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.     

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.     

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.     

Cytochemical localization of glycolate oxidase in microbodies of Klebsormidium

The filamentous green alga Klebsormidium flaccidum A.Br. was fixed with glutaraldehyde, incubated in a cytochemical medium designed to detect glycolate-oxidase activity, and prepared for electron microscopy. Heavy deposits of stain were observed in microbodies following incubation with either glycolate or L-lactate as substrate, but not after incubation with D-lactate or H₂O. When Chlamydomanas reinhardi Dangeared cells were treated in the same way, their microbodies did not appear stained. The results establish that in Klebsormidium glycolate-oxidase occurs in microbodies (peroxisomes), as it does in angiosperms; also, they emphasize the dichotomy between those green algae which contain glycolate-oxidase and those, such as Chlamydomonas, which possess the mitochondrial enzyme glycolate dehydrogenase.     

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.     

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.     

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.     

Regulation and compartmentalization of β‐lactam biosynthesis

Penicillins and cephalosporins are β-lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l -α-aminoadipic acid, l -cysteine and l -valine to form the tripeptide δ-l -α-aminoadipyl-l -cysteinyl-d -valine catalysed by the non-ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two-component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.     

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     

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.     

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.     

Spheroplast formation and partial purification of microbodies from hydrocarbon-grown cells ofCladosporium resinae

Summary Cells ofCladosporium resinae form greater numbers of microbodies when grown onn-alkanes than when grown on glucose. To facilitate isolation of microbodies, hydrocarbon-grown cells were spheroplasted. Of four spheroplasting agents and five osmotic supports examined, best results were obtained after a 4-h incubation with Novozym 234 plus chitinase and with 0.8 M sorbitol as osmotic support. Equal numbers of spheroplasts were obtained at pH 5.8 and at pH 7.0. Catalase was used as a marker for microbodies and cytochrome-c oxidase as a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. Microbodies were extremely fragile; of eight spheroplast disruption techniques attempted, the best yield of microbodies was obtained using a Teflon homogenizer for 5 min. Microbodies were partially purified by differential and density gradient centrifugation. Best results were obtained with discontinuous Percoll gradients which yielded a fraction enriched in microbodies and one enriched in mitochondria.     

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.     

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.     

Tubuläre Einschlüsse in Microbodies vonSaccharomycopsis (Candida) lipolytica-Protoplasten

Zusammenfassung Auf Ultradünnschnitten Glutaraldehyd-OsO₄ oder KMnO₄ fixierterSaccharomycopsis lipolytica-Zellen, die in einem Medium mit Laktat als C-Quelle wuchsen, werden durchschnittlich 3–4 Anschnitte von Microbodies gefunden. Ihre Anzahl erhöht sich etwa um das Dreifache, wenn n-Hexadecan die C-Quelle ist.Nach der Umwandlung der Zellen zu Protoplasten mit Hilfe von Enzymgemischen vonHelix pomatia enthalten die Microbodies tubuläre Einschlüsse. Diese Tubuli, deren äußerer Durchmesser etwa 25 nm beträgt, haben eine zentrale Verdichtung und erreichen Längen bis zu 0,8 m. Sie sind in Bündeln oder Schichten angeordnet. Die Tubuli bilden sich möglicherweise durch Assemblierung von Enzymproteinen, wenn die Zellen während der Umwandlung zu Protoplasten mit hypertonischen Lösungen osmotisch geschockt werden.

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Tubular inclusions within microbodies ofSaccharomycopsis (Candida) lipolytica-Protoplasts

Summary Electron micrographs ofSaccharomycopsis lipolytica-cells grown in a medium with lactate as carbon source and fixed with glutaraldehyde-OsO₄ or KmnO₄ show 3–4 profiles of microbodies per section in average. The number of microbodies is about three times greater when cells are cultivated in a medium with n-hexadecane. The spherical to avoid microbodies (0.3–0,8 m in diameter) have a homogeneous matrix.After conversion of cells into protoplasts by the aid of snail gut juice tubular inclusions occur in microbodies. The single tubulus has an outer diameter of about 25 nm, a length up to 0.8 m and contains a central rod. Several tubules form bundles or layers. It is suggested that the tubules could be enzyme protein assembling by osmotic shock with hypertonic solutions during preparation of protoplasts.

     

Separation of mitochondria from microbodies of Pisum sativum (L. cv. Alaska) cotyledons

A method is described for separating mitochondria from microbodies in cotyledon preparations of Pisum sativum L. cv. Alaska. Pure and intact mitochondria were obtained on a continuous: discontinuous sucrose density gradient as shown by marke-enzyme assay and electron microscopy. Manipulation of sucrose-gradient construction to widen the distance between organelles provided a quick method for the separation of the mitochondria from the microbodies. The shorter time of exposure of mitochondria to centrifugation and osmotic stress produces mitochondria free of contamination.     

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.     

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbody defective mutants of arabidopsis

In germinating fatty seedlings, microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”