Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.     

The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells

Synopsis The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback.Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase was determined using H₂O₂ generated by the enzyme, D-alanine as a substrate, and cerous ions for the formation of an electron-dense precipitate. Both enzymes appeared to be located in microbodies. The combined presence of these enzymes characterizes the microbodies as peroxisomes. Biochemically and cytochemically, no urate oxidase or glycolate-oxidizing L--hydroxy acid oxidase could be demonstrated.Stereological analysis of the epithelia lining the renal tubules showed that the fractional volume of the microbodies is 5 to 10 times higher in the cells of the second proximal tubules than in the other nephronic segments or the ureter. The fractional volume of the microbodies was similar in kidneys of freshwater and seawater fishes.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

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.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

THE OCCURRENCE AND STRUCTURE OF MICROBODIES : A Comparative Study

Livers from chickens, rats, mongrel dogs, Dalmatian dogs, and man have been examined in the electron microscope in order to compare the microbodies with the known content of uricase. It is concluded that microbodies with inclusion are present in rats, mongrel dogs, and, although the inclusion generally is smaller, in Dalmatian dogs. The inclusion has a characteristic structural appearance. These species (rat, dog) have uricase. Chickens and man lack both the enzyme uricase and the microbody inclusion. This evidence and that from previously published electron micrographs in the literature on microbodies support the notion of a positive correlation between uricase and microbodies with an inclusion. It is recommended that the term "uricosome" be used for such microbodies that have an inclusion of the appearance here described.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons

Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence.     

Fine-structural characterization of plant microbodies

Summary Morphology and distribution of the relatively less well known organelles of plants have been studied with the electron microscope in tissues fixed in glutaraldehyde and postfixed in osmium tetroxide. An organelle comparable morphologically to the animal microbody and similar to the plant microbody isolated by Mollenhauer et al. (1966) has been encountered in a variety of plant species and tissues, and has been studied particularly in bean and radish roots, oat coleoptiles, and tobacco roots, stems and callus. The organelle has variable shape and is 0.5 to 1.5 in the greatest diameter. It has a single bounding membrane, a granular to fibrillar matrix of variable electron density, and an intimate association with one or two cisternae of rough endoplasmic reticulum (ER). Microbodies are easily the most common and generally distributed of the less well characterized organelles of plant cells. It seems very probable that they contain the enzymes characteristic of animal lysosomes (containing hydrolases) or animal microbodies (containing catalase and certain oxidases). Spherosomes are also possible sites of enzyme activity but are not as common or as widely distributed as microbodies. For this reason it appears likely that the particles designated as plant lysosomes, spherosomes, peroxisomes, etc., in some of the cytochemical and biochemical studies on enzyme localization will prove to be microbodies.Variations in the morphology and ER associations of microbodies in tissues of bean and radish are described and discussed. Crystal-containing bodies (CCBs) are interpreted as a specialized type of microbody characteristic of metabolically less active cells. Stages in the formation of CCBs from microbodies of typical appearance are illustrated for Avena.The general occurrence of microbodies in meristematic and differentiating cells and their close association with the ER suggest that they may play active roles in cellular metabolism. The alterations in their morphology and numbers that are observed in certain differentiating cells suggest further that the enzyme complements and metabolic roles of microbodies might change during cellular differentiation. If so, microbodies could be the functional equivalent of both microbodies and lysosomes of animal cells.NASA Predoctoral Trainee.Public Health Service Postdoctoral Fellow.     

ULTRASTRUCTURAL AND ENZYMATIC EVIDENCE FOR THE PRESENCE OF MICROBODIES IN THE FUNGUS ACHLYA

Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

SECRETION OF ACID HYDROLASES AND ITS INTRACELLULAR SOURCE IN TETRAHYMENA PYRIFORMIS

Axenic Tetrahymena pyriformis, syngen 1, mating type II cells were grown in Cox's defined medium. When washed and transferred into nonnutrient dilute salt solution or resuspended in the defined medium, the intact cells secrete acid hydrolases into the medium. Cells starving in the salt solution release in 5 hr about two-thirds of their β-glucosidase, β-N-acetylglucosaminidase, α-glucosidase, and amylase activities, about one-third of their deoxyribonuclease and phosphatase activities, smaller amounts of ribonuclease, and only a negligible fraction of their proteinase activity and protein content. During this period there is practically no change in the enzyme activities (except for a sudden increase of ribonuclease activity) and protein content of cells and medium together. Cells resuspended in the nutrient medium secrete enzymes as do the starved cells, but replace this loss, so that there is a continuous increase of the activities in the total system. According to isopycnic centrifugation experiments performed in sucrose gradients, the source of the hydrolases is a special population of lysosomes which disappear from the cells during starvation. This population equilibrates in the high density region of the gradients and contains the various acid hydrolases in about the proportion in which these enzymes appear in the medium.     

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.     

Glycolate oxidase content of microbodies as affected by nitrate

Glycolate oxidase is loosely held by microbodies obtained from etiolated barley (Hordeum vulgare L.) leaves depleted of nitrate. Defined centrifugation conditions cause the complete detachment of the enzyme from the microbodies. Addition of nitrate to these plants brings about a greater retention of glycolate oxidase by the microbodies. Synthesis of a nitrate-induced protein seems to be responsible for the enhanced retention of glycolate oxidase. Catalase, on the contrary, is strongly attached to the microbodies under all nutritional and experimental conditions considered.     

Trypanosoma brucei: preparation and some properties of a multienzyme complex catalysing part of the glycolytic pathway

After grinding Trypanosoma brucei with alumina or silicon carbide, it is possible to prepare a multienzyme complex which catalyses the breakdown of glucose to l-glycerol-3-phosphate and 3-phosphoglycerate. The complex sediments with the postnuclear large granule fraction which pellets at 14,500g; it is also eluted in the void volume during Bio-Gel A-5m column chromatography of a cell homogenate. During isopycnic sucrose gradient centrifugation, the multienzyme complex bands at a density of 1.22 g/ml. Because this is the density of T. brucei microbodies, and because Triton X-100 treatment of the material greatly enhances the activities of its component enzymes, we conclude that the multienzyme complex is probably located in the microbodies of the bloodstream long slender forms of T. brucei.     

Charakterisierung der Microbodies aus Spadix-Appendices von Arum maculatum L. und Sauromatum guttatum Schott

Summary When the sections of the spadix appendix of Arum are incubated in a medium containing diaminobenzidine and H₂O₂, only the membrane of microbodies is stained. On the other hand, microbodies of Sauromatum show a stained matrix as usual. Catalase-containing cell organelles isolated from spadix appendices of Arum show the same typical membrane staining as the microbodies in situ do. Thus the identity of these organelles with microbodies seems to be proved. After anthesis the microbodies in situ usually do not give a positive reaction for catalase with diaminobenzidine and H₂O₂. However, cytochemical and biochemical tests for catalase on microbodies isolated during this stage of development clearly demonstrate the presence of this enzyme. Uricase is localized in the microbodies of Arum as well as catalase. No malate dehydrogenase, peroxidase, and allantoinase could be found in the microbodies. Before anthesis the microbodies of spadix appendices of Arum have an equilibrium density in aqueous sucrose of 1.22 gcm^-3. After anthesis the density changes into 1.23 to 1.24 gcm^-3.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.