1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 10(5)g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH- and NADPH-cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH-cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.
1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl(2). 2. These homogenates contained no detectable cytochrome c oxidase, succinate-cytochrome c oxidoreductase, succinate-ferricyanide oxidoreductase, l(+)-lactate-cytochrome c oxidoreductase or catalase. Cytochromes a+a(3) and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH- and NADPH-cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 10(5)g-min left 80-90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S(1)) together with 65 and 56% of the NADH- and NADPH-cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S(1) revealed the presence of a class of small particles containing NADPH-cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ;promitochondria' as described by other workers.
1. Subcellular fractionation of sphaeroplasts produced at different stages during the first 4h of respiratory adaptation of anaerobically grown glucose-de-repressed Saccharomyces carlsbergensis gave mitochondrial fractions that contained all the detectable c- and a-type cytochromes. 2. The rates of cytochrome formation were studied; individual cytochromes were produced at different rates so as to give respiratory chains having widely differing cytochrome ratios. A CO-reacting haemoprotein other than cytochrome a(3) also increased throughout 8h of respiratory adaptation. 3. Even after short periods of aeration, organisms contained mitochondria in which cytochrome-cytochrome interactions and the reaction of cytochrome a(3) with O(2) proceeded at rates almost as fast as in organelles from aerobically grown cells. 4. The technique of flow-flash photolysis enabled kinetic resolution of the reoxidation of cytochromes a(3) and a to be achieved and their individual contributions to extinction changes in the Soret region were assessed. The ratio cytochrome a(3)/cytochrome a increased over the early stages of adaptation.
Spheroplasts of glucose grown and n-hexadecane-grown Candida stellatoidea were prepared using snail-enzyme or Zymolyase-5000 and the resultant cell extracts fractionated on sucrose or metrizamide gradients. Organelles from n-hexadecane-grown cells were more fragile than those from glucose-grown cells and organelle integrity was maintained only after spheroplast formation using Zymolyase-5000. Isopycnic density gradient centrifugation through metrizamide gradients yielded more complex distributions and markedly higher percentage sedimentabilities of marker enzymes than with sucrose gradients. The zone containing cytochrome c oxidase and all tricarboxylic acid cycle enzymes assayed was readily identified. The density of microbodies appears to be similar to that of mitochondria on either gradient material; on metrizamide a second catalase peak at p = 1.07 g ml-l was also observed. This zone was shown by electron microscopy to contain organelles up to 1 pm diameter, and activities of carnitine acetyltransferase and long chain alcohol and aldehyde dehydrogenases. The first enzyme was located mainly in zones containing mitochondria and microbodies; the last two enzymes were multilocational and of differing distributions, but were found mainly in mitochondria1 and microsomal fractions. The possibility that cells grown on n-hexadecane contain two populations of microbodies is discussed. Most lysosomes were disrupted on sucrose gradients but sedimented to a density of 1-12 g ml-1 on 'metrizamide gradients.
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