Heterogeneity of mitochondrial particles in fresh and wounded tissues of sweet potato roots

A mitochondrial fraction prepared from fresh tissue of sweetpotato root was subjected to sucrose density gradient centrifugation.The distribution of cytochrome oxidase activity, after the centrifugation,showed the presence of at least three kinds of mitochondrialparticles which differed in their sedimentation velocity. Byrepeating the sucrose density gradient centrifugation, it wasdemonstrated that they are not interconvertible. There seemedto be no difference in the distribution between cytochrome andsuccinate oxidase activities. In the case of malate or succinatedehydrogenase activity, however, the greater the sedimentationvelocity of the particle, the greater was the dehydrogenaseactivity per unit of cytochrome oxidase activity. Some changesin the distribution of cytochrome oxidase activity in responseto aging of the tissue slices were observed. ¹This paper constitutes Part 62 of the Phytopathological Chemistryof Sweet Potato with Black Rot.     

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 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.     

Inducible beta-oxidation pathway in Neurospora crassa.

An inducible beta-oxidation system was demonstrated in a particulate fraction from Neurospora crassa. The activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. The induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. Since an acyl-coenzyme A (CoA) dehydrogenase was detected instead of acyl-CoA oxidase, the former enzyme seems to catalyze the first step of the beta-oxidation sequence in N. crassa. After isopycnic centrifugation in a linear sucrose gradient, the intracellular organelles housing the fatty acid degradation pathway cosedimented (1.21 g/cm3) with the glyoxylate bypass enzymes isocitrate lyase and malate synthase and were clearly resolved from both mitochondrial marker enzymes (1.19 g/cm3) and catalase (1.26 g/cm3). On the basis of biochemical as well as morphological properties, these particles from N. crassa have recently been designated as glyoxysome-like particles (G. Wanner and T. Theimer, Ann. N.Y. Acad. Sci. 386:269-284, 1982). The failure to detect catalase, urate oxidase, and acyl-CoA oxidase indicate that these glyoxysome-like microbodies in N. crassa lack peroxisomal function and thus are clearly different from the various microbodies reported so far to contain a beta-oxidation pathway.     

Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody.

Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.     

Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Localization of Sorbitol Oxidase in Vacuoles and Other Subcellular Organelles in Apple Cotyledons

To investigate the function and subcellular localization ofsorbitol oxidase, free cells, protoplasts and isolated vacuolesof apple cotyledons (Malus pumila Mill. var. domestica Schneid.)were examined by differential and sucrose density gradient centrifugation.Twenty percent of the activity of sorbitol oxidase in the wholetissue was contained in the subcellular fraction (d=1.06) whichcorresponded closely to the main peaks of activity and proteinafter the recentrifugation of the 150,000?g pellet of rupturedvacuoles with a linear sucrose density gradient. The enzymethus appears to be derived from the tonoplast membrane. Thistonoplast membrane-bound sorbitol oxidase may play an importantrole in the transport of vacuolar sorbitol into the cytoplasm,rather than in the transport of sorbitol into the vacuole. About10% of the enzyme activity also occurred in the subcellularfraction having a density of 1.12–1.16, which coincidedwith the peaks of acid phosphatase and ATPase activities. Thereforesorbitol oxidase may also be associated with the plasma membrane.Furthermore, 30–40% of its activity was located in theinterspace between the cell wall and the plasma membrane, orperhaps attached weakly to them. These results suggest thatsorbitol is transported into the cytoplasm by being convertedto glucose by sorbitol oxidase. ¹ This paper is contribution A-138 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

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.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

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.     

The hatching of schistosome eggs

A method is described for the isolation of peroxisomes from mitochondria-enriched fractions obtained from both species of nematodes. The distributions of these organelles are characterized after density gradient centrifugation in sucrose or Percoll by urate oxidase and catalase activities. The possession of peroxisomes may be part of an important defence mechanism in parasites.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.     

Biochemical studies on biogenesis of mitochondria in wounded sweet potato root tissue I. Time course analysis of increase in mitochondrial enzymes

Cytochrome oxidase and succinate dehydrogenase activities increasedduring aging of sliced tissue for sweet potato root after respectivelag phases of about 8 and 10 hr. The increase in the formerwas stepwise. On the other hand, malate dehydrogenase activityincreased slowly without a lag phase. Spectrophotometric determinationof heme contents in mitochondria indicated that the hemes increasedafter a lag phase for at least 8 hr during aging Treatment of tissue slices with cycloheximide at a concentrationof 10^–6 M prior to aging resulted in an extension of thelag phase in the increase of cytochrome oxidaseactivity andin complete inhibition of the increase of malate dehydrogenaseactivity. The antibiotic, at a concentration of 10^–5 M,completely suppressed the increases. Chloramphenicol (6 ? 10^–3M) also blocked the increases, except for that in malate dehydrogenaseactivity at an early stage of aging (Received December 22, 1970; )     

Microbodies from spirogyra: organelles of a filamentous alga similar to leaf peroxisomes

Organelles from Spirogyra cells were separated on a linear sucrose gradient. After centrifugation, most of the protein was found in the top fraction. Two minor protein peaks at density (g/cm(3)) 1.17 and 1.21 were due to chloroplast particles and mitochondria, respectively. Although there was an extremely low concentration of protein at density 1.25 g/cm(3), a major part of the activity of glycolate oxidase was found in this region. The enzyme was able to transfer electrons to O(2) and only lost 12% of its activity in the presence of 1 mm cyanide. As documented by electron micrographs, microbodies moved to density 1.25 g/cm(3) during centrifugation. This observation, as well as the fact that high activities of hydroxypyruvate reductase and catalase were also found at the same density, suggest that the microbodies from Spirogyra are similar to those in green leaves of higher plants.     

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.     

Effect of antibiotics on biogenesis of mitochondria during aging of sliced sweet potato root tissue

The number of mitochondrial particles, and the activities ofperoxidase and cytochrome oxidase increased during aging ofsliced sweet potato root tissue. Blasticidin S inhibited allthese processes, indicating the occurrence of de now synthesisof protein. Chloramphenicol suppressed the synthesis of cytochromeoxidase and the reproduction of mitochondria but did not suppressperoxidase synthesis. Cylcoheximide suppressed peroxidase synthesisleaving the activities of cytochrome oxidase synthesis and mitochondrialreproduction unimpaired. A differentiation of ribosomal andmitochondrial protein syntheses is suggested. Mitomycin G and5-iododeoxyuridine inhibited the reproduction of mitochondriabut not the synthesis of enzymes. ¹This paper constitutes Part 76 of the Phytopathological Chemistryof Sweet Potato with Black Rot. (Received January 4, 1969; )     

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.     

The Intracellular Location of Particulate-Bound Polyphenol Oxidase in Avocado Mesocarp

Polyphenol oxidase of avocado mesocarp exists in a supernatant and a participate fraction. Isolation conditions were sought where maximal polyphenol oxidase activity could be retained in the particulate fraction. More polyphenol oxidase was associated with the 270–2000 g pellet than with the 2000–12,000 g pellet. Analysis of the particulate polyphenol oxidase on linear sucrose density gradient revealed two relatively heavy peaks. The data showed that the major part of polyphenol oxidase was not a constituent of chloroplasts, chromoplasts or mitochondria. The closeness of the positions of polyphenol oxidase and of catalase in both the green and the yellow zones of the mesocarp established that most of the particulate polyphenol oxidase of avocado is associated with microbodies.     

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.     

Ultrastructural localization of urate oxidase in nodules of Sesbania exaltata,Glycine max,and Medicago sativa

Summary The localization of urate oxidase (=uricase, E.C. 1.7.3.3) was determined cytochemically in nodules of Sesbania exaltata (Raf.) Cory, soybean (Glycine max [L.] Merr.) and alfalfa (Medicago sativa [L.]), using the precipitation of peroxide (produced during the oxidation of urate) by cerium chloride. Cerium perhydroxide reaction product was noted only in the microbodies, a localization consistent with biochemical fractionation studies on urate oxidase. Urate oxidase was present not only in the uninfected cells of the cortical tissue, but also in both infected and interstitial cells in the central tissue, suggesting that at least this enzyme of ureide metabolism is not confined to interstitial cells. Urate oxidase cytochemistry of nodules from alfalfa (Medicago sativa L.), an amide producer, also resulted in microbody staining but the microbodies were infrequently noted in cell profiles.