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.     

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.     

OBSERVATIONS ON THE MICROBODIES IN THE GENUS TILLANDSIA (BROMELIACEAE)

Organelles morphologically similar to microbodies have been found in several tissues of atmospheric species of Tillandsia from different habitats. The presence of catalase was demonstrated by the DAB reaction thus confirming the microbody nature of these organelles. They are a feature of the Tillandsia species with normal photosynthetic carbon fixation and with CAM. Their size is consistently small. The nucleoid observed in the microbodies shows a characteristic morphology which has not been reported before within other plant microbodies. This nucleoid is composed of minute tubular structures, for which the authors here propose a three-dimensional arrangement.     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.     

Physiological role of microbodies in the yeast Trichosporon cutaneum during growth on ethylamine as the source of energy,carbon and nitrogen

Compartmentation of the metabolism of ethylamine in Trichosporon cutaneum X4 was studied in cells, grown on this compound as the sole source of energy, carbon, and nitrogen. Transfer experiments indicated that an amine oxidase is involved in the early metabolism of ethylamine. The synthesis of this enzyme was induced by primary amines and was subject to partial carbon catabolite repression. Repression by ammonium ions was not observed. Adaptation of glucose-grown cells to growth on ethylamine was associated with the development of many microbodies, which developed from already existing organelles present in the inoculum cells and multiplied by division. Cytochemical experiments indicated that the organelles contained amine oxidase and catalase. Therefore, they were considered to play a key role in the metabolism of ethylamine. The physiological significance of the microbodies was investigated by fractionation studies of homogenized protoplasts from ethylamine-grown cells by differential- and sucrose-gradient centrifugation of subcellular organelles. Intact microbodies were only obtained when the isolation procedure was performed at pH 5.8 in the absence of Mg²⁺-ions. Analysis of the different fractions indicated that the key enzymes of the glyoxylate cycle, namely isocitrate lyase and malate synthase, cosedimented together with catalase and amine oxidase. In addition, activities of malate dehydrogenase, glutamate:oxaloacetate aminotransferase (GOT) and (NAD-dependent) glutamate dehydrogenase were detected in these fractions. Electron microscopy revealed that they mainly contained microbodies. Cytochemical experiments indicated that the above enzymes were all present in the same organelle. These findings suggest that microbodies of ethylamine-grown T. cutaneum X4 produce aspartate, so allowing NADH generated in the oxidation of malate by malate dehydrogenase to be quantitatively reoxidized inside the organelles in a series of reactions involving GOT and glutamate dehydrogenase. Aspartase and fumarase were not detected in the microbodies; activities of these two enzymes were present in the cytoplasm.Abbreviations ABTS 2,2-Azino-di(3-ethylbenzthiazoline sulfonate [6]) - DTT dithiothreitol - GOT glutamate:oxaloacetate aminotransferase - DTNB 5,5-dithiobis-2-nitrobenzoate - DAB diaminobenzidine - BSPT 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-t-styryl-sH-tetrazolium chloride - PF convex fracture face - EF concave fracture face     

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.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

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.     

Immobilization of yeast microbodies and the properties of immobilized microbody enzymes

Summary Yeast microbodies isolated from methanol-grown cells of Kloeckera sp. No. 2201 were immobilized by two types of entrapping techniques: photocrosslinking of liquid oligomers of suitable photosensitive resins and crosslinking of albumin molecules with glutaraldehyde. The apparent activities of catalase, alcohol oxidase, and D-amino acid oxidase in the gel-entrapped microbodies were 40–50, 70–80, and ca. 50% respectively as compared with those in the free microbodies. Alcohol oxidase in the immobilized microbodies, similarly to that in free ones, oxidized methanol, ethanol, n-propanol, n-butanol, n-amyl alcohol, and benzyl alcohol. Some properties of catalase and alcohol oxidase in the microbodies immobilized by the above-mentioned techniques were studied in comparison with those of the enzymes in the free microbodies.     

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Solubilization of enzymes from glyoxysomes of maize scutellum

Glyoxysomes isolated from maize scutella (Zea mays L.) were subjected to several disruptive treatments (osmotic shock, resuspension in an alkaline medium, addition of detergent). The damaged glyoxysomes were centrifuged at 89,500g for 40 minutes and several enzymic activities (isocitratase, malate synthetase, catalase, citrate synthetase, malate dehydrogenase) were measured in the supernatant fraction and in the pellet. Isocitratase is the most easily released of all glyoxysomal enzymes closely followed by malate synthetase. Citrate synthetase is in all instances the most insoluble enzyme. All of the enzymes had higher specific activities in the supernatant than in the pellet. These findings suggest that in corn scutellum glyoxysomes none of these enzymes is truly membrane-bound.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Activities and subcellular localization of enzymes responsible for lipolysis and gluconeogenesis during the germination of Brassica campestris cv. esculenta seeds

During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.     

Cytochemischer Nachweis von Katalase in Microbodies bei Acetabularia mediterranea

Summary Direct evidence for the occurrence of microbodies in the marine green algae Acetabularia mediterranea is presented. The microbodies were characterized by means of their marker-enzyme catalase, (EC 1.11.1.6), the presence of which was demonstrated by electron microscopic cytochemical techniques. The function of microbodies in the metabolism of the Acetabularia cell is discussed.     

Isolation of microbodies from, sweet potato root tissue and their development during aging of slices

Microbodies were isolated from, sweet potato root tissue bydifferential and linear sucrose density gradient centrifugation.When the tissue was homogenized in the presence of PolyclarAT, the microbodies sedimented together with the mitochondriathrough the sucrose gradients. The microbodies had a densityof 1.25 g/cm³, and contained catalase and urate oxidase, butnot malate dehydrogenase, isocitrate lyase, glycolate oxidase,hydroxypyruvate reductase and the cyanide-insensitive palmitoylCoA-oxidation system. A small amount of o-diphenol oxidase alsoseemed to be present. Catalase, but not urate oxidase, activity in the crude extractincreased during aging of the sliced tissue. A similar resultwas obtained with the microbody fraction after linear sucrosedensity gradient centrifugation. We propose that microbodiescontaining only catalase develop during aging of sliced sweetpotato root tissue. ¹ This work was supported in part by a Grant-in-Aid (No. 311908)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received June 20, 1979; )     

Histochemical and biochemical evidence for the presence of microbodies in phytophthora palmivora.

Using the diaminobenzidine (DAB) reaction catalase activity could be demonstrated histochemically in cytoplasmic structures of Phytophthora palmivora bearing general ultrastructural features of microbodies. These socalled U-bodies sediment together with the catalase activity in Ficoll-Sorbitol-Sucrose gradients following prior purification by differential centrifugation.     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.