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.     

Glyoxysomes in megagamethophyte of germinating ponderosa pine seeds

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated.     

Cytochemical localization of malate synthase in amphibian fat body adipocytes: possible glyoxylate cycle in a vertebrate

The adipocytes of amphibian abdominal fat bodies contain typical microperoxisomes, as indicated by their fine structure. Electron microscopic cytochemistry showed that these organelles contain the enzymes catalase, typical for peroxisomes, and malate synthase. The latter is an enzymatic component characteristic of the glyoxylate cycle, a biochemical pathway known to exist in plant glyoxysomes (peroxisomes). This metabolic pathway makes possible the net conversion of lipid to carbohydrate. Toad adipocytes may represent yet another example of vertebrate peroxisomes which contain one of the marker enzymes (malate synthase) characteristic of the glyoxylate shunt.     

Determination of malate synthase activity in polyacrylamide gels

It is shown that the generation of the insoluble precipitate, copper ferrocyanide, provides a distinct, rapid, and sensitive method for localizing malate synthase in polyacrylamide gels. Both enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, can now be specifically stained using the same polyacrylamide gel system. This can contribute substantially to elucidating the developmental or adaptive patterns of the glyoxylate cycle and in determining the degree of homology of glyoxylate cycle enzymes from different organisms or organelles. These techniques also provide a simple method for establishing the occurrence of isoenzymes. Finally, the ability to detect a reaction capable of reducing ferricyanide to form cooper ferrocyanide suggests that this technique may be useful for localization of many of the enzymes which generate CoASH (18,19).     

Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found.The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway.Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: I. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.     

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.     

Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)     

Enzymes of the tricarboxylic acid cycle in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate pathway were investigated in adults and infective larvae of Ancylostoma ceylanicum and Nippostrongylus brasiliensis, and their activities were compared with those obtained in rat liver. A complete sequence of enzymes of the TCA cycle, with most of them showing activities quite similar to those in the rat liver homogenate, was detected in adults of both species. All the enzymes except fumarase and malate dehydrogenase were located predominantly in mitochondria where they showed a variable distribution of activities between the soluble and the membranes fractions. Malate dehydrogenase and fumarase were found in both the mitochondria and the 9,000-g supernatant fraction. Succinyl CoA synthetase, which was present in minimum activity, appeared rate limiting. Enzymes of the glyoxylate pathway, particularly isocitrate lyase, seemed to aid the functioning of the Krebs cycle by allowing the formation of succinate from isocitrate. The infective larvae of both species also were found equipped with all the enzymes of the Krebs cycle. Nonetheless, only isocitrate lyase of the glyoxylate pathway could be detected in these parasites.     

Intact Cell Transformation of Saccharomyces cerevisiae by Polyethylene Glycol

The activities of isocitrate lyase and malate synthase—the key enzymes in the glyoxylate cycle—were found to be fairly high in n-alkane-, acetate-, and propionate-grown cells of Candida tropicalis compared with those in glucose-grown cells. In fact, the results of immunochemical studies showed that the increases in the enzyme levels resulted from increases in the amounts of the enzyme proteins. But the increases in these enzyme activities were not always coincident with the appearance of peroxisomes. Isocitrate lyase and malate synthase were purified from a peroxisome-containing particulate fraction of alkane-grown cells and from whole cells grown on glucose, acetate and propionate. The respective enzymes showed no significant differences in immunochemical properties, specific activities, molecular masses of active forms and subunits, on patterns of limited proteolysis with proteases, but the malate synthases of alkane- and propionate- grown cells showed higher Km values for acetyl-CoA than the enzymes of glucose- and acetate- grown cells. The results indicated that the synthesis of the key enzymes in the glyoxylate cycle did not necessarily have to be coincident with the development of peroxisomes in this yeast.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Role of malate synthase in citric Acid synthesis by maturing cotton embryos: a proposal

Cotton embryos from 34 to 54 days after anthesis were analyzed for organic acids, and enzymes associated with organic acid metabolism. During this developmental period, embryos accumulated citrate. Malate synthase activity appeared at 46 days after anthesis and increased rapidly to 54 days. Of other enzymes examined, only citrate synthase activity increased during this period. As isocitrate lyase activity was absent from cotton embryos during maturation, an alternative source of glyoxylate would be required for in vivo malate synthase activity. Of several metabolic sources tested, glycine was converted to glyoxylate via a transamination reaction.     

Ultrastructure and isolation of glyoxysomes (microbodies) in zoospores of the fungusentophlyctis sp.

Summary A correlative approach, involving light and electron microscopic, cytochemical, and biochemical techniques, was used to study the structure and function of microbodies in zoospores ofEntophlyctis sp. The same population of microbodies already existing in the zoosporangium appeared to be segregated into zoospore initials during cytoplasmic cleavage. Microbodies laid at the anterior end of zoospores and were part of an organized assemblage of organelles, the microbody-lipid globule complex. In the microbody-lipid globule complex, endoplasmic reticulum occurred on the surface of the lipid globules toward the zoospore's exterior, and the microbody, subtended by mitochondria, was appressed to the opposite surface of the lipid globule. The organization of the microbody-lipid globule complex changed as the zoospore swam and encysted. As lipid globules coalesced, the microbody-lipid globule complex became disorganized. After lipid globule coalescence was completed, the microbody-lipid globule complex regained its order, and several microbodies were clustered adjacent to a single lipid globule. The microbodies persisted even in the encysted zoospore, but they were found on all sides of the lipid globule.Microbodies isolated from zoospores contained catalase as well as malate synthase and isocitrate lyase, two enzymes of the glyoxylate cycle. When zoospores encysted greater activities of these glyoxylate cycle enzymes could be detected. The presence of glyoxylate cycle enzymes and the close association between the microbody and lipid globule suggest that microbodies function as glyoxysomes in zoospores and encysted zoospores. The functional significance of the morphological organization of the microbody-lipid complex is discussed in terms of energy production and the conversion of storage lipid into structural components of the cell.     

Studies on the Respiratory Physiology of Euglena gracilis Cultured on Acetate or Glucose

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO₂ fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis

Summary The distribution of glyoxylate-cycle enzymes between microbodies and mitochondria was examined in ethanol-grown Aspergillus tamarii Kita. Particulate activities of catalase and the two glyoxylate by-pass enzymes, malate synthase and isocitrate lyase, were localized in the microbodies. The microbodies had a buoyant density of about 1.23 g cm^-3 after isopycnic centrifugation in linear sucrose gradients. Particulate activities of the other two glyoxycitrate synthase, together with that of succinate dehydrogenase were restricted to the mitochondria, which had a buoyant density of about 1.20 g cm^-3. Catalase also appeared to be localized in a second particle, perhaps the microbody inclusions or the Woronin bodies, having a buoyant density of about 1.26 g cm^-3.     

Purification of peroxisomal malate synthase from alkane-grown Candida tropicalis and some properties of the purified enzyme

Malate synthase, one of the key enzymes in the glyoxylate cycle, was purified from peroxisomes of alkane-grown yeast, Candida tropicalis. The enzyme was mainly localized in the matrix of peroxisomes, judging from subcellular fractionation followed by exposure of the organelles to hypotonic conditions. The molecular mass of this peroxisomal malate synthase was determined to be 250,000 daltons by gel filtration on a Sepharose 6B column as well as by ultracentrifugation. On sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis, the molecular mass of the subunit of the enzyme was demonstrated to be 61,000 daltons. These results revealed that the native form of this enzyme was homo-tetrameric. Peroxisomal malate synthase showed the optimal activity pH at 8.0 and absolutely required Mg²⁺ for enzymatic activity. The K _m values for Mg²⁺, acetyl-CoA and glyoxylate were 4.7 mM, 80 M and 1.0 mM, respectively.     

Regulation of Glyoxylate Metabolism in Escherichia coli K-12

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the "glycolate form" of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.     

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A- gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.     

Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of alpha-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes

A comparative study of the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing alpha-ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.