlamydomonas reinhardtii ------------------------- viii Page l8 I8 24 24 29 3O 30 31 37 40 45 54 LIST OF FIGURES (continued) Figure ll 12 l3 l4 l5 l6 17 18a 18b 19a 19b 20a 20b 21 Revised DEAE-cellulose fractionation of P-glycolate phosphatase from Chlamydomonas reinhardtii ............. The pH profile of the ammonium sulfate fractions of P- glycolate phosphatase from Chlamydomonas reinhardtii before and after dialysis assayed withTS mM MgCl2 ...... The pH profile of the ammonium sulfate fraction of P- glycolate phosphatase from Chlamydomonas reinhardtii before and after dialysis assayed'withoutMgCl2 -------- The pH profile of fractions from the DEAE-cellulose fractionation of P-glycolate phosphatase from Chlam - domonas reinhardtii before and after dialysis With 5 mM MgCl2 -------------------------------------------------- The pH profiles of phosphate released from several sub- strates assayed with an ammonium sulfate fraction from Chlamydomonas reinhardtii ------------------------------ The effect of increasing ionic strength on P-glycolate Phosphatase from Chlamydomonas reinhardtii ------------- The pH profile of NADPH:glyoxylate reductase from Chlamydomonas reinhardtii ------------------------------ The rate of NADPH:glyoxylate reductase from Chlamydo- monas reinhardtii with different glyoxylate concentra- tion --------------------------------------------------- Lineweaver-Burk plot for NADPH:glyoxylate reductase---- The rate of NADPH:glyoxylate reductase from Chlamydo- monas reinhardtii with different NADPH concentrations-- Briggs-Haldane plot of NADPH:glyoxylate reductase ------ TEAE-cellulose fractionation of NADPH:glyoxylate reduc- tase from Chlamydomonas reinhardtii -------------------- A second TEAE-cellulose fractionation of NADPH:glyoxy- late reductase from Chlamydomonas reinhardtii ---------- AOA inhibition of NADPH:glyoxylate reductase from Chlamydomonas reinhardtii .............................. ix Page 55 58 59 60 63 67 76 77 77 78 78 83 83 87 ADA Bicine BSA Ches DCPIP DEAE DTT EDTA HBA Hepes HPMS MTT NBT Pi PP, PMS ribulose-P2 TEAE Tris LIST OF ABBREVIATIONS (aminooxy)acetic acid N,N-bis[2-hydroxyethyl]glycine bovine serum albumin 2-[N-cyclohexylaminolethane sulfonic acid 2,6-dichlorophenol-indophenol diethylaminoethyl dithiothreitol ethylenediaminetetraacetic acid 2-hydroxy-3-butynoic acid N-Z-hydroxyethylpiperazine-NLZ-ethanesulfonic acid o-hydroxypyridine methane sulfonic acid 3-[4,5-dimethylthiazol-Z-yl]-2,5-diphenyl tetrazolium bromide nitro blue tetrazolium inorganic phosphate pyrophosphate phenazine methosulfate ribulose bisphosphate tetraethylaminoethyl Tris(hydroxymethyl)aminomethane INTRODUCTION Photorespiration occurs in all green plants as an accompaniment to photosynthesis. Some of the newly fixed carbon dioxide is imme- diately lost by this process which is different from mitochondrial respiration and electron transport. Photorespiration is defined as light-dependent 02 uptake and concurrent CD2 release, taking place in a photosynthetically active organism (1,2). In plants, photorespiration occurs due to the reaction of ribu- lose-P2 carboxylase/oxygenase, the principal COZ-fixing enzyme of photosynthesis, with 02 instead of C02. Ribulose-P2 reacts with 02 to produce 2-P-glycolate and 3-P-glycerate (Figure l). The P-glycolate is dephosphorylated to glycolate by a specific phosphatase present in the chloroplasts. Glycolate is oxidized in the peroxisomes to glyoxy- late by glycolate oxidase, which reduces 02 to H202. The catalase present converts the H202 to 802 and H20. The glyoxylate is trans- aminated to form glycine, which is metabolized in mitochondria. Glycine is decarboxylated to C02, methylene-tetrahydrofolic acid, and NH3, with energy conservation by the electron transport system.’ This reaction is responsible for the CO2 release of photorespiration (Figure l). The methylene-tetrahydrofolic acid reacts with another glycine to form a serine. The serine may return to the peroxisome, where it ar-KETO WARM: cumulus 90°" P. ) um NADPH CHLOROPLASTS F. 9,0,. 900“ t I 900“ “MAME L cup: cram cuo / \ r A cum I"? Sucrose .— .—TrhIo-P “Pm—“Mam A LASM mums CYTOP W( m am PM W PEROXISOMES cm, (T 00. I O MALATE - ______ .._-- MALATE QHOH cooH x’uooow 0 CH3“! *0: cfllm [I L k. mo km on , o; l/ [W02 -NAu-l bigot—Hp,“ A) ”a *9” OXALAcsTATE OXAUCETATE do A / Lt GLUTAMATE .._A GUJ'TAMATE ....... MN GUJTAMATE \ l \ mKETo- _____ _-____, ___. 5‘ GLUTARATE LGLUTARATE GLU‘MRATE—— \\ \ MATE ———————— _. AS’ARTATE \‘ l I” J I I 1 1 , serum: —~ mum—oxen: I I I . I IR ' ' 3ATP MITOCHONDRIA mt, tic! ’I been fig)" KM it GLUTAMATE "Am r- a_ KETO- co.+ can: + NH, MTARATE METHYL Aocsmns’ "“3“ ‘Hooou Figure l. Oxidative photosynthetic carbon cycle in higher plants. This figure is from a review by N.E. Tolbert (2). 3 is converted to hydroxypyruvate, which is reduced to glycerate, which in turn can be phosphorylated to enter the photosynthetic reductive carbon cycle (1). Estimations for CO2 loss during photorespiration range up to 50% of that fixed. One way to estimate photorespiration is by the CO2 compensation point, which is the level to which the plant will reduce the external CO2 in a closed system. The photorespiratory, or oxidative photosynthetic carbon cycle, also occurs in green algae, with some differences (3). The C02 compensation point is generally lower than in higher plants (4,5). This may be because the algae excrete part or all of the glycolate rather than metabolize it. The reason for this is not known. The magnitude of glycolate excretion may be very high. A major difference of glycolate metabolism in algae is in its oxidation. Many unicellu- lar green algae do not contain a glycolate oxidase. They appear to have a glycolate dehydrogenase instead, which does not couple directly to 02. This enzyme is present in low activity, and as currently understood, cannot account for the metabolism of all glycolate formed. A high rate of glycolate metabolism is not necessary, however, since so much glycolate is excreted. The other enzymes of the oxidative photosynthetic carbon cycle have always been assumed to be the same in algae as in higher plants. The goal of the thesis project was to understand more of the differences in glycolate metabolism between unicellular green algae and the more thoroughly investigated system from higher plants. The alga used was Chlamydomonas reinhardtii. 4 Chlamydomonas reinhardtii is a member of the division Chloro- phycophyta, order Volvocales of the plant kingdom. It has a single chloroplast and a flagellum at one end. It also has a heavy cell wall with a glycoprotein layer and no cellulose (6). It is easy to grow and has been used for many previous studies. The enzymes of glycolate metabolism from this organism were partially purified and examined. Glycolate dehydrogenase was studied first (Chapter I). Since this enzyme is also a D-lactate dehydro- genase, this latter activity was also studied (Chapter 2). The enzymes producing glycolate, P-glycolate phosphatase and NADPH:glyoxy- late reductase, were also studied for possible clues to glycolate excretion (Chapters 3 and 4). A large increase in glycolate excretion resulting from added aminooxyacetate was also looked at (Chapter 5). CHAPTER I GLYCOLATE DEHYDROGENASE LITERATURE REVIEW Glycolate is the central product produced during photorespira- tion, and hence the past usage of the term, "the glycolate pathway." P-Glycolate is biosynthesized from ribulose-P2 by the reaction with O2 catalyzed by ribulose-P2 carboxylase/oxygenase, followed by hydrolysis by P-glycolate phosphatase. The resulting glycolate is oxidized in higher plants by glycolate oxidase. This oxidase is present in peroxisomes, is FMN-dependent, and during the reaction 02 is taken up with the production of H202. Many of the first studies on photosyn- l4 thetic products labeled from H C03' were done with algae, as well as higher plants, and glycolate was always observed as a product. An equal distribution in the 14 C between the two carbon atoms was always observed with plants or algae (7). Glycolate was early shown to be excreted into the medium by algae (8) and attempts to find glycolate oxidase in algae failed, although other enzymes of the glycolate pathway were present (9). A. Presence of Glycolate Dehydrogenase in Algae Several groups have studied a glycolate-utilizing system in algae which was first called a glycolate oxidase (l0,ll). Lord and Merritt (10) made a preparation of Chlorella pyrenoidosa in which they 5 6 observed a glycolate-dependent formation of a phenylhydrazone, which could be followed spectrophotometrically. The formation of glyoxylate phenylhydrazone is one assay for glycolate oxidase. They obtained a stoichiometric relationship between glycolate used and glyoxylate formed. Another assay for glycolate oxidase is the reduction of the dye, 2,6-dichlorophenol-indophenol (DCPIP), which can substitute for the 02 as electron acceptor in an anaerobic assay. Using this assay, Zelitch and Day (11), found activity in Chlamydomonas reinhardtii and Chlorella pyrenoidosa. They showed approximate stoichiometry between dye reduced and glyoxylate formed and demonstrated 14 C-glyoxylate production from 14C-glycolate. In 1969, Nelson and Tolbert (12) reported that Chlamydomonas reinhardtii contained a glycolate:DCPIP reductase that was not direct- ly linked to 02. They proposed that this was a different enzyme from the previously described higher plant glycolate oxidase. The amount of activity in the cells was affected by the CO2 level with which the cells were grown. Codd gt_al, (13) also found that Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Euglena gracilis have a glycolate:DCPIP reductase that was not affected by, and did not use, 02. In 1970, Nelson and Tolbert (14) further characterized from Chlamydomonas the glycolate:DCPIP reductase, which they called glyco- late dehydrogenase. This is the name that will be used throughout this thesis to refer to this activity. Nelson and Tolbert (l4) ob- tained an extract by incubating the algae with 1% Triton X-100 for one hour in the cold. The debris was centrifuged out and a 35-50% ammo- nium sulfate fraction was prepared. The enzyme could use D-lactate 7 60% as well as glycolate, but it used L-lactate only at a much lower rate. It could also use glyoxylate, and DL-hydroxybutyrate, but not DL-glycerate, DL—o-phenyl-lactate, P-glycolate, glycine or malate. The pH optimum was at pH 8.5-9.0. The enzyme had a Km for glycolate of 0.2 mM and 1.5 mM for D-lactate. It was able to couple both to DCPIP and PMS, but not to 02, ferricyanide, FMN, FAD, NAD+, NADP+, methylene blue, glutathione, nitrate, or cytochrome c. The enzyme was sensitive to sulfhydryl inhibitors, but not to EDTA. KCN inhibited the algal enzyme, but had no effect on the peroxisomal glycolate oxidase from leaves. B. Distribution of Glycolate Dehydrogenase Nelson and Tolbert (14) also looked at the distribution of the enzyme in several species. They found no D-lactate-dependent or CN- sensitive glycolate-dependent DCPIP reduction in the three species of higher plants tested. These plants had an activity that could utilize L-lactate and glycolate. This was the glycolate oxidase. However, in the four species of green algae tested, the glycolate or D-lactate: DCPIP reduction was present and was 100% inhibited by 2 mM KCN. This was the glycolate dehydrogenase. With Euglena results were obtained indicative of the presence of both of these enzymes. In l973 a larger survey of the distribution of the two enzymes was published (15). The criteria described above were used to dis- tinguish between the activities of the two enzymes. All the lower land plants, and aquatic angiosperms contained the oxidase. Some green algae contained the oxidase, the rest the dehydrogenase. This difference in enzyme content did not strictly correlate with the 8 unicellularity or multicellularity of the algae. The distribution seemed most to relate to a phylogenetic scheme based on certain cytological structures visible at mitosis. This hypothesis has not been confirmed because some of the algae with glycolate dehydrogenase had the higher plant type of cytostructure (16). C. Cellular Location of Glycolate Dehydrogenase The glycolate oxidase of higher plants is present in leaf peroxi- somes (1). It is compartmentalized there with catalase, which can immediately react with the H202 formed. Stabenau (17) made a sucrose gradient of the homogenate from Chlamydomonas, and got a main peak of catalase which was centrifuged into the bottom of the gradient, whereas the rest of the catalase, the cytochrome oxidase, hydroxy- pyruvate reductase, malate dehydrogenase and glycolate dehydrogenase all centrifuged together. From the quality of this work it is hard to determine if organelle separation was really obtained, and if these enzymes are really mitochondrial. However, in 1976, glycolate de- hydrogenase was located in the outer mitochondrial membrane of EELEEE‘ domonas by electron microscopy with a cytochemical stain (18). D- Lactate dependent activity was also found in the same place. No stain was deposited in the peroxisomes. Paul and Volcani (19) found the enzyme located in the mitochon- dria of a thin-walled Chlamydomonas mutant. This enzyme was a glyco- latezcytochrome c reductase and was sensitive to antimycin and 2- heptyl-4-hydroxyquinoline-N-oxide, which are mitochondrial electron transport inhibitors. Rotenone had no effect. 9 Glycolate dehydrogenase has also been found in the mitochondria of diatoms (20,21) and other algae (22). In blue-green algae (pro- karyotes) the glycolate dehydrogenase is associated with the thylakoid membranes (23,24,25). Euglena gracilis contains a glycolate dehydrogenase in the mito- chondria (26) sensitive to antimycin A, but insensitive to rotenone. Electron transfer to Euglena cytochrome c was demonstrated. Mitochon- dria from heterotrophically grown Euglena show a difference spectrum with glycolate (27) with peaks for cytochromes b, c and a, indicating that glycolate donates electrons to the whole transport chain to con- serve energy. Similar results were seen in a diatom (21). Some diatom enzymes are atypical in using L-lactate instead of D-lactate (20,28). Euglena grown autotrophically with air, contain a glycolate oxidizing activity in peroxisomes as well as mitochondria (29). "Glycolate dehydrogenases" with different properties have been found in E, coli (30) and human liver (31). 0. Regulation of Glycolate Dehydrogenase It was shown early that Chlamydomonas grown on 5% C02 had less glycolate dehydrogenase than air grown cells. These cells also are known to excrete more glycolate (12). Cooksey (32) indicated that nutritional nitrogen limitation was a cause of lower enzyme levels. Nitrogen limitation also caused repression of Euglena glycolate dehydrogenase (27). This suggested that this enzyme is necessary mainly when the growth limiting factor is carbon, either to conserve the needed fixed carbon, or to somehow participate in HC03' uptake. 10 P-glycolate phosphatase levels also may decrease during growth on high 002 (12). This makes sense since P-glycolate should be formed in lower amounts, because high C02 should outcompete 02 for the ribulose- P2 carboxylase/oxygenase reaction. P-Glycolate phosphatase is synthesized during regreening of Euglena, even when ribulose-P2 synthesis is artificially inhibited (33). Euglena grown on 5% C02 lose the glycolate dehydrogenase activity in the peroxisomes, but not in the mitochondria (34). E. Yellow Chlorella mutant In 1971, Kowallik and Schmid (35) described a yellow Chlorella vulgaris mutant that, along with the wild type Chlorella vulgaris, had glycolate-dependent 02 uptake. This was demonstratable both with whole cells and with an ammonium sulfate fraction from an extract prepared in a French press. These fractions also had anaerobic DCPIP reduction that was dependent on glycolate. These activities were stimulated by FMN. The rates were in the range of 0.2-20 nmol-min'l-mg 1 protein' . Especially in the yellow mutant, the 02 uptake was stimu- lated by blue light. Chlorella pyrenoidosa and several other green algae did not exhibit this glycolate-dependent 02 uptake. In fact Chlorella pyrenoidosa was one of the first algae in which glycolate dehydrogenase was discovered (10). Kowallik and Schmid concluded that Chlorella vulgaris, but not Chlorella pyrenoidosa had glycolate oxidase instead of glycolate dehydrogenase. This would be interest- ing, if true, since these two algae are closely related, and would indicate a transition between two systems for oxidizing glycolate during photorespiration. 11 MATERIALS AND METHODS A. Algae Chlamydomonas reinhardtii, Dangeard (-) strain, was obtained from the Type Culture collection of the University of Texas at Austin as catalog #90. Chlorella vulgaris 211-llh, catalog #263, was from the same source. The yellow Chlorella mutant, 211-1lh/20, from the Algal Collection of the Institute of Applied Microbiology. University of Tokyo, catalog #C-425, was the gift of S. Miyachi. B. Growth of Algae The green cells were grown at 23-28°C in the medium of Orth, Tolbert and Jiminez (36) in aerated (several m1/min) flat Erbach flasks on a shaker, under lighting of 500 uEinstein-m'1-s'] from cool white fluorescent tubes. The wild type Chlorella were grown on a medium containing 50% less NH4+. The yellow Chlorella were grown in the dark at 30°, in a medium containing: 0.81 g KN03, 0.47 9 NaCl, 0.25 g MgSO4-7H20, 0.44 g NaH2P04-2H20, 0.36 g NazHP04-12H20, 0.022 g CaC12°6H20, 0.0013 g ZnSO4-7H20, .0002 g MnClzo4H20, 1 drop 5% w/v FeCl3, 7.0 g of glucose in 1 liter of H20, pH of 6.0 (Miyachi, per- sonal communication). Cells were harvested by centrifugation after about 5 days of growth, washed once with distilled water, and centrifuged in weighed Corex tubes at 10,000 rpm, and the wet weight was recorded. They were used immediately. C. Assay for Glycolate Dehydrogenase Homogenization procedures will be described in the Results. The assay was the same as one of the assays for glycolate oxidase. The 12 reduction of 2,6-dich10rophenolindophenol (DCPIP), used as an electron acceptor, was followed spectrophotometrically at 600 nm in 50 mM NaPPi pH 8.7. The endogenous rate with enzyme but without substrate was subtracted. Because the oxidized dye is colored, and has low solubility, it can only be present in the reaction at less than saturating amounts. The assay for glycolate oxidase was run in an anaerobic Thunberg cuvette with glycolate in the side arm. The cuvette was degassed 10 times with N2 to remove 02 to prevent the competing reaction with 02 to form H202 which can in turn reoxidize any reduced DCPIP. The glycolate oxidase assay was modified for glycolate dehydrogenase. In 0.9 ml total volume there were 700 pl of 50 mM NaPPi buffer, pH 8.7, containing 1.87 x 10'4 M DCPIP and 200 u] extract or H20. This was incubated at 30° for 15 min, while measur- ing any endogenous rate, then the reaction was started by addition of 100 pl of 0.125 mM Na glycolate. The rate was followed at 600 nm on a Beckman spectrophotometer. With glycolate dehydrogenase the reaction can be run aerobically because there is no reaction with 02. The extinction coefficient for DCPIP is 21.5 at pH 8.7 (37). The enzyme in homogenates was stable on ice for several hours, but the reaction rate in the assay was far from linear, and rapidly decreased. Initial rates were determined. The assay was not entirely satisfactory for homogenates, since apparently something occurred as the reaction proceded to either destabilize the enzyme or to reoxidize the dye. This occurred more in the crude extracts than in partially purified preparations. Although assays in air or N2 gave the same result, the anaerobic assays were more linear in the crude extracts. 13 The glycolate dehydrogenase preparation, which was partially purified by ammonium sulfate fractionation appeared to be stable for at least several weeks in the -18° freezer, and to several freeze-thaw cycles. The rates were also linear longer than in crude extracts. D. Other Methods and Materials Protein was determined by a modified Lowry assay procedure (38). Biochemical reagents were purchased from the Sigma Chemical Company, with the exception of the DEAE-cellulose (DE52, microgranu- lar) which was from Whatman, and the ammonium sulfate which was a special enzyme grade from Schwarz/Mann. RESULTS A. Glycolate Dehydrogenase-Crude Extract Following the work of Nelson and Tolbert (14), the principal method used at first to obtain a preparation containing the dehydro- genase was to incubate the cells in a small volume with 1% Triton X- 100. This gave on the average 6 nmolomin'l-ml'], 0.003 units-mg protein-1, or 0.06 units-gram wet weight-1. A unit is defined as 1 umole DCPIP reduced per min. By the sonication apparatus, with bursts totalling 5 min, an activity of glycolate dehydrogenase as high as 0.6 1-mg protein"1 nmol-min' was obtained. Very often, however, this method yielded no activity. No activity was observed in the super- natant medium after incubation of the cells with phospholipase C or after osmotic shock treatment. Since glycolate dehydrogenase may occur in the mitochondrial membrane, an attempt was made to obtain a crude organelle preparation from the cells by grinding them in a 14 mortar and pestle lined with nylon mesh, followed by a differential centrifugation. The cells were apparently too tough to be opened by this approach. Treatment with cellulase and hemicellulase before the grinding also did not work, because Chlamydomonas cell walls do not contain cellulose (6). Part of the problem was that the enzyme existed at a low level in the crude extract, and the endogenous rate could be 50% of the enzymic rate. Several methods to partially purify the enzyme were tried. The method of Nelson and Tolbert (14), ammonium sulfate precipitation between 35-50% saturation, gave 2 to 3-fold purification, which is similar to their results, but the yield was only about 20%. After this procedure, the enzyme appeared to be attached to chlorophyllous matter that was of all sizes, and which could not be fractionated with ammonium sulfate. Polyethylene glycol will also precipitate a protein fraction with the enzyme. Purification was not helped by the presence of polyvinylpolypyrolidone, different amounts of Triton X-100, or several concentrations of salts in the extract. B. Various Extraction Methods for Glycolate Dehydrogenase and P- glycolate Phosphatase Cells were centrifuged from their culture medium, washed with 20 mM Tris, pH 7.7, centrifuged, and incubated in the various procedures at 3:1 (v:w) for 30 min, with stirring, in the cold. Nearly every procedure caused P-glycolate phosphatase activity (Chapter 3) to be solubilized (Table 1). Whereas KCl or detergent alone or together removed the phosphatase from the cell, salt alone did not remove the dehydrogenase, and it reduced the yield by detergents by 50%. These 15 .oo_-x couwcp an cmuomcpxm mam: mmmumcamogq manpouxpmnd coy -cwmuoca me.»-=as.m_os: o_m.o can mmmcwmoccxsmc mumpooxpm cow wncwwuoca as. .c.5. ..051 _ m_o.o mpmewxocaa< .oole coavcp an umpumcuxm uczosm m mommpcmucmq mam m waszz mmmpmggmogd o m_ mm as oo_ «a ma Aoo_v mom_oospo-a ommcmmocvxcoo o o o o oo_ ONN _m Aoopv moa_oo»_w Fux+ Fox+ om: ¢ 1.0% deoxycholate; late. Chlam do- 1+.- 0 0.3% deoxycho- 19 aeration stream, and low C02 during growth is the air level (0.03- 0.06% 002). It was not clear whether the level of C02 during growth of Chlamydomonas had an effect upon the amount of P-glycolate phos- phatase. The high CD2 cells had about half the activity of glycolate dehy- drogenase and P-glycolate phosphatase on a per g wet weight, mg pro- tein, or mg chlorophyll basis (Table 3). There was about twice as much material (wet weight) of cells grown on high CO2 so the total amounts of each enzyme per culture came out about the same for enzyme preparations. 0. Yellow Chlorella Mutant No evidence for a glycolate-dependent 02 uptake or glycolate oxidase was found in extracts from Chlorella vulgaris, either the yellow mutant or the wild type. Neither the crude, French press extract nor the ammonium sulfate fraction prepared by the methods of Kowallik and Schmid (37), had any activity for glycolate oxidase (Table 4). However, glycolate-dependent, DCPIP reduction was present, which by the criteria of Frederick, Gruber, and Tolbert (15), one would call glycolate dehydrogenase. The activity was not inhibited by 02, was inhibited by cyanide, and used D-lactate as well as glycolate much more readily than L-lactate as substrate. The activity was not stimulated by FMN. In these respects the glycolate-oxidizing system in Chlorella vulgaris was similar to the Chlamydomonas reinhardtii enzyme. It is possible to consider that glycolate dehydrogenase is part of an electron transport chain in the mitochondria, and under the 20 mo.o o.~ N.o o.N mo.o m.o no.0 m.m Nou sac: EF< mFPmu umummcp pcwmcmumo o.m om o.m mm o o m.o mm moo cow: LF< mP—mu cmpmuwcom .< F news. ucmmpoca mE.PoE: P pm: .Fosn ices. npzmwmz — news. -cwmpoca news._-u;mwm3 mE.~oE: F pm: m.FoE: mmmumgamocd mum_ou»_und mmmcmmocnxcmo omeooxpw moo saw: no cw< saw: czoco Pwpucmccwmc mucosou>2mpcu cw mmmpmgqmogd mumpouxpwnd new mmmcmmogoxsoo compouapu mo >p_>wuo< m m4m5 noesmoos mo; Frzsqoeopgu .mmppmcmmco somewam mo cowpmmaewcpcmo acmwcmcm xuwmcmu mmocoam w weaned 41 w-CHLOROPHYLL (ma/ml) K) Si 1.0 i“) Q OE it“ :i 5 12 ..—-J : _____ 8% Q: .00 F". cu is ‘ T5 8 o to (o —— CATALAS‘E (,umo/e - min”’-m/"’) ---CYTOCHROMEI C OXIDASE (nmo/e min (ml Figure 9 42 . -Fe.me mm mm: mza we» .F-PE.m: mm mm: ammmm on“ cw sz mo covumcpcwocou on» F.2owuwuum on cue: mum; mum—compo mgp mo monogamoema on» men mammnucmcma cw mgdnEsz nemev N.om Aeowv m.m~ Aaoav a.o~ Assay o.,~ oooooe_-o Assay m.mN Assay m.m~ “somv e.m~ Aaoo_v e.m~ ooepoospo Flewmyoca mE.F-:wE.PoEc we; see emz mza emz eoaoceee oz oomcomsem mwuuemgcwmc mmcoEonAEm_;u Eoce mmmcmmoeuxcmo mpm_ouxpw coo mamm< deuo co pmz co pummem m m4m P-glyco- late phosphatase activity, -~1 protein concentration (A280). 56 separate the phosphatase with a 4-fold purification, most of the activity was lost (Table 10). Other methods were tried to purify this phosphatase without success. This included having 2 mM MgCl2 present throughout the DEAE- column chromatography. Sephadex G-200 chromatography caused a 99% loss in activity from the previous step. The enzyme activity does not bind to carboxymethyl-cellulose column with 10 mM Bicine at pH 8.0 with a 60% loss of activity. Hydroxylapatite chromatography in Hepes buffer also did not work. A similar failure to purify the P-glycolate phosphatase due to loss of activity has been experienced with the enzyme from tobacco leaves (56). D. Search for Isozymes of P-Glycolate Phosphatase from Chlamydomonas reinhardtii It has been reported that there are two isozymes of P-glycolate phosphatase in pea leaves (59). These forms had been separated both by DEAE-cellulose chromatography and by polyacrylamide gel electro- phoresis. DEAEcellulose chromatography in a Tris-citrate buffer from 10 to 250 mM was tried on (NH4)ZSO4 fractions from Chlamydomonas ex- tracted by both sonciation and detergent treatment. Both times one major peak of activity was found halfway into the gradient and some- times a tiny amount of activity was seen at the end of the gradient, that had disappeared by the time the fractions were reassayed (Figure 10). If there were two isoenzymic forms, one was present in vanish- ingly small amounts. Polyacrylamide gels (7%) were run on fractions from both column experiments, and on the material applied to the columns, as well as on the main peak. The material from the possible 57 second peak was also used, but no activity was seen on the gradients. The gels were stained for protein by Coomassie blue, and for phos- phatase activity by lead precipitation of the liberated phosphate from P-glycolate (56). In all cases only one band at an Rf of 0.4 was visualized by the activity stain. The phosphatase was not the major protein band in those gels run on the DEAE-cellulose pools. E. pH Profile of Activity of P-Glycolate Phosphatase from Chlamydomonas reinhardtii The deoxycholate extract of Chlamydomonas cells grown with air had a pH optimum at pH 8, which was not the same as that for the enzyme from pea, spinach, or tobacco leaves (see section A). The ammonium sulfate precipitate had a pH optimum of 8.7, which was the same as that for glycolate dehydrogenase. When the (NH4)ZSO4 fraction was dialyzed against 2 mM MgC12, the activity at pH 8.7 was about 60% lost (Figure 12) and a broad pH profile was obtained for the remaining activity with an apparent peak between pH 7 and 8. This change during dialysis occurred in the presence of added MgClz. In the absence of MgCl2 during dialysis, the same pH shift occurred (Figure 13), without much loss of maximum activity. The maximum activity was, however, less without M9012 and in the range seen in the sample dialyzed with MgClZ. The pH profile of the low remaining enzyme activity after DEAE chromatography was also assayed with Mg++. Activity extended over a broad range between 6.5 to 8.7 (Figure 14), and was similar in both dialyzed and undialyzed samples. Whatever factor or reason for the activity maximum to peak in the basic range seems to have been lost from the enzyme either by dialysis or chromatography on DEAE-cellu- lose. 58 .s E /.O~ $ 0 g o o d’ :1' 0 ° ’1,’H‘:F‘ it: !\i» o v‘f’ '0 .“\f ‘/ i/o' ' dialyzed -\ LUO.5’ o o L? 0: g a a 53 pH Figure 12 The pH profile of the ammonium sulfate fraction of P-glycolate phosphatase from Chlamydomonas reinhardtii before and after dialysis assayed with 5 mM M9012. The buffers used were 50 mM succinate, Bis-tris, Hepes, Bicine, and Ches. 59 'oi lama/e :mInT’m/T’ 3} Figure 13 The pH profile of the ammonium sulfate fraction of P-glycolate phosphatase from Chlamydomonas reinhardtii before and after dialysis assayed without M6012. o° undialyzed O o O I dialyzed ' .3 o o o .0 O . I o O 0 . o o . ,0 oo . o o o O O o o o .' Q 6 7 8 9 Conditions are the same as in Figure 12. ‘1’! Vi: n...“ .. m at. 6O .5- .4. undialyzed P l\ o ..o. o . I \E' _ ’-"~ 1 I 53 //////////’.. . A .5; o o i ES "0 o o-41_8 $9 '2’ o/ . 1” \ O dialyzed \ . E \ 3. /. O Figure 14 The pH profile of fractions from the DEAE-cellulose fractiona- tion of P-glycolate phosphatase from Chlamydomonas reinhardtii before and after dialysis with 5 mM MgC12. Conditions are the same as in Figure 12. 61 Although citrate shifted the pH optimum of the higher plant enzyme (56), added 1 mM citrate had no effect on the ammonium sulfate fraction after dialysis with Mg++. The ammonium sulfate fraction containing the P-glycolate phos- phatase from high CO2 grown cells was also tested. There was no difference in the enzyme purification characteristics from the air grown cells, and the pH profiles were the same. F. Substrate Specificity of P-Glycolate Phosphatase There existed the possibility that what was being assayed was not a specific P-glycolate phosphatase as exists in higher plants, but alkaline or acid phosphatases with residual activity with P-glycolate. However, the activity of the phosphatase was specific for P-glycolate at pH 8 (Table 11). No acid phosphatase would be detectable, because the assays were run above that pH range. In Figure 15 are the pH profiles for the various phosphatases present in the ammonium sulfate fraction from air grown Chlamydomonas. The peak of P-glycolate phosphatase activity between pH 8 and 9 was not due to alkaline phosphatase, which utilizes p-nitrophenyl-P (Figure 15a). There was an acid phosphatase which corresponded to a D-3-P-glycerate phosphatase (Figure 15b), but it had its maximum activity at or below pH 5 and could not contribute to P-glycolate hydrolysis above pH 7. The activity with ATP was negligible across the pH range. A peak of activity with pyrophosphate was seen at pH 7.5 which disappeared upon dialysis and was dependent on Mg++. The P- glycerate activity was also dependent on Mg++, but neither pyrophos- phate, ATP, o-carboxyphenyl-P or p-nitrophenyl-P were sensitive to Mg++ or dialysis. 62 TABLE 11 Substrate Specificity of P-Glycolate Phosphatase from Chlamydomonas reinhardtii Substrate Relat1ve%Act1v1ty P-Glycolate 100 D-3-P-glycerate B-Glycerol-P Fructose-6-P 6-P-gluconate o-carboxyphenyl-P ADP NO-bOOOO p-nitrophenyl-P Potassium Pyrophosphate 1.0 ATP 0 Assayed with 0.6 units of a partially purified enzyme (DEAE pool). All substrates were 30 mM. Assays were for P. release in 20 mM Bicine at pH 8.0. 1 63 Figure 15 The pH profiles of phosphate released from several substrates assayed with an ammonium sulfate fraction from Chlamydomonas rein- hardtii. a) H is activity with P-glycolate, and H is with the alkaline phosphatase substrate, p-nitrophenyl-P. b)00 is activity with 3-P- glycerate, and E1 is with the acid phosphatase substrate, 0- carboxyphenyl-P. 64 . . _ 2 I . I kETEEGBES. T \E.\ 3E. BE: Figure 15 65 G. Attempts at Reactivation of P-Glycolate Phosphatase Because P-glycolate phosphatase activity was lost after dialysis or DEAE-cellulose chromatography (Table 10), the possibility of the loss of an essential cofactor was explored. Various compounds were tested for their effect on enzyme activity in an ammonium sulfate fraction, a Sephadex G-50 preparation, and a dialyzed ammonium sulfate fraction of P-glycolate phosphatase from air grown Chlamydomonas. l i! mM DTT, 1 mM isocitrate, and 1 mM L and DL proline had no effect. 1 i Dialysate, with and without EDTA when added.back to the enzyme, had no effect. ‘ An ammonium sulfate precipitate was resuspended in 10 mM EDTA and dialyzed against 20 mM Bicine at pH 8.0 with 9 mM EDTA, and then dialyzed extensively against Bicine without EDTA. It was then assayed with various divalent cations which activate the higher plant enzyme. None of the cations tested worked as well as MgClz. The optimum Mg++ concentration was between 0.5-5.0 mM (Table 12). It can be seen from Figure 16 that 100 mM KCl stimulated the P- glycolate phosphatase activity slightly, but was inhibitory at higher concentrations. Ammonium sulfate at a similar concentration also stimulated activity. P-glycolate phosphatase from blood is markedly stabilized by KCl (62). H. Stability of P-Glycolate Phosphatase The more purified enzyme, past the ammonium sulfate preparation step, was not stable to storage when frozen. Also the crude enzyme preparations could be stored only a week, but the loss of activity in the crude and purified preparations could have been due to different 66 TABLE 12 Effect of Some Salts on Dialyzed P-Glycolate Phosphatase from Chlamydomonas reinhardtii Addition ”Hufio],m,n-1.§?-i'4 No addition 0.05 0 5 mM MnCl2 0.12 0.07 5 mM ZnCl2 0.09 0.02 5 mM CoCl2 0.15 0.08 5 pM MgCl2 0.14 0.01 50 pM MgCl2 0.54 0.48 0.5 mM MgCl2 0.73 0.72 5 mM MgCl2 0.84 0.72 50 mM MgCl2 0.67 0.47 67 R 8 no added Mg++ ,umo/e - min "’m/ ‘l 9 F3 IOO K C/ (mM) Figure 16 The effect of increasing ionic strength on P-glycolate phos- phatase from Chlamydomonas reinhardtii. 68 causes. Since it has been reported that the tobacco enzyme was stabilized by citrate (56), citrate was tried as a stabilizer for the enzyme from Chlamydomonas. Neither 10 mM citrate or isocitrate slowed down the loss of activity at 50°C of an ammonium sulfate fraction at pH 8.0, with 2 mM MgC12. The 5 time of inactivation was 4 min. These acids also did not prevent loss of activity on purification when present in buffers. Other compounds were tested for their effect on the storage of the enzyme and its stability to freeze-thawing (Table 13). Aliquots of a DEAE-cellulose pooled enzyme fraction frozen in the presence of various compounds was later assayed for activity. With no additions, 45-65% of the activity was lost after freeze-thawing. Including 5 mM DDT not only seemed to inhibit the reaction, it also caused a loss of most of the activity on storage. Both 2.5 mM MgCl2 and 25% glycerol protected the activity with glycerol being slightly more effective. In the presence of 2% BSA, activity was slightly stimulated, and was not lost on freeze-thawing. The activity actually increased with ammonium sulfate over the course of the experiment. 69 TABLE 13 Stability of P-Glycolate Phosphatase from Chlamydomonas reinhardtii to Freeze-thawing Treatment Freeze-thaw before 1 2 none 100% (100%) 34% (54%) -- (53%) 25% Glycerol 186% (90%) 127% (83%) 115% (107%) 5 mM DTT 66% (47%) 5% (7%) 5% ( %) 60% Saturated (NH4)ZSO4 85% (7 %) 144% (108%) 212% (118%) 2.5 mM MgCl2 127% (81%) 97% (65%) 107% (82%) 10 mM citrate 129% (79%) 107% (51%) 90% (65%) 1 mg/ml BSA 202% (133% 132% (113%) 132% (121%) Activities are expressed in % relative to the control from the first day of the experiment. The treatments were done with an enzyme after a DEAE cellulose chromatography had 0.06 units/m1. In a second experiment (results in parentheses) the preparation had 0.07 units/ml. CHAPTER IV NADPH:GLYOXYLATE AND NADH:HYDROXYPYRUVATE REDUCTASES FROM CHLAMYDOMONAS REINHARDTII LITERATURE REVIEW F Glyoxylate reductase activity was first described by Zelitch and Ochoa (63) in 1953 in extracts of spinach leaves. These extracts used glyoxylate to oxidize NADH and the enzyme was named by them "glyoxy- late reductase" (EC 1.1.1.26). About the same time Vennesland's group (64) described an activity with NADH which converted hydroxypyruvate to D-glycerate, which they called D-glycerate dehydrogenase. This enzyme was purified to a crystalline form from both spinach and tobacco leaves and found to use only NADH at pH 6.5. In 1962 Zelitch and Gotto (65) showed that two activities, one for NADH and one for NADPH could be separable by ammonium sulfate fractionation. The NADH- linked activity had a high Km for glyoxylate, but the NADPH-linked activity had a lower Km for this substrate. Both activities from spinach or tobacco leaves had a pH optimum at around 6-6.5. In 1970 the properties of spinach leaf “glyoxylate (NADH) reductase" were extensively described in a series of papers from Kohn's lab (66,67, 68,69). The crystalline enzyme is available commercially. In 1970 Tolbert et_al, (70) showed that the two activities were located separately in the spinach leaf. The "glyoxylate (NADH) re- ductase" activity was located in the peroxisomes and was more specific 70 71 for hydroxypyruvate (Km = 0.12 mM) than for glyoxylate (Km = 15-35 mM) and was also specific for NADH. The NADPH activity was located in the chloroplasts and was not active with hydroxypyruvate. It had a Km for glyoxylate of 0.13 mM. They suggested that the peroxisomal activity previously called "glyoxylate reductase" be renamed "hydroxypyruvate reductase" (or D-glycerate dehydrogenase) and that the name "glyoxy- late reductase" be reserved for the chloroplastic NADPH specific activity. This is the nomenclature that will be used here. In spinach leaves the activity of the hydroxypyruvate reductase l 1 Fr; “‘ “*1fizn TIN ' was 10 nmol-min' -g of tissue' with hydroxypyruvate as substrate, and the activity of glyoxylate reductase (NADPH) with glyoxylate was 0.2 1-g of tissue'1 (70). to 0.3 nmol-min' It is highly debatable whether any substantial glyoxylate pool remains in the peroxisome. It is a highly reactive substance and should be immediately removed by the specific transaminases present to form glycine (1). It if were not, it would immediately be decar- boxylated with any excess H202. It has been argued that 10% of the glyoxylate is decarboxylated in this way in leaf tissue (71), and that this is an important source of photorespiratory C02. Glyoxylate is also the source of oxalate in plants, and it is well known (72) that spinach plants grown on poor nitrogen containing soil have more oxalate-salts presumably since there is insufficient NH3 to trans- aminate glyoxylate to glycine. Others have suggested the accumulation of oxalate may be because of necessity to chelate metal ions and detoxify the plant (72). In any case, in green unicellular algae such as Chlamydomonas, which do not have glycolate oxidase in their peroxisomes, and which 72 apparently metabolize very little glycolate to glyoxylate, the ne- cessity for glyoxylate reductase cannot be explained in this way. When the algae are grown on low nitrogen containing media (32), they can simply excrete the excess glycolate. The NADPH:glyoxylate reduc- tase would provide a mechanism for utilization of excess photoreducing power as NADPH, if a source of glyoxylate were present. These algae grow best in lower light intensities and so perhaps they are very sensitive to the effects of high light, in which excess reducing power may be generated. The peroxisomal hydroxypyruvate reductase is thought to act in the oxidative photosynthetic carbon cycle to generate glycerate which can be phosphorylated and enter the reductive photosynthetic carbon cycle in the chloroplasts. This would return 3 of every 4 carbon atoms which left the chloroplasts as glycolate due to the unavoidable side reaction of ribulose-P2 carboxylase/oxygenase with 02. The low activity of NADPH:glyoxylate reductase does not seem to have a very vital role, and there is no known source of glyoxylate in the chloroplasts. A terminal oxidase system has been proposed with a cycle between glycolate oxidase in the peroxisomes and glyoxylate reductase in the chloroplasts (1). Such a cycle would link NADPH oxidation to H202 production in the peroxisomes. No physiological evidence for such a cycle has been found. Yokota and Kitoaka (72) presented evidence that in Euglena both activities (glycolate dehydro- genase and glyoxylate NADPH reductase) were present in the mitochon- dria. They disrupted mitochondria and added both rotenone and glyco- late and got NADPH oxidation. They took this as evidence for the shuttle in Euglena mitochondria. The glycolate oxidizing system is 73 different in Euglena from both higher plants and from algae and cyano- bacteria as is discussed elsewhere. Another hypothesis for the presence of glyoxylate reductase is as a scavenger for the highly reactive glyoxylate should it be formed by any mechanism in the sensitive chloroplast. MATERIALS AND METHODS Chlamydomonas reinhardtii were grown with air or 5% C02 and harvested as described in Chapter 1. The 0.3% deoxycholate extracts were prepared as described in Chapter 1. The ammonium sulfate frac- tionation and cellulose column preparation were also done as described previously. Both NADPH:glyoxylate reductase and NADH:hydroxypyruvate reductase were assayed at 30° and at 340 nm in a Beckman spectrophoto- meter in 1 m1 total volume. The assay contained: 0.7 ml of 0.2 M K phosphate at pH 6.2, 0.05 ml of 5 mg NADPH/ml, enzyme and water to 0.9 ml, and the reaction was started with 0.1 m1 of 20 mM sodium glyoxy- late. Hydroxypyruvate reductase was assayed the same way, except NADH and lithium hydroxypyruvate were used. RESULTS A. Presence of Glyoxylate Reductase and Hydroxypyruvate Reductase in Chlamydomonas reinhardtii 1 of NADPH: There was approximately 0.4 pmol-min'1-mg chlorophyll" glyoxylate reductase in the algal extracts. Hydroxypyruvate reductase was about 10 times more active in these algae than the glyoxylate reductase in both air and C02 grown cells (Table 14). This difference was pronounced in the experiment shown, but was not always this great. 74 TABLE 14 NADPH:Glyoxylate Reductase and NADH:Hydroxypyruvate Reductase in Air and 5% C02 Grown Chlamydomonas reinhardtii Air-grown_ 5% C0 -grown umol.min - wet weig t'1 NADPH:Glyoxylate 0.19 (0.02)* 0.8 (0.06) reductase NADH:Hydroxypyruvate 1.3 (0.14) 1.3 (0.10) reductase *Numbers in parentheses are nmol-min'l-mg protein']. 75 A reproducible difference in the level of glyoxylate reductase between air and 5% C02 grown cells was observed. The high-CO2 grown cells had 2-4 fold more NADPH:glyoxylate reductase than did the air grown cells. B. Direction of Glyoxylate Reductase Assay Glyoxylate reductase was extractable by the same 0.3% deoxycho— late extract which contained glycolate dehydrogenase and P-glycolate phosphatase. An attempt was made to cause glycolate reductase to :""f"“.“.:! V proceed in the reverse direction as a glycolate dehydrogenase to see if it could account for glycolate oxidation in Chlamydomonas. The reaction was tried at pH 9.5 in 0.1 M Ches buffer with 1 mg NADP+ and 12.5 mM glycolate in 1 m1. No reaction was seen, and addition of phenylhydrazine did not establish a reaction rate. In other ways glycolate dehydrogenase and NADPH:glyoxylate reduc- tase appeared to be two different enzymes. Most of the glyoxylate reductase was found in the 45-60% saturated (NH4)2504 precipitate compared with 30-45% for the glycolate dehydrogenase. These results do not rule out the possibility that glyoxylate reductase may act as a dehydrogenase in some unknown manner jg_yjyg, C. Assay Conditions for NADPH:Glyoxylate Reductase from Chlamydo- monas reinhardtii The conditions for assaying NADPH:glyoxylate reductase did not differ from those reported for the spinach enzyme (70). The pH optimum was at approximately pH 6.0 to 6.3 (Figure 17). Both sub- strates were inhibitory at high concentrations (Figures 18a and 19a), and the concentrations used for the standard assay were at the maximum of these curves (2 mM glyoxylate and 0.25 mM NADPH). 76 6 5 LIIi4 <3 0:2- 9.4.2 \X /’ ‘~:E'\L1i c i e a pH Figure 17 The pH profile of NADPH:glyoxylate reductase from Chlamydo- monas reinhardtii. The rates are relative. <>- 0.07 M KPi; -*- 0.14 M KPi; -0- 0.07 M KPPi; -><- 0.14 mM KPPi. 77 RI 9 ole/m1 ’5 RA IE (n o n o n 1 L// _|_J I 2 5 4 5 75 GL YOXYLA TE (mM) I T 188.3% 9 ’ D b C b I I l l I l I I I n l l -2 2 4 6 8 IO 12 GLYOXYLATE (mm-’1 Figure 18 a. The rate of NADPH:glyoxylate reductase from Chlamydomonas reinhardtii with different glyoxylate concentrations. b. Lineweaver-Burk plot for NADPH:glyoxylate reductase. The concentration of NADPH was 0.245 mM. 78 50‘ 8 E20 E ,0 .2' .‘ NADPHIm/W 20- b IO 100 200 500 400 500 V/S Figure 19 The rate of NADPH:glyoxylate reductase from Chlamydomonas a. reinhardtii with different NADPH concentrations. The glyoxylate concentration was held at 2 mM. b. Briggs-Haldane plot of NADPH:glyoxylate reductase. The glyoxylate concentration was held at 1 mM. flChAfl.Js W . i. 79 The assay was proportional to the amount of enzyme added. In some cases the small rate of nonenzymatic oxidation of NADPH with glyoxylate present was significant relative to enzymatic rates and the endogenous rate without enzyme had to be subtracted. The non-enzy- matic rate may amount to 1-3 nmol-min'i. Concentrations of potassium phosphate up to 0.13 M did not cause any change in the activity (Figure 17). 0. Purification of NADPH:Glyoxylate Reductase from Chlamydomonas reinhardtii The best method for solubilizing this enzyme from the cells was extraction with 0.3% deoxycholate, although 0.5% Triton X-100 was nearly as good (Table 15). Times between 20-60 min incubation with deoxycholate gave approximately equivalent results. These detergents were preferable to sonicating or using the French press to disrupt the whole cell because the chlorophyll and much of the protein in the cell was not solubilized. The next step in the purification procedure after extraction from the algal cells was precipitation by 0.5% protamine sulfate of protein and nucleic acids which were discarded after centrifugation (Table 16). The supernatant was fractionated by ammonium sulfate precipita- tion, and the 45-60% pellet with the activity was resuspended in 5 mM glycylglycine buffer pH 8.7. The enzyme preparation was then dialyzed with several changes of a 50-fold dilution of the buffer. The hydroxy- pyruvate reductase activity was lost at this dialysis step. The glyoxylate reductase could not be absorbed onto DEAE-cellulose. A TEAE-cellulose column, equilibrated with the 5 mM glycylglycine r4 a- ““-—n s ‘1 80 TABLE 15 Extraction Methods for NADPH:Glyoxylate Reductase from Chlamydomonas reinhardtii Method nmol-mineiigv;:{ weight-1 Buffer only 3 0.5% Triton X-100 547 0.15% Deoxycholate 735 0.5 M KCl 153 1.0 M KCl 468 0.3% Deoxycholate 813 2-3 9 of cells were resuspended in the extraction medium (6 mls) and at 0 min and subsequent times aliquots were removed and centrifuged in an Eppen- dorf Microfuge for 1/2 min. The supernatant was immediately removed and later assayed. Negligible activities were seen at 0 min. The time shown is the activity after 20 min. 81 Z «Juana! m.¢o m.— er.m Nv— om.o xsqmgmopmaocsu mmo—zpqu-maoo< owc_ooam e_oa> _- soa>aoo< m— MJm2 02000 127 Table V. Effect of 02 And Aminooxyacette 0n 14C Excretion by Chlamydomonas. Air-Grown Cells 5% COZ-Grown Cells 9; Concentration 0 1 mM AOA 0 1 mM AOA % 14C excreted 0 (N2) 1.3 5.1 2.0 21% (air) 1.0 8.4 8.0 100% 3.0 24.9 16.4 Air-grown cells were 3 days old and entering the stationary phase. 4.8 13.7 22.7 COZ-grown cells were 5 days old and in the stationary phase. Values are % of total 14C fixed that was excreted after a period of 20 min of photosynthesis with 3 mM NaH14CO3. Since the experiments were run in the light and in tubes open to the air strictly anaerobic conditions were not obtained when gassing with N2. Figure I. Figure 2. Figure 3. Figure 4. 1283 Rate of 14C02 Fixation And Glycolate Excretion By Chlamydomonas Grown On Air When Treated With 1 mM Aminooxyacetate. A 2% Suspension containing 4 x 104 cells/mm3 from the log phase of growth were given 1 mM NaH14CO3 at zero time - total 14C fixed; --- 14C excreted; open circles no treatment; closed circles 1 mM AOA was added 2 min prior to addition of NaH14co3. Rate of 14C02 Fixation And Glycolate Excretion By Chlamydomonas Grown With 5% C02 when treated With 1 mM Aminooxyacetate. Experimental conditions similar to those in figure 1 except that the experiment were run at a different periods with NaH14C03 of lower specific activity. Increase In pH By Air-Grown Chlamydomonas Cells In Water In The Light Upon Addition of NaHCO3. The horizontal dashed line indicates the pH of a NaHCO3 solution. The first Pka of a bicarbonate solution is 6.3 and the second is 9.4. The pH before adding NaHC03 was about 5.5 from the natural acidity of the algal suspension. pH was measured by electrodes in the algal suspension. --- 0.5% cells in 1 mM NaHC03; ———- 1% cells in 1 mM NaHCO3; -——— 1% cells in 10 mM NaHC03. The pH increase with a 2% cell suspension and 10 mM NaHC03 was similar to that with 1% cells and 10 mM NaHCO3. Effect of DCMU on Photosynthetic 14002 Fixation and Excretion of 14C By Air-Grown Chlamydomonas. "‘c fixation (cpm x lo6 - ml") .5 N 129 '4C Excreted Time control lmM AOA min ‘70 Va 2 I2 4.5 5 L7 l2.9 IO 2.2 20.7 20 32 25.8 30 4.2 30.3 ' I //’. /Q2/+AOA v’/ ' ng” Excreted l4C // _____._o . —-—-Q-"“"""'—-?--— 1 2 5 IO 20 30 Minutes after adding NaH l4co3 130 5 T "‘C fixation (0pm x l05 - ml") 03 O .b I +AOA Total '40 O "‘C Excreted Time control lmM AOA min 0/0 0/o 2 0 ID 5 0.7 7.5 ' IO 2.8 l6.6 20 80 26.8 30 89 BID ,0 +AOA ,/ // ’D // /,V Excreted /// / a // ’do /' "_..—-’ // ”,2—0’ . 40"?” 1 1 2 5 IO 20 30 Minutes after adding NaH "‘CO3 131 lo/o CGIIS IO mM NaHCO3 l°/o Cells lmM NaHCO33 I r’osvo Cells /’ lmM NCIHCO3 l l l .IO . I5 20 * Minutes N 05L. '40 fixation (cpm x IO6 - ml") 152 4- Total ”C 3‘ 2 ,’ “02‘ I “0 / I / I, .o 2' , +ocmu/ I, o’// . I . / I II I- I l _.....—--O "9' I A 1 J l o 2 .5 l0 . 20I4 25 Minutes after adding NaH CO3 N D % fixed '4C excreted STRTE UN V. BRQRIE S :9I 9||||7 ||||||9||9|||||||9 9|