A glyoxysomal role for microbodies in germinating conidia ofBotryodiplodia theobromae

Microbodies increase in number during germination of conidia ofBotryodiplodia theobromae and display an intimate association with lipid bodies. Activities of the glyoxylate bypass enzymes, isocitrate lyase and malate synthase, the tricar☐ylic acid cycle enzymes, malate dehydrogenase and citrate synthase, and the enzymes of β-oxidation, crotonase and thiolase, increased during germination. In germinating conidia, isociatrate lyase, malate synthase, and catalase were localized in microbodies, which had an equilibrium density of 1.223 g cm⁻³ after isopycnic centrifugation on sucrose density gradients. Malate dehydrogenase and citrate synthase, together with succinate dehydrogenase, were confined to the mitochondria, which had an equilibrium density at 1.185 g cm⁻³. Thiolase and β-acyl-CoA dehydrogenase activities were present in both mitochondria and microbodies, but a third enzyme of β-oxidation, crotonase, was found only in the mitochondria. This distribution of enzymes between the mitochondria and microbodies (glyoxysomes) of a germinating fungal spore is different from that found in germinating seeds of vascular plants.     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

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.     

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.     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

Isolation and characterization of organelles from soybean suspension cultures

Whole homogenates from cells of Glycine max grown in suspension culture were centrifuged on linear sucrose gradients. Assays for marker enzymes showed that distinct peaks enriched in particular organelles were separated as follows: endoplasmic reticulum (density 1.10 g/cm³, NADH-cytochrome-c reductase), Golgi membranes (density 1.12 g/cm³, inosine diphosphatase), mitochondria (density 1.18—1.19 g/cm³, fumarase, cytochrome oxidase) and microbodies (density 1.21—1.23 g/cm³, catalase). In cells which had ceased to grow (stationary phase) only a single symmetrical catalase peak at density 1.23 g/cm³ was observed on the sucrose gradient. During the phase of cell division and expansion a minor particulate catalase component of lighter density was present; its possible significance is discussed.     

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.     

Fractionation of the enzymes of the barley aleurone layer: Evidence for a soluble mode of enzyme release

Summary Aleurone cells of barley (Hordeum vulgare L.) contain microbodies as determined by histochemical localization with diaminobenzidine. These microbodies can be isolated from both water and gibberellic acid (GA₃) treated cells and identified on sucrose density gradients as glyoxysomes on the basis of their buoyant densities (1.25 g cm^-3) and their enzyme complement. Fractionation of aleurone layer homogenates by differential centrifugation after varying times of exposure to GA₃, however, does not indicate the presence of a discrete secretory vesicle containing either -amylase or -1,3-glucanase. Cytological evidence also suggests that at least -1,3-glucanase is not released from these cells by means of a discrete secretory vesicle.Supported by National Science Foundation Grant GB-27468. The assistance of Dr. William Breidenbach in the assay of glyoxysomal enzymes is gratefully acknowledged.     

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Microbodies isolated from sporangia of the aquatic fungus $\text{Blastocladiella emersonii}$ have a mean buoyant density of 1.222 g/cm³ after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm³ while the spore's single mitochondrion has a buoyant density of 1.219 g/cm³. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.     

Glycolate metabolism and subcellular distribution of glycolate oxidase in Spatoglossum pacificum (Phaeophyceae, Chromophyta)

Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same fraction at a density of 1.23 g cm^?3 with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm^?3 with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants.     

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes

Three DNA buoyant density species (nuclear, 1.692 g cm⁻³; mitochondria 1.705 g cm⁻³; and proplastid, 1.713 g cm⁻³) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm⁻³ species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

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.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

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

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

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.     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.