1887

Abstract

Cells of the yeast were transformed to true protoplasts by means of snail gut enzyme. The plasma membrane of purified protoplasts was labelled either by [H]dansyl chloride or by Na I. The crude lysate of osmotically ruptured protoplasts was fractionated by two subsequent centrifugations on sucrose density gradients. The two protein peaks obtained at densities of 1·16 and 1·13 g cm were identified according to their characteristic markers (radioactivity, cytochrome oxidase, oligomycin-sensitive ATPase at pH 8.5, and sterol content) as mitochondrial and plasma-membrane fractions, respectively. The ATPase activity associated with the plasma-membrane fraction exhibited a pH optimum at 6.5, was insensitive to oligomycin and was inhibited by vanadate. It can therefore be used as an intrinsic plasma-membrane marker.

Adherence of silica microbeads to the protoplasts effectively increased the density of the plasma membrane so that it could be obtained in a purified state by a single density gradient centrifugation. A third ATPase, presumably associated with the tonoplast membrane, was also demonstrated.

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1984-03-01
2021-10-28
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