Susumu Kawamoto

Localization of carnitine acetyltransferase in peroxisomes and in mitochondria of n-alkane-grown candida tropicalis

The profuse appearance of peroxisomes (microbodies) in the n-alkane-grown cells of various yeasts has been demonstrated [ 1,2] . Subsequent isolation of the peroxisomes from n-alkane-grown Candida tropicalis was accomplished and the localization of the following enzymes was confirmed in the isolated peroxisomes: Catalase, D-amino acid oxidase, isocitrate lyase, malate synthase, NADP-linked isocitrate dehydrogenase, uricase and fatty acid P-oxidation system [3-51 . In spite of the fact that isocitrate lyase and malate synthase, the key enzymes of glyoxylate cycle essential for gluconeogenesis in alkane-utilizing yeasts [6] , were located in the peroxisomes, the other glyoxylate cycle enzymes common to the TCA cycle, malate dehydrogenase, citrate synthase and aconitase, were detected exclusively in the mitochondria [3] . Furthermore, the fatty acid /I-oxidation system, which participates in acetyl-CoA production from alkane substrates, was essentially peroxisome-associated under the experimental conditions employed [S] . These results strongly indicate that cooperation of peroxisomes with mitochondria is necessary for the complete operation of the glyoxylate cycle and that the acetyl-CoA utilized by citrate synthase in the mitochondriamust be supplied from the peroxisomes. The purpose of this report is to provide evidence for the localization of carnitine acetyltransferase (EC 2.3.1.7, acetyl-CoA:carnitine O-acetyltransferase) (CAT) both in the peroxisomes and in the mitochondria of alkane-grown C. tropicalis. The possible

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 long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid β-oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via β-oxidation to yield energy and cell constituents.

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

SummaryMicrobial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.