Subcellular Fractionation of Candida stellatoidea after Growth with Glucose or n-Hexadecane

Jenkins, Richard O.; Cartledge, Trevor G.; Lloyd, David

doi:10.1099/00221287-129-4-1171

Cited by 6 publications

(7 citation statements)

References 26 publications

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“…2 and Table 1). The localization of ADH and ALDDH in both peroxisomes and in mitochondria has so far been shown only by YAMADA et al (1980) andJENKINS et al (1983) for other Cundida yeasts. For the FAOD this question has yet to be clarified.…”

Section: Discussionmentioning

confidence: 96%

Subcellular organization of alkane oxidation in the yeast Candida maltosa

Mauersberger¹,

Kärgel²,

Matyashova³

et al. 1987

J. Basic Microbiol.

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After spheroplast lysis and differential centrifugation the alkane monooxygenase system consisting of cytochrome P‐450 and the NADPH‐cytochrome P‐450 reductase of alkane‐grown Candida maltosa cells is enriched in the microsomal fraction. This membrane fraction is nearly free of intact mitochondria (cytochrome oxidase) and peroxisomes (catalase), but contains considerable amounts of plasma membrane fractures (azide insensitive, vanadate‐sensitive Mg2+‐ATPase) as demonstrated by biochemical an electron microscopic examinations. By means of sucrose density gradient centrifugation it was possible to separate the cytochrome P‐450 containing membranes ( = 1,11 g/cm3) from the plasma membranes ( = 1,18 g/cm3). Therefore the cytochrome P‐450 alkane monooxygenase system is most likely localized in the endoplasmic reticulum of the yeast cells. For the following enzymatic steps of terminal alkane oxidation to the corresponding fatty acid a quite different subcellular distribution was observed. The fatty alcohol oxidase and aldehyde dehydrogenase activities are mainly localized in the mitochondrial peroxisomal membrane fraction. During the oxidation of n‐alkanes by yeast cells the fatty alcohol should be regarded as an intracellular transport from between the cytochrome P‐450 containing endoplasmic reticulum and the sites of its further oxidation in peroxisomes and mitochondria.

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Section: Discussionmentioning

confidence: 96%

Subcellular organization of alkane oxidation in the yeast Candida maltosa

Mauersberger¹,

Kärgel²,

Matyashova³

et al. 1987

J. Basic Microbiol.

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“…Enzymic disruption of cells Yeast cell suspensions (2 x 109 cells/ml) in 100 mMpotassium phosphate buffer (pH 7.2) containing 100 mM-EDTA and 100 mM-2-mercaptoethanol were digested with 100 units [1 unit will give AA800 of 0.001 unit/min at pH 7.5 and 25°C, with a suspension of S. cerevisiae (3 mg dry wt.) as substrate in a 3 ml reaction mixture (1 cm light path)] of zymolyase 60000 (Kirin Brewery, Tokyo, Japan) by the method of Jenkins et al (1983). Sphaeroplasts were harvested by centrifugation at 600 g (ra.…”

Section: Mechanical Disruption Of Cellsmentioning

confidence: 99%

Cytochrome P-450-dependent 14 α-demethylation of lanosterol in Candida albicans

Hitchcock

Brown

Evans

et al. 1989

Biochemical Journal

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A novel assay for cytochrome P-450-dependent 14 alpha-sterol demethylase of the important opportunistic fungal pathogen, Candida albicans, is described. The enzyme was assayed in microsomal preparations (microsomes) by measuring the incorporation of [14C]lanosterol into (4,14)-desmethylated sterols. The efficacy of different cell-breakage methods was compared; desmethylated-sterol biosynthesis was maximal when cells were broken with a Braun disintegrator. The solubilization of [14C]lanosterol with detergent in the assay system was essential for enzyme activity, which was enhanced considerably when microsomes were gassed with O2. Under these conditions, there was a reciprocal relationship between the amount of radioactivity incorporated into desmethylated sterols and that lost from lanosterol. The major radiolabelled desmethylated sterol was ergosterol. The enzyme had an apparent Km of 52.73 +/- 2.80 microM and an apparent Vmax of 0.84 +/- 0.14 nmol/min per mg of protein (n = 3). Enzyme activity was decreased greatly when microsomes were treated with CO or the triazole antifungal ICI 153066.

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“…3 c). Poor separation between peroxisomes and mitochondria was also observed in C. stellatoidea by Jenkins et al (1983).…”

Section: Metabolism Of Putrescine By C Boidiniimentioning

confidence: 73%

Partial Purification of a Peroxisomal Polyamine Oxidase from Candida boidinii and its Role in Growth on Spermidine as Sole Nitrogen Source

Haywood

Large

1984

Microbiology

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Candida boidinii grows well on spermidine as sole nitrogen source, but poorly on spermine. Cells grown on spermidine, cadaverine, putrescine and 1,3-diaminopropane contained a polyamine oxidase which attacks spermine and spermidine at the secondary amino groups, forming putrescine and a product thought to be 3-aminopropionaldehyde. The enzyme was synthesized before growth began when C. boidinii that had been grown in medium containing glucose + ammonium was transferred to medium in which spermidine replaced ammonium.Other enzymes increasing in specific activity during this adaptation were catalase, benzylamine oxidase and NAD-dependent glutamate dehydrogenase. The polyamine oxidase was purified to 50% homogeneity, but was too unstable to obtain completely pure. It had a pH optimum of 10.0, and could be stabilized by addition of inert protein. It oxidized spermine, spermidine, N 1acetylspermidine, N-n-butylpropylamine, di-n-butylamine and di-n-hexylamine. It did not oxidize di-n-propylamine, diethylamine or N 1 ,N8-diacetylspermidine. Apparent K , values were determined for the active substrates. The enzyme was potently inhibited by quinacrine and by divalent cations. The stoicheiometry of the enzyme reaction was established using di-nbutylamine as substrate. The enzyme has a molecular weight in the range 80000 to 110000. Putrescine (the oxidation product of spermidine) was not oxidized by cell-free extracts, but evidence of aminotransferase activity was found. The oxidation/transamination product of putrescine, 4-aminobutyraldehyde (1 -pyrroline), was oxidized by extracts and a scheme is presented by which spermidine could be catabolized. Polyamine oxidase was shown to cosediment with NAD-dependent glycerol 3-phosphate dehydrogenase and catalase in sucrose gradients after mechanical breakage of spheroplasts, and is thus a peroxisomal enzyme. Polyamine oxidase was present in some other yeasts when grown on spermidine, C. nagoyaensis, Hansenula polymorpha and Trichosporon melibiosaceum, but absent from C . steatolytica, Pichia pastoris and Sporopachydermia cereana. These latter yeasts probably contained an enzyme resembling benzylamine/putrescine oxidase which attacks the primary amino groups of spermidine.Abbreuiarion : ABTS, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate).

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Subcellular Fractionation of Candida stellatoidea after Growth with Glucose or n-Hexadecane

Cited by 6 publications

References 26 publications

Subcellular organization of alkane oxidation in the yeast Candida maltosa

Subcellular organization of alkane oxidation in the yeast Candida maltosa

Cytochrome P-450-dependent 14 α-demethylation of lanosterol in Candida albicans

Partial Purification of a Peroxisomal Polyamine Oxidase from Candida boidinii and its Role in Growth on Spermidine as Sole Nitrogen Source

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