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The occurrence in yeast Saccharomyces cerevisiae of cytoplasmic granules which resemble microbodies

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... Unlike the situation for other yeasts and filamentous fungi, peroxisomes of S. cerevisiae have not yet been characterized in great detail. Avers and Federman first demonstrated the presence of peroxisomes in yeast cells (2). Under the conditions chosen for growth and isolation, however, peroxisomes appeared to be rare, small, and very fragile. ...
... Catalase is the prototypical marker enzyme for peroxisomes, identifying the organelle in animals, plants, and a variety of unicellular organisms (5,25). The existence of catalase-containing peroxisomes in S. cerevisiae was first reported by Avers and Federman (2). Subsequently, it was found that this yeast contains two catalase isozymes: an atypical organelle-associated catalase A and a typical soluble catalase T (33,34). ...
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Toisolate peroxisomes fromSaccharomyces cerevisiae ofaquality sufficient forinvitro import studies, we optimized theconditions forcell growth andforcell fractionation. Stability oftheisolated peroxisomes was monitored bycatalase latency andsedimentability ofmarkerenzymes. Itwasimproved by(i) using cells that wereshifted tooleic acidmediumafter growth tostationary phaseinglucose precultures, (ii) shifting thepH from7.2to6.0during cell fractionation, and(iii) carrying outequilibrium density centrifugation with Nycodenz containing 0.25M sucrose throughout thegradient. A concentrated peroxisomal fraction wasused forinvitro import ofcatalase A.After 2hofincubation, 62%ofthecatalase wasassociated with, and16% wasimported into, theorganelle ina protease-resistant fashion. We introduced immunofluorescence microscopy forS.cerevisiae peroxisomes, using antibodies against thiolase, whichallowed ustoidentify even theextremely smallorganelles inglucose-grown cells. Peroxisomes frommediacontaining oleic acidwere larger insize, weregreater innumber, andhadamoreintense fluorescence signal. Theperoxisomes were located, sometimes inclusters, inthecell periphery, often immediately adjacent totheplasmamembrane. Systematic immunofluorescence observations ofglucose-grown S.cerevisiae demonstrated thatallsuchcells contained atleast oneandusually several verysmall peroxisomes despite theglucose repression. Thisfind- ingfits a central prediction ofourmodelofperoxisome biogenesis: peroxisomes formbydivision of preexisting peroxisomes; therefore, every cell musthaveatleast oneperoxisome ifadditional organelles areto beinduced inthatcell. Thebiogenesis ofmicrobodies (peroxisomes, glyoxy- somes, andglycosomes) ischaracterized bycertain features thatdistinguish themfromother cell organelles. Ineucary- otes, peroxisomes arenearly ubiquitous organelles that are surrounded byasingle bilayer membrane. Matrix andmem- brane proteins ofperoxisomes aresynthesized onfree poly-
... Unlike the situation for other yeasts and filamentous fungi, peroxisomes of S. cerevisiae have not yet been characterized in great detail. Avers and Federman first demonstrated the presence of peroxisomes in yeast cells (2). Under the conditions chosen for growth and isolation, however, peroxisomes appeared to be rare, small, and very fragile. ...
... Catalase is the prototypical marker enzyme for peroxisomes, identifying the organelle in animals, plants, and a variety of unicellular organisms (5,25). The existence of catalase-containing peroxisomes in S. cerevisiae was first reported by Avers and Federman (2). Subsequently, it was found that this yeast contains two catalase isozymes: an atypical organelle-associated catalase A and a typical soluble catalase T (33,34). ...
Article
Full-text available
To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell.
... In yeast, the morphology of microbodies was described for the first time in S. cerevisiae by Avers and Federman (1968). However, it took almost 20 years before collaborative efforts of the groups of Veenhuis and Kunau demonstrated that peroxisomes play a crucial role in oleate metabolism in S. cerevisiae and that, consequently, these organelles are massively induced during growth of yeast on oleate as the sole carbon and energy source (Veenhuis et al. 1987). ...
Article
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Lipid droplets (LDs) and peroxisomes are central players in cellular lipid homeostasis: some of their main functions are to control the metabolic flux and availability of fatty acids (LDs and peroxisomes) as well as of sterols (LDs). Both fatty acids and sterols serve multiple functions in the cell-as membrane stabilizers affecting membrane fluidity, as crucial structural elements of membrane-forming phospholipids and sphingolipids, as protein modifiers and signaling molecules, and last but not least, as a rich carbon and energy source. In addition, peroxisomes harbor enzymes of the malic acid shunt, which is indispensable to regenerate oxaloacetate for gluconeogenesis, thus allowing yeast cells to generate sugars from fatty acids or nonfermentable carbon sources. Therefore, failure of LD and peroxisome biogenesis and function are likely to lead to deregulated lipid fluxes and disrupted energy homeostasis with detrimental consequences for the cell. These pathological consequences of LD and peroxisome failure have indeed sparked great biomedical interest in understanding the biogenesis of these organelles, their functional roles in lipid homeostasis, interaction with cellular metabolism and other organelles, as well as their regulation, turnover, and inheritance. These questions are particularly burning in view of the pandemic development of lipid-associated disorders worldwide.
... Occurrence of microbodies in yeast was first demonstrated by Avers and her co-workers (1,2,18) in various strains of Saccharomyces cerevisiae. They detected several enzymes including catalase in the microbody fraction from the yeast. ...
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A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.
... The techniques employed have generally involved differential and/or density gradient centrifugation. It is mainly the separation and characterization of organelles such as mitochondria (Schatz, 1963;Schatz & Klima, 1964;Perlman & Mahler, 1g70), vacuoles (Matile & Wiemken, 1967) and peroxisomes (Avers & Federman, 1968) which have been reported. ...
Article
Plasma membranes were isolated from the yeast and mycelial forms of Candida albicans as described previously (Marriott, 1975) and examined for the presence of several enzymes. Measurement of specific activities showed enrichment of Mg2+-dependent and Ma+/K+-stimulated Mg2+-dependent adenosine triphosphatase and mannan synthetase, in the plasma membrane fractions from both morphological forms of the organism. However, acid and alkaline phosphatase, NADH oxidase and 5'-nucleotidase showed no such specific location.
... However, until recently, evidence for the presence of peroxisomes in S. cerevisiae has been scant and controversial. Early reports of the presence of catalase, isocitrate lyase, and malate synthase in peroxisomes (3,4,47) were disputed. The glyoxylate cycle enzymes were reported to be soluble (18,48), while catalase A was reported to be vacuolar (58). ...
Article
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Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.
... In yeasts, microbodies were first described by Avers [6]. In the intervening years much progress has been made towards our understanding of the mechanisms involved in biogenesis and metabolic function of these organelles [4,[7][8][9][10]. ...
Article
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Abstract In wild-type Hansenula polymorpha the proliferation of peroxisomes is induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per−; 13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim−; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss−; two complementation groups). In addition, several conditional Per−, Pim− and Pss− mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.
... However, until recently, evidence for the presence of peroxisomes in S. cerevisiae has been scant and controversial. Early reports of the presence of catalase, isocitrate lyase, and malate synthase in peroxisomes (3,4,47) were disputed. The glyoxylate cycle enzymes were reported to be soluble (18,48), while catalase A was reported to be vacuolar (58). ...
Article
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.
Article
This chapter outlines the breakdown of sugars by yeasts. To continue biosynthetic processes necessary for growth, yeasts obtain energy from sugars by breaking them down. The energy set free is stored as the “high energy” phosphate derivative adenosine 5’-triphosphate (ATP) that is synthesized as the sugar is catabolized. In catabolism, glycosidic bonds are hydrolyzed to yield component monosaccharides. A low concentration of oxygen is often important for obtaining a high yield of ethanol from certain sugars. Particularly in initial and terminal reactions, differences are found among different yeasts, and such relatively minor biochemical differences are often of considerable practical importance. The information provided in this chapter is based chiefly on studies yeast such as, Saccharomyces cerevisiae, Saccharomyces uvarum, Candida utilis, and Kluyveromyces fragilis. However, the ability of yeasts to utilize sugars is not only of potential value, it can also be a nuisance. Yeasts are notorious as spoilers of foods that contain a high concentration of one or more sugars, such as honey, maple syrup, sugar cane, and confectionery.
Article
The subcellular distribution of catalase A in the yeast Saccharomyces cerevisiae has been investigated. The enzyme was found to be bound to large particles, whereas most of the activity of catalase T was located in a 38 000 X g supernatant. Under various isolation conditions catalase A always showed a distribution among subcellular fractions virtually identical to that of two markers for vacuoles, proteinase B and alpha-mannosidase. More than 80 percent of the catalase A activity of a crude vacuole fraci-onercent of the catalase A activity of a crude vacuole fraction has been detected in purified vacuoles. Malate synthase, isocitrate lyase and glyoxylate reductase (NADP), three peroxisomal markers, showed a subcellular distribution significantly different from that of catalase A. It is concluded from these results that catalase A is specifically associated with the vacuoles of yeast. Like vacuoles, "peroxisomal" fractions isolated from yeast spheroplasts as described by Avers[1] contain only one catalase protein, catalase A. It could be shown by isopycnic and sedimentation velocity separations of crude mitochondrial fractions that catalase A in "peroxisomal" fractions is accompanied by considerable activities of proteinase B and alpha-mannosidase. From all our results it seems that the catalase-active particles isolated under such conditions are not typical peroxisomes but vesicles formed from vacuoles during the isolation procedure.
Article
Cells of 3 yeast species capable of assimilating methanol have been examined by electron microscopy. When grown on methanol as the sole source of carbon and energy they contained many microbodies. Cells grown on glucose or ethanol either did not contain such bodies at all, or only to a limited extent.
Article
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Nine strains of methanol-utilizing yeasts belonging to the genera Candida, Hansenula, Kloeckera, Pichia, and Torulopsis were examined with respect to the interrelationship between their catalase content and ultrastructure. Methanol-grown cells of all the yeasts tested showed higher catalase activities than the respective ethanol- and glucose-grown cells. In connection with this, occurrence of a specific organelle surrounded by a single-unit membrane ("microbodies") was observed only in the methanol-grown cells. Several morphological differences were observed between the microbodies of methanol-utilizing yeasts and those of hydrocarbon-utilizing yeasts such as Candida tropicalis. That is, microbodies of methanol utilizers were large in size, existed in closely associated forms, and had crystalloid structures. Localization of catalase activity in these microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidene. Especially, 3,3'-diaminobenzidine reaction product accumulated heavily in crystalloids of yeast microbodies.
Article
To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell.
Article
Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3 %. As shown by electron microscopy the peroxisomes were 0.4-0.6 μm in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electròphoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.
Article
The Saccharomyces cerevisiae POT1 gene is, as are other yeast peroxisomal protein genes, inducible by fatty acids and repressible by glucose. We have now found that it is also induced during the stationary phase of the culture. To investigate these three regulatory circuits, we have studied the mRNA levels of regulatory mutants as well as the changes in chromatin structure upon gene activation. We conclude that the regulation of transcriptional activity in glucose repression, oleate induction, and stationary phase induction follow different molecular mechanisms. We suggest that this multiplicity of regulatory mechanisms may represent a general rule for the yeast peroxisomal protein genes.
Article
Peroxisomes (microbodies), one of the subcellular organelles, have been shown to have indispensable functions in the metabolism of n-alkanes, fatty acids, methanol, and several nitrogen-containing compounds in eukaryotic microorganisms after we described the appearance of large numbers of the organelles in an n-alkane-assimilating yeast, Canadida tropicalis. It has been suggested that peroxisomes proliferate by division from already existing peroxisomes and their characteristic proteins, which are encoded by nuclear genes, have been shown to be specifically localized after synthesis outside peroxisomes. These physiological phenomena can be controlled simply by changing the carbon and/or nitrogen source for growth, making microorganisms an excellent target to investigate the biogenesis of peroxisomes in eukaryotic cells.This article deals with the functions of peroxisomes in alkane metabolism by yeasts, together with discussion about the mechanisms of their development and degradation as well as the properties and application of peroxisomal enzymes.
Article
The observation that peroxisomes of Saccharomyces cerevisiae can be induced by oleic acid has opened the possibility to investigate the biogenesis of these organelles in a biochemically and genetically well characterized organism. Only few enzymes have been identified as peroxisomal proteins in Saccharomyces cerevisiae so far; the three enzymes involved in β-oxidation of fatty acids, enzymes of the glyoxylate cycle, catalase A and the PAS3 gene product have been unequivocally assigned to the peroxisomal compartment. However, more proteins are expected to be constituents of the peroxisomes in Saccharomyces cerevisiae. Mutagenesis of Saccharomyces cerevisiae cells gave rise to mutants unable to use oleic acid as sole carbon source. These mutants could be divided in two groups: those with defects in structural genes of β-oxidation enzymes (fox-mutants) and those with defects in peroxisomal assembly (pas-mutants). All fox-mutants possess morphologically normal peroxisomes and can be assigned to one of three complementation groups (FOX1, 2, 3). All three FOX genes have been cloned and characterized. The pas-mutants isolated are distributed among 13 complementation groups and represent 3 different classes: peroxisomes are either morphologically not detectable (type I) or present but non-proliferating (type II). Mislocalization concerns all peroxisomal proteins in cells of these two classes. The third class of mutants contains peroxisomes normal in size and number, however, distinct peroxisomal matrix proteins are mislocalized (type III). Five additional complementation groups were found in the laboratory of H.F. Tabak. Not all PAS genes have been cloned and characterized so far, and only for few of them the function could be deduced from sequence comparisons. Proliferation of microbodies is repressed by glucose, derepressed by non-fermentable carbon sources and fully induced by oleic acid. The regulation of four genes encoding peroxisomal proteins (PAS1, CTA1, FOX2, FOX3) occurs on the transcriptional level and reflects the morphological observations: repression by glucose and induction by oleic acid. Moreover, trans-acting factors like ADR1, SNF1 and SNF4, all involved in derepression of various cellular processes, have been demonstrated to affect transcriptional regulation of genes encoding peroxisomal proteins. The peroxisomal import machinery seems to be conserved between different organisms as indicated by import of heterologous proteins into microbodies of different host cells. In addition, many peroxisomal proteins contain C-terminal targeting signals. However, more than one import route into peroxisomes does exist. Dissection of the import mechanism in a genetically well suited organism like Saccharomyces cerevisiae together with further characterization and functional assignment of the PAS gene products will provide insight into the biogenesis of peroxisomes. Moreover, these studies will lead to a good model system for elucidation of the mechanisms underlying human peroxisomal disorders.
Article
The descriptive ultrastructure of a large variety of fungal species either grown under in vitro culture conditions or in situ during infection of the host has been reported in detail. Morphologic characterization of yeasts, dimorphic fungi, and filamentous fungi has been done using scanning electron microscopy to elaborate the surface structure, transmission electron microscopy to reveal the internal subcellular organelles, and freeze-fracture electron microscopy to display some intramembranous molecular structures (2, 3, 5, 6, 12, 17, 28, 31, 45, 50, 61, 64, 66, 70, 75, 83, 85, 112, 113, 124). For a long time the detailed description of fungal substructure has been hampered by the lack of adequate methodology. From the early days of explorative research, especially the field of transmission electron microscopy has been encumbered with difficulties of clear-cut visualization, hence obscuring the interpretation of the observations (17).
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Saccharomyces cerevisiae contains two genes, CIT1 and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942, 1986). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CIT1 product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikingly at their amino termini. The amino terminus of the CIT1 primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases. This extension consists of a typical mitochondrial targeting motif. The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E. coli and porcine enzymes. The CIT2-encoded extension is not homologous to that of CIT1, resulting in a nonmitochondrial localization of the product. The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences. The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.
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In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected.
Article
1. Sphaerosomes (oil droplets) extracted and isolated from tobacco endosperm contain over 90% of the total endosperm lipids. About 99% of the dry matter of these organelles consist of reserve lipids. 2. Upon germination 90% of the lipid reserves are mobilized within 96 hours. The hydrolysis of the neutral fat is achieved by lipolytic activity present in sphaerosomes. This activity could be demonstrated in sphaerosomes of resting seeds. 3. A large fraction of the total activities of proteases, acetyl esterase, phosphatase, RNase and DNase present in the endosperm extract is localized in sphaerosomes. These enzyme activities are present in resting seeds. Protease, acetyl esterase and phosphatase activities increase temporarily in the course of germination. 4. The sphaerosomes of tobacco endosperm are interpreted as lysosomes in which not only the accumulation and mobilization of the reserve lipids take place, but also the breakdown of autophagocytized cytoplasmic material. The morphological and functional relations between sphaerosomes and other types of vacuoles are discussed. 5. Enzymes of the glyoxylic acid cycle present in the endosperm extract from germinating seeds can be sedimented together with mitochondrial enzymes. Upon centrifugation of the mitochondrial fraction in density gradients of sucrose mitochondria can be separated from glyoxisomes. 6. In addition to isocitrate lyase and malate synthetase catalase is associated with glyoxisomes. The former enzymes are present almost exclusively in the fraction of glyoxisomes whereas about 90% of the latter occur in the soluble fraction. 7. The function of glyoxisomes in the conversion of fatty acids into sugar is discussed.
Article
A procedure was described for the isolation of mutants affected in the regulation of catalase activity. Two such mutants, cgr 1 and cgr 2 were obtained. Both of them show catalase activity that is resistant to repression by glucose, but is sensitive to anoxia to the same extent as the wild type.
Article
Mit Hilfe der cytochemischen Methode vonFahimi (1968) wurde in den Zellen der PilzeNeurospora crassa, Rhizopus nigricans undSaccbaromyces cerevisiae das Enzym Katalase in distinkten Granula nachgewiesen. Die Spezifitt der Frbereaktion wurde durch ihre Hemmbarkeit mit 3-Amino-1,2,4-triazol gezeigt.In the cells of the fungiNeurospora crassa, Rhizopus nigricans, andSaccharomyces cerevisiae catalase was detected in distinct granula with the cytochemical procedure ofFahimi (1968). The specificity of the staining reaction was demonstrated by the inhibitory action of 3-amino-1,2,4-triazole.
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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 μm 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 μm 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.
Chapter
In addition to the cisternae and tubules that comprise the endomembrane system just given attention, several major types of vesicles are involved in the secretory processes of cells. One of these, the lysosome, has already received mention as being part of GERL (Chapter 6) and still earlier in connection with endocytosis and receptor sites in the discussion of membranes (Chapter 1, Sections 1.2.1 and 1.2.2). Here even more clearly than in the membranous organelles, secretion is revealed as the underlying mechanism for most cellular functions, for these little bodies carry the secreted products to where the action is finally to take place. But much more is involved in the functioning of these vesicular particles than mere conduction of finished products, as becomes evident as the lysosome, peroxisome, spherosome, and others of lesser importance are discussed.
Chapter
Feinstrukturelle Besonderheiten der Pilzzelle sind, von wenigen Ausnahmen abgesehen, bisher nicht bekannt geworden. Wenn trotzdem eine gesonderte Darstellung erfolgt, so liegt hierfür eine Rechtfertigung vielleicht darin, daß die Pilzzelle eine gewisse Mittelstellung zwischen tierischer (Glykogenspeicherung, Chitinsynthese, Harnstoffbildung, Kohlenstoff-Heterotrophie, Vitaminbedürftigkeit, Centriolenbesitz) und pflanzlicher (Zellpolarität, Spitzenwachstum, Zellwandbau, Querwandbildung) Zelle einnimmt und berufen scheint, in zunehmendem Maße zur Lösung einer Reihe von Fragen der modernen Zellforschung (Regulationsphänomene, Genese und Dynamik intraplasmatischer Membranen, genetische Kontrolle physiologischer Prozesse) beizutragen.
Chapter
In yeasts proliferation of microbodies (glyoxysomes/peroxisomes) is largely prescribed by the growth environment and due to the fact that the organelles harbour essential enzymic functions required for the metabolism of the carbon and/or nitrogen source. Presently activities of more than 20 different enzymes have been shown to reside in yeast microbodies namely catalase, several H202-producing oxidases and enzymes involved in 8-oxidation, glyoxylate cycle enzymes, dehydrogenases, an amino transferase and a transketolase. The functioning of these organelles often requires extensive metabolic interactions with other cell compartments such as mitochondria, microsomes or the cyrosol. All available evidence indicates that yeast microbodies do not arise from the ER or de novo but develop from already existing organelles. Synthesis of microbody-matrix enzymes appears to be mainly controlled at the transcriptional level and takes place on cytosolic polysomes. Microbody-protein subunits (both matrix and membrane proteins) are synthesized in their mature form and are imported post-translationally. Topogenic signals for directing yeast microbody proteins to their target organelle are contained within their structure and are probably universal. Yeast microbodies have been shown to be acidic in nature with an internal pH of 5.8 – 6.0 whereas the cytosolic pH is approximately 7.1. This electrochemical proton gradient — generated by a membrane bound H+-ATPase — may play an important role in different transport processes across the peroxisomal membrane including uptake of matrix proteins and transport of low molecular weight compounds such as substrates and/or metabolic intermediates.
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Lysosomen und Peroxysomen bilden zwei Klassen von Organellen, welche zunächst aus Zellen der Rattenleber, später aus anderen tierischen Geweben und Organismen isoliert wurden. In den Lysosomen sind saure hydrolytische Enzyme lokalisiert (Leitenzym: saure, unspezifische Phosphatase). Die Peroxysomen enthalten Enzyme des Peroxydstoffwechsels (Leitenzyme: Urikase, Katalase).
Article
Peroxisomes are nearly ubiquitous organelles of eukaryotic cells. The original biochemical characterization led to the discovery of their H2O2 metabolism (H2O2-producing oxidases and catalase) and this in turn was used as a definition of these subcellular structures. Peroxisoines are the major subgroup of the morphologically defined microbodies. Current knowledge has revealed that the H2O2 metabolism is just one aspect of the metabolic functions associated with peroxisomes. Due to the diversity of enzymatic reactions discovered in peroxisomes, they are now viewed as multi-purpose organelles. However, despite their remarkable biochemical heterogeneity, all peroxisomes share the capability to degrade fatty acids via the P-oxidation pathway. Another notable property is the inducibility of peroxisomal proliferation and peroxisomal metabolic pathways. Both diversity of metabolic functions and inducibility of peroxisomal proliferation and metabolism are especially pronounced in eukaryotic microorganisms such as yeasts and filamentous fungi. In these microorganisms, the final size, volume fraction, and enzyme pattern of peroxisomes can be drastically changed by varying growth conditions. Proliferation of fungal peroxisomes is under the control of glucose repression and de-repression as well as induction by distinct carbon and nitrogen sources. Their properties make peroxisomes rather unique among organelles, which in general have retained one major and mostly constitutive function throughout evolution. Recently, the yeast Saccharomyces cerevisiae has been more and more frequently used as a eukaryotic model to study basic functions and structures of eukaryotic cells. Its genetic and biochemical attributes make it an attractive organism for analyzing complex processes.
Article
Peroxisomes are characterized, among plastids and mitochondria, in the photosynthetic lamellae of the leaf of Polytrichum commune gametophytes. The significance of such a functional association in the haploid phase of a Bryophyte is briefly discussed from an evolutionary standpoint.
Article
Numerous microbodies developed at the logarithmic phase of growth in cells of Candida tropicalis, when cultures were grown in a medium containing normal alkanes. From the homogenate of protoplasts prepared from 16 hr cells of Candida, biologically active microbodies were isolated by discontinuous sucrose density gradient centrifugation. The fractions recovered in the middle and lower density regions of the gradient were associated with substantially high activities of three peroxisomal marker enzymes, catalase, D amino acid oxidase, and urate oxidase, while a mitochondrial marker enzyme, cytochrome oxidase, was only slightly detected. Electron microscopic studies revealed that these fractions were largely comprised of morphologically well preserved microbodies with a smaller number of more or less degraded organelles, but were virtually devoid of mitochondria. The microbody fractions contained an appreciable amount of DNA.
Article
An ultrastructural examination of conidiogenesis in Drechslera sorokiniana reveals that conidia develop enteroblastically through channels in the conidiogenous cell wall. These channels probably form by autolysis of the outer wall layers. The data support earlier concepts based on light-microscopic studies of conidium ontogeny in this and other developmentally related species of hyphomycetes. The surface morphology and relationship of wall layers of the conidium and conidiogenous cell at various stages of development are illustrated by scanning and transmission electron microscopy, respectively. This information is summarized in a diagrammatic interpretation of conidiogenesis. Cytodifferentiation during conidium formation and conidiogenous cell proliferation is also examined. A possible association between organelle migration in developing conidiogenous cells and fascicles of microfibrils, proposed in an earlier paper, is discussed. A suggestive explanation is presented for the accumulation of microbodies in conidium initials and apices of proliferating conidiogenous cells. Layers of endoplasmic reticulum which are terminally hypertrophied and juxtaposed to the plasma membrane of developing conidiogenous cells are also noted.
Article
One of the most striking features of alkane-grown yeast cells is conspicuous appearance of peroxisomes in harmony with a high level of catalase. This unique phenomenon was first demonstrated in the authors′ laboratory, and the metabolic functions of peroxisomes in yeasts utilizing alkanes has been estabilished with intact peroxisomes isolated by density gradient centrifugation. The organelles participate in the degradation of fatty acids derived from alkanes to C2-units and the synthesis of gluconeogenic intermediates from C2-units. The abundant appearance of peroxisomes in alkane-utilizing cells has allowed successful production of several useful enzymes including catalase, D-amino acid oxidase, uricase, acyl-CoA oxidase etc. Yeast cells will be an excellent system for investigation the functions and development of peroxisomes because biogenesis of the organelles is induced only by transferring the cells into alkane medium from glucose or ethanol medium.
Article
Four newly synthesized molecules derived from pyrazole-pyrimidine were assayed on Botrytis cinerea Micheli, Fusarium moniliforme Sheld and Pythium ultimum Trow. All proved effective in inhibiting the growth of the phytopathogens at all of the test concentrations (10, 20, 50, 100 μg/ml). The most effective compound was 1-(3)nitrophenyl - 6 - trifluoromethylpyrazolo[3,4 - d]pyrimidine 4(5H)-thione (CF33). Ultrastructural studies on P. ultimum treated with CF33 revealed alterations in the normal hyphal shape and, at high concentration, plasmolysis and damage to the wall texture was observed. At 20 μg/ml different vesicles were seen in the cytoplasm: some appeared quite dense, and specific cytochemical reactions indicated that they were most likely peroxysomes; other vesicles seem to be vacuoles of varying content. In some cases there was disintegration of the nuclear envelope. The effects on membrane lipids and interference in protein synthesis are hypothesized as possible mechanism of action of the molecule.
Article
Recently the cytology and biochemistry of peroxisomes has attracted increasing interest. These organelles were discovered in 1954 in mouse kidney cells and called ‘microbodies’ by electron-microscopists, their functions in mammalian and plant cells have been investigated extensively [1].
Article
A method of hemoproteid-deficient mutants isolation is described. 13 mutants lacking catalase belonging to 9 complementation groups were isolated. Mutants representing 7 loci were also unable to form cytochromes.Addition of -aminolevulinic acid does not restore catalase activity. Normal mendelian segregation in crosses with wild strain was observed only in three mutants. The rest give 4:0 segregation. After ethidium bromide treatment and induction of rho — strains normal segregation was observed in all mutants. The masking effect of normal mitochondrial activity on gene expression in mutant strains is discussed.
Article
Microbodies are distinctive organelles which occur in a wide variety of plant cells. In this review emphasis is placed on the ultrastructure, cytochemistry, function, and development of a) microbodies (peroxisomes) in leaves of angiosperms and b) microbodies (glyoxysomes) in lipid-storing organs of seeds following germination. Information regarding the occurrence, fine structure, and possible role of microbodies in achlorophyllous tissues of higher plants is also considered, as is the distribution of these organelles in various taxa of the plant kingdom.
Article
Catalase activities of the cells growing onn-alkanes of various strains ofCandida yeasts wer markedly higher than those of the cells growing on glucose, ethanol or acetate. In connection with this, electron-microscopical studies revealed abundant appearance of specific microbodies having homogeneous matrix surrounded by single unit membrane in the hydrocarbon-growing cells. Localization of catalase activity in the microbodies, in addition to the mitochondria, was confirmed by cytochemical treatment of the cells with 3,3-diaminobenzidine reagent.
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Article
A number of biochemical properties of mitochondria from a cytoplasmic petite mutant ofSaccharomyces cerevisiae with an extremely high adenine plus thymine content have been studied. When such particles are isolated by means of standard procedures developed for use with wild-type yeasts they are grossly contaminated by non-mitochondrial membrane fragments. Further enrichment of mitochondria is achieved by non-equilibrium centrifugation in sucrose gradients. Throughout this purification procedure the particles can be shown to retain an outer limiting, as well as a non-cristate inner membrane. In many of their morphological and physical features (size, shape, buoyant density) they resemble mitochondria isolated from the wild type. Although enzymes of the respiratory chain are absent from the mutant particles, their content ofl-malate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and ATPase is comparable to that found in the wild type. The mitochondrial ATPase in this mutant strain is cold stable, oligomycin insensitive, Dio-9 sensitive, and susceptible to inhibition by the F1 inhibitor of beef heart. The enzyme can be rendered cold labile by its detachment from the membrane, followed by fractionation with protamine sulfate and ammonium sulfate. The existence of mutant particles that are incapable of function in oxidation and phosphorylation but resemble their functional homologues in many other ways raises the possibility that mitochondria are required in the cellular economy for purposes not directly linked to oxidative phosphorylation and electron transport. This hypothesis has led us to suggest that, contrary to models currently under discussion, mitochondria did not evolve as a consequence of endosymbiosis. We propose as an alternative that the mitochondrial organelle evolved as a means of improvement of existing subcellular structures in the primordial (perhaps eukaryotic) cell. Partial autonomy may thus constitute a relatively recent modification; the present-day mitochondrial genome had its origin in nuclear DNA and may have been amplified in a manner not unlike the amplification of ribosomal RNA cistrons in developing oocytes ofXenopus.
Article
Differential and density-gradient centrifugation studies have estaiblished an associatiotn between allaintoinase and peroxisomes from the liver of the frog Rana pipiens. The presence of allantoincase in the peroxisome indicates. a iricolytic function for this orgainelle in the liver of amphibians.
Article
Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.
Article
Crosses were made between haploid wild-type and suppressive petite strains of bakers' yeast to obtain zygotes for analysis of mitochondrial heterogeneity. Wild-type x petite zygotes contained about 40% noncristate mitochondria when immediate mating mixtures were examined. The frequency of defective mitochondria had decreased to an average of 9.2% in 1-week-old zygote isolate cultures, and to 4.4% in slant cultures 1.5 years after initial zygote isolation. The latter value was not significantly different from values obtained with wild x wild zygotes of either age. The noncristate mitochondria were of two types: one lacking inner membrane invaginations or elaborations and the other containing concentrically arranged loops of inner membrane. The significance of these two types of respiration-deficient mitochondria is unknown. The gradual decrease in frequency of noncristate mitochondria, perhaps due to selection pressures in mixed chondriomes, was discussed as a further indication of the semiautonomous nature of the yeast organelle.
Article
Studies of mitochondrial biogenesis in yeast have been hampered by a lack of suitable membrane markers in anaerobically grown cells subsequently grown in air. Cytochrome c peroxidase activity and subcellular location was studied to determine whether it would be a useful marker for an analysis of mitochondrial formation. Cytochemical tests revealed enzyme reaction product on all mitochondrial membranes in aerobically grown wild-type cells. Anaerobically grown wild-type and all petite cultures contained cytochrome c peroxidase cytochemical reaction deposits on abundant cytoplasmic membranes and on the few mitochondrial profiles which also were seen in the electron photomicrographs. Biochemical studies corroborated the cytochemistry because mitochondrial fractions were greatly enriched in cytochrome c peroxidase activity for aerobically grown wild-type cultures, but petite and anaerobically grown wild-type cultures showed higher enzyme activities in supernatant fractions than was present in the corresponding particulate fractions after differential centrifugation. Evidence from low-temperature microspectroscopy, spectrophotometric assays of mitochondrial enzyme activities, and electron microscopy showed mitochondrial formation during the time required for preparation and lysis of spheroplasts from anaerobically grown cultures. The data were interpreted as indicating that cytochrome c peroxidase was an oxygen-inducible enzyme, and that there was a developmental relationship between enzyme-reactive membranes of mitochondria and cytoplasm during the period of respiratory adaptation.
Article
Six particulate preparations isolated from rat liver under different experimental conditions were analyzed biochemically and examined in the electron microscope. The results confirm the lysosomal nature of the pericanalicular dense bodies and demonstrate that the microbodies are the bearers of urate oxidase, catalase, and D-amino acid oxidase. Catalase, representing a major component of the particles, and D-amino acid oxidase appear to be associated with the structureless "sap" of the particles, urate oxidase with their crystalloid core or with their outer membrane.
Article
Differential and density equilibrium centrifugation have established the presence of α-hydroxy acid oxidase in microbodies of the kidney of the rat. The enzyme has been demonstrated in cells of the distal convoluted tubule by a microscopic cytochemical method. This enzyme, like certain others in microbodies, produces hydrogen peroxide.
Article
1.1. The development of A-type zonal rotors with transparent end plates has made possible the direct determination of sedimentation coefficients of organelles down to the size of mitochondria.2.2. The sedimentation rates of the particles migrating through density gradients were measured from visual observations through the end plates of the rotor with a travelling microscope. Equilibrium densities of the particles were determined in the same rotors.3.3. Plastids were extracted from bean leaves (Phaseolus vulgaris var. Improved Tendergreen) which had been etiolated or exposed to 5 h illumination. The calculated sedimentation coefficient of the plastids from etiolated plants was 5111 000 ± 2000 S and that of plastids from partially greened plants was 666 000 ± 2000 S.4.4. The feasibility of using sedimentation coefficients as biophysical parameters of organelle differentiation is discussed.
Article
Investigations of various enzymic activities, respiratory capacity, and cytochrome content have been carried out on mutants of yeast which had: p genes resulting in the inability to utilize nonfermentable carbon sources for growth; the cy1 gene resulting in a partial deficiency of cytochrome c; the “loss” of the cytoplasmic factor (ϱ−) necessary for the synthesis of cytochromes a + a3 and b; and various combinations of these determinants.p4ϱ+ strains respired (but “ineffectually”) and had low concentrations of cytochromes a + a3 and b. p5ϱ+ strains were deficient in respiration and cytochromes a+a3. p1ϱ+, p6ϱ+, p7ϱ+, Pϱ− an all pϱ− strains were deficient in respiration and cytochromes a + a3 and b.Numerous strains, which had various alterations in the content of cytochromes a + a3, b, c, and c1 + b2, could be obtained by using various combinations of the p/P genes, cy1/CY gene and ϱ+/ϱ− cytoplasmic factor.The frequently occuring deficiency of cytochromes a + a3 and b, is discussed in relationship to mitochondrial structure.
Article
Duell, E. A. (Western Reserve University, Cleveland, Ohio), Sakae Inoue, and Merton F. Utter. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88 1762–1773. 1964.—Functionally intact mitochondria can be obtained in good yields by osmotic disruption of spheroplasts formed from yeast by treatment with an enzyme mixture from the snail digestive tract. The useful range of this method is extended greatly by pretreatment of the yeast cells with 2-mercaptoethylamine and ethylene-diaminetetraacetate. The concentration of the yeast suspension can be increased greatly, the incubation period can be shortened considerably, and the requirement for log-phase cells is obviated. Mitochondria prepared by this method have been compared with those obtained by mechanical disruption in terms of respiratory control, specific activity on a wide range of oxidizable substrates, heterogeneity during centrifugation, and structures observed by electron microscopy. In all cases, the mitochondria obtained from spheroplasts appear to be much less damaged by the preparative procedures.
Article
Avers, Charlotte J. (Rutgers, The State University, New Brunswick, N. J.), Cynthia R. Pfeffer, and Martha W. Rancourt. Acriflavine induction of different kinds of “petite” mitochondrial populations in Saccharomyces cerevisiae. J. Bacteriol. 90 481–494. 1965.—Mutant frequencies induced by 1 or 2 hr in 16 and 64 μg/ml of acriflavine were significantly higher during acceleration and log-phase exposures than during lag or stationary phases. From these induced petites, 59 colonies were selected at random and established in pure culture. All strains were analyzed histochemically for mitochondrial cytochrome oxidase and succinic dehydrogenase (SDH) reactions. On the basis of counts of stained mitochondria per cell obtained by light microscopy, four different cell phenotypes were recognized among the mutant strains: (i) reduced cytochrome oxidase, wild-type SDH; (ii) reduced cytochrome oxidase, high SDH; (iii) absent cytochrome oxidase, high SDH; and (iv) absent cytochrome oxidase, wild-type SDH. The last group was the most common, characterizing 43 of the 59 strains. Electron microscopy showed differences in mitochondrial ultrastructure for the various cell phenotype classes. Electron histochemical localizations showed cytochrome oxidase reaction product only on mitochondrial membranes of respiration-competent cells. Both reactive and unreactive mitochondria occurred in the same cell in mutants with partial respiratory competence. Different mitochondrial subpopulation mixtures characterized the mutant strains, many of which had at least two kinds of respiratory-competent types per chondriome. The diverse chondriomes comprised a stable feature of the mutants, since they have been maintained unchanged during serial transfer for more than 1 year in culture. Together with earlier reports of at least two kinds of mitochondria in wild-type cells, the evidence indicated that mitochondria were capable of regulating some portion of their phenotype. The recognition of mitochondrial phenotypes was proposed as an initial step in a formal analysis of organelle heredity.
Article
Histochemical localizations of cytochrome oxidase, succinic dehydrogenase, DPNH-tetrazolium reductase, and TPNH-tetrazolium reductase activities revealed at least two kinds of mitochondria in the intracellular population. The total chondriome in stationary phase cells contains about 45 mitochondria, all with cytochrome oxidase activity. But, only about 30 mitochondria per cell were active for dehydrogenase or reductases. The differences in mitochondrial enzyme activities persisted throughout the growth cycle, showing different numbers of active mitochondria and different rates of their increase and decrease for all four enzyme systems. Manometric data verified the differences between cytochrome oxidase and succinic dehydrogenase for the earlier phases of the growth cycle. In histochemical counts, zero values for all four enzymes occurred in late acceleration phase, but persisted into log phase only for the tetrazolium reductases. Both cytochrome oxidase- and succinic dehydrogenase-active mitochondria began to increase in numbers at the inception of log phase, but at very different rates. The demonstration of more than one kind of mitochondrion in the common nucleocytoplasmic system of a single cell was considered to be evidence of some measure of autonomous control of the mitochondrial phenotype.
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