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.
Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H(+)-ATPase (0.9-1.1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca(2+)- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.
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