植物中草酸积累与光呼吸乙醇酸代谢的关系

对几种C3 和C4 植物中草酸含量及相应的乙醇酸氧化酶活性测定结果表明 :叶片光呼吸强度及其关键酶活性大小与草酸积累量没有相关性 ;植物根中均能积累草酸 ,但未测出乙醇酸氧化酶活性。烟草根、叶中的草酸含量在不同生长时期差异明显 ,且二者呈极显著正相关 (y =2 .5 6 5lnx 2 .137,r =0 .749,P <0 .0 0 1) ,说明根中草酸可能来自叶片。氧化乙醇酸的酶的活性与氧化乙醛酸的酶的活性呈极显著线性正相关 (y =0 .2 41x 0 .0 0 6 ,r=0 .96 7,P <0 .0 0 0 1) ,进一步证实是乙醇酸氧化酶催化了两种底物的反应。烟草在不同生长期叶片中草酸总含量变化与相应的乙醇酸氧化酶活性变化亦没有相关性 ;低磷胁迫可显著诱导烟草根叶中的草酸形成和分泌 ,但并未影响乙醇酸氧化酶活性 ,进一步证明草酸积累与该酶活性大小无关     

Compartmentation studies on spinach leaf peroxisomes : evidence for channeling of photorespiratory metabolites in peroxisomes devoid of intact boundary membrane

In concurrence with earlier results, the following enzymes showed latency in intact spinach (Spinacia oleracea L.) leaf peroxisomes: malate dehydrogenase (89%), hydroxypyruvate reductase (85%), serine glyoxylate aminotransferase (75%), glutamate glyoxylate aminotransferase (41%), and catalase (70%). In contrast, glycolate oxidase was not latent. Aging of peroxisomes for several hours resulted in a reduction in latency accompanied by a partial solubilization of the above mentioned enzymes. The extent of enzyme solubilization was different, being highest with glutamate glyoxylate aminotransferase and lowest with malate dehydrogenase. Osmotic shock resulted in only a partial reduction of enzyme latency. Electron microscopy revealed that the osmotically shocked peroxisomes remained compact, with smaller particle size and pleomorphic morphology but without a continuous boundary membrane. Neither in intact nor in osmotically shocked peroxisomes was a lag phase observed in the formation of glycerate upon the addition of glycolate, serine, malate, and NAD. Apparently, the intermediates, glyoxylate, hydroxypyruvate, and NADH, were confined within the peroxisomal matrix in such a way that they did not readily leak out into the surrounding medium. We conclude that the observed compartmentation of peroxisomal metabolism is not due to the peroxisomal boundary membrane as a permeability barrier, but is a function of the structural arrangement of enzymes in the peroxisomal matrix allowing metabolite channeling.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

Travels in a world of small science*

As a boy, I read Sinclair Lewis's Arrowsmithand dreamed of doing research of potential benefit to society. I describe the paths of my scientific career that followed. Several distinguished scientists served as my mentors and I present their profiles. Much of my career was in a small department at a small institution where independent researchers collaborated informally. I describe the unique method of carrying on research there. My curiosity about glycolate metabolism led to unraveling the enzymatic mechanism of the glycolate oxidase reaction and showing the importance of H₂O₂ as a byproduct. I discovered enzymes catalyzing the reduction of glyoxylate and hydroxypyruvate. I found α-hydroxysulfonates were useful competitive inhibitors of glycolate oxidase. In a moment of revelation, I realized that glycolate metabolism was an essential part of photorespiration, a process that lowers net photosynthesis in C₃ plants. I added inhibitors of glycolate oxidase to leaves and showed: (1) glycolate was synthesized only in light as an early product of photosynthetic CO₂ assimilation, (2) the rate of glycolate oxidation consumed a sizable fraction of net photosynthesis in C₃ but not in C₄ plants, and (3) that glycolate metabolism increased greatly at higher temperatures. For a while I studied the control of stomatal opening in leaves, and this led to the finding that potassium ions are a key solute in guard cells. I describe experiments that show that when photorespiration rates are high, as occurs at higher temperatures, genetically increasing leaf catalase activity reduces photorespiration and increases net photosythetic CO₂ assimilation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cadmium induces senescence symptoms in leaf peroxisomes of pea plants

The effect of growing pea (Pisum sativum L.) plants with a toxic CdCl₂ concentration (50 µm ) on the metabolism and proteolytic activity of leaf peroxisomes was studied. In peroxisomes purified from plants treated with cadmium, an increase in the total protein concentration and in the activity and protein level of the photorespiratory enzyme glycolate oxidase was found. The glyoxylate cycle enzymes, malate synthase and isocitrate lyase, whose activity is normally very low in leaf peroxisomes, were enhanced by Cd treatment. The activity of the endogenous proteases of leaf peroxisomes was determined. Two leucine‐aminopeptidase isozymes (AP1‐AP2) were detected, and their activity was slightly higher in Cd‐treated plants. Five endopeptidases (EP1‐EP5) were present in pea leaf peroxisomes, and in plants grown with Cd the activity of isozymes EP1‐EP4 was increased. The ultrastructural analysis of pea leaves showed that Cd produced a disorganization of the chloroplast structure, with an increase in the number of plastoglobuli, and the formation of vesicles in the vacuoles. Taken together, these results indicate that Cd induces senescence symptoms in leaf peroxisomes, and probably a metabolic transition of leaf peroxisomes into glyoxysomes, and suggest that the peroxisomal proteases could participate in the metabolic changes produced by Cd.     

Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae.

Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.     

Synthesis of oxalic Acid by enzymes from lettuce leaves

A rapid purification of lactate dehydrogenase and glycolate oxidase from lettuce (Lactuca sativa) leaves is described. The kinetics of both enzymes are reported in relation to their possible roles in the production of oxalate. Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model is proposed in which glycolate oxidase in the peroxisomes and lactate dehydrogenase in the cytosol are involved in the production of oxalate. The effect of pH on the balance between oxalate and glycolate produced from glyoxylate suggests that in leaves lactate dehydrogenase may function as part of an oxalate-based biochemical, pH-stat.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled--others remain

The most prominent role of peroxisomes in photosynthetic plant tissues is their participation in photorespiration, a process also known as the oxidative C2 cycle or the oxidative photosynthetic carbon cycle. Photorespiration is an essential process in land plants, as evident from the conditionally lethal phenotype of mutants deficient in enzymes or transport proteins involved in this pathway. The oxidative C2 cycle is a salvage pathway for phosphoglycolate, the product of the oxygenase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), to the Calvin cycle intermediate phosphoglycerate. The pathway is highly compartmentalized and involves reactions in chloroplasts, peroxisomes, and mitochondria. The H2O2-producing enzyme glycolate oxidase, catalase, and several aminotransferases of the photorespiratory cycle are located in peroxisomes, with catalase representing the major constituent of the peroxisomal matrix in photosynthetic tissues. Although photorespiration is of major importance for photosynthesis, the identification of the enzymes involved in this process has only recently been completed. Only little is known about the metabolite transporters for the exchange of photorespiratory intermediates between peroxisomes and the other organelles involved, and about the regulation of the photorespiratory pathway. This review highlights recent developments in understanding photorespiration and identifies remaining gaps in our knowledge of this important metabolic pathway.     

S-Nitrosylated proteins in pea (Pisum sativum L.) leaf peroxisomes: changes under abiotic stress

Peroxisomes, single-membrane-bounded organelles with essentially oxidative metabolism, are key in plant responses to abiotic and biotic stresses. Recently, the presence of nitric oxide (NO) described in peroxisomes opened the possibility of new cellular functions, as NO regulates diverse biological processes by directly modifying proteins. However, this mechanism has not yet been analysed in peroxisomes. This study assessed the presence of S-nitrosylation in pea-leaf peroxisomes, purified S-nitrosylated peroxisome proteins by immunoprecipitation, and identified the purified proteins by two different mass-spectrometry techniques (matrix-assisted laser desorption/ionization tandem time-of-flight and two-dimensional nano-liquid chromatography coupled to ion-trap tandem mass spectrometry). Six peroxisomal proteins were identified as putative targets of S-nitrosylation involved in photorespiration, β-oxidation, and reactive oxygen species detoxification. The activity of three of these proteins (catalase, glycolate oxidase, and malate dehydrogenase) is inhibited by NO donors. NO metabolism/S-nitrosylation and peroxisomes were analysed under two different types of abiotic stress, i.e. cadmium and 2,4-dichlorophenoxy acetic acid (2,4-D). Both types of stress reduced NO production in pea plants, and an increase in S-nitrosylation was observed in pea extracts under 2,4-D treatment while no total changes were observed in peroxisomes. However, the S-nitrosylation levels of catalase and glycolate oxidase changed under cadmium and 2,4-D treatments, suggesting that this post-translational modification could be involved in the regulation of H(2)O(2) level under abiotic stress.     

Peroxisomes in the Alga Vaucheria are Neither of the Leaf Peroxisomal Nor of the Glyoxysomal Type*,1

Microbodies were isolated from the freshwater alga Vaucheria sessilis as well as from a marine Vaucheria. The organelles equilibrated on sucrose gradients at densities 1.23 g . cm^?3 and 1.24g . cm^?3, respectively. On electron micrographs they showed an ovoid or spheroid shape with a diameter of 0.5 to 0.8 μm. Besides catalase, the peroxisomes of both algae possess glycolate oxidase and glutamate-glyoxylate aminotransferase, but no other leaf-peroxisomal enzymes. Instead, the enzymes malate synthase and isocitrate lyase, which are markers of glyoxysomes in higher plants, are constituents of the peroxisomes in the marine as well as in the freshwater alga. Citrate synthase, aconitase, malate dehydrogenase and enzymes of the fatty acid β-oxidation pathway are located exclusively in the mitochondria. Therefore, the peroxisomes from Vaucheria do not belong to either the type of leaf peroxisomes or to the type of glyoxysomes.     

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Metabolism of glycolate and glyoxylate in intact spinach leaf peroxisomes

Intact and broken (osmotically disrupted) spinach (Spinacia oleracea) leaf peroxisomes were compared for their enzymic activities on various metabolites in 0.25 molar sucrose solution. Both intact and broken peroxisomes had similar glycolate-dependent o₂ uptake activity. In the conversion of glycolate to glycine in the presence of serine, intact peroxisomes had twice the activity of broken peroxisomes at low glycolate concentrations, and this difference was largely eliminated at saturating glycolate concentrations. However, when glutamate was used instead of serine as the amino group donor, broken peroxisomes had slightly higher activity than intact peroxisomes. In the conversion of glyoxylate to glycine in the presence of serine, intact peroxisomes had only about 50% of the activity of broken peroxisomes at low glyoxylate concentrations, and this difference was largely overcome at saturating glyoxylate concentrations. In the transamination between alanine and hydroxypyruvate, intact peroxisomes had an activity only slightly lower than that of broken peroxisomes. In the oxidation of NADH in the presence of hydroxypyruvate, intact peroxisomes were largely devoid of activity. These results suggest that the peroxisomal membrane does not impose an entry barrier to glycolate, serine, and O₂ for matrix enzyme activity; such a barrier does exist to glutamate, alanine, hydroxypyruvate, glyoxylate, and NADH. Furthermore, in intact peroxisomes, glyoxylate generated by glycolate oxidase is channeled directly to glyoxylate aminotransferase for a more efficient glycolate-glycine conversion. In related studies, application of in vitro osmotic stress to intact or broken peroxisomes had little effect on their ability to metabolize glycolate to glycine.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Conversion of serine to glycerate in intact spinach leaf peroxisomes: role of malate dehydrogenase

In photorespiration, leaf peroxisomes convert serine to glycerate via serine-glyoxylate aminotransferase and NADH-hydroxypyruvate reductase. We isolated intact spinach leaf peroxisomes in 0.25 M sucrose, and characterized their enzymatic conversion of serine to glycerate using physiological concentrations of substrates and coenzymes. In the presence of glycolate (glyoxylate), and NADH and NAD alone or together in physiological proportions, the rate of serine-to-glycerate conversion was enhanced and sustained by the addition of malate. The rate was similar at 1 and 5 mM serine, but was two to three times higher in 50 mM than 5 mM malate. In the presence of NAD and malate, there was 1:1 stoichiometric formation of glycerate and oxaloacetate. Addition of 1 or 5 mM glutamate resulted in a negligible enhancement of the conversion of hydroxypyruvate to glycerate. Intact peroxisomes produced glycerate from either serine or hydroxypyruvate at a rate two times higher than osmotically lysed peroxisomes. These results suggest that under physiological conditions, the peroxisomal malate dehydrogenase operates independent of aspartate-alpha-ketoglutarate aminotransferase in supplying NADH for hydroxypyruvate reduction. This supply of NADH is the rate-limiting step in the conversion of serine to glycerate. The compartmentation of hydroxypyruvate reductase and malate dehydrogenase in the peroxisomes confers a higher efficiency in the supply of NADH for hydroxypyruvate reduction under a normal, high NAD/NADH ratio in the cytosol.     

Effects of water stress on glycolate metabolism in the leaves of rice seedlings (Oryza sativa)

To investigate the effects of water stress on glycolate metabolism, seedlings of a drought-tolerant cultivar (N-22) and a susceptible cultivar (Jaya) of Oryza sativa L. were subjected to water stress for 5, 8 or 10 days. Increasing the duration of water-deficit-stress produced a proportional decrease in relative water content and leaf water potential, reduced glycolate content and catalase (EC 1.11.1.6) activity, but increased glycolate oxidase (EC 1.1.3.1) activity, hydrogen peroxide and glyoxylate contents in the leaves of both cultivars. In a radiotracer experiment, with increasing duration of water stress, the proportion of label increased in 3-phosphoglycerate, glycolate, glycine and serine. The drought-tolerant cultivar (N-22) was affected less than the susceptible cultivar (Jaya). The glycolate pathway metabolism is discussed in relation to photorespiration and the effects of water stress.     

茉莉酸甲酯对水稻幼苗光呼吸代谢的影响

经2．5×10-4mol／L茉莉酸甲酯（MJ）处理后的水稻幼苗,在处理后第2天即表现出RuBP加氧酶活性的明显升高,至处理后第4天,叶片中乙醇酸氧化酶的活性也升高,同时叶片中的乙醇酸累积量也明显高于对照。经α-HPMS预处理后的幼苗叶片材料中,这种乙醇酸累积量升高的程度更大,表明2．5×10-4mol／LMJ处理能促进水稻幼苗光呼吸的增强,但2．5×10-7mol／LMJ处理对叶片中乙醇酸的累积和光呼吸过程中的有关酶系没有影响。     

Mitochondrial glycolate oxidation contributes to photorespiration in higher plants

The oxidation of glycolate to glyoxylate is an important reaction step in photorespiration. Land plants and charophycean green algae oxidize glycolate in the peroxisome using oxygen as a co-factor, whereas chlorophycean green algae use a mitochondrial glycolate dehydrogenase (GDH) with organic co-factors. Previous analyses revealed the existence of a GDH in the mitochondria of Arabidopsis thaliana (AtGDH). In this study, the contribution of AtGDH to photorespiration was characterized. Both RNA abundance and mitochondrial GDH activity were up-regulated under photorespiratory growth conditions. Labelling experiments indicated that glycolate oxidation in mitochondrial extracts is coupled to CO(2) release. This effect could be enhanced by adding co-factors for aminotransferases, but is inhibited by the addition of glycine. T-DNA insertion lines for AtGDH show a drastic reduction in mitochondrial GDH activity and CO(2) release from glycolate. Furthermore, photorespiration is reduced in these mutant lines compared with the wild type, as revealed by determination of the post-illumination CO(2) burst and the glycine/serine ratio under photorespiratory growth conditions. The data show that mitochondrial glycolate oxidation contributes to photorespiration in higher plants. This indicates the conservation of chlorophycean photorespiration in streptophytes despite the evolution of leaf-type peroxisomes.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.