Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae

Abstract

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.

Sterol synergism in yeast.

“…For every milliliter of spheroplast suspension, 15 ml of buffer containing 0.3 mg of concanavalin A per ml was then added. After standing for [10][11][12][13][14][15] min at room temperature, spheroplasts were centrifuged at 2°C for 1 min in a desk-top clinical centrifuge. The pellet was resuspended in buffer containing 10 mM Tris, 5 mM MgSO4, 0.6 mM p-phenylmethylsulfonyl fluoride, and DNase (8.5 mg for every 0.5 ml of original suspension).…”

The Plasma Membrane of Candida albicans: Its Relevance to Transport Phenomenon

“…It is fairly well established in yeast and fungi where it acts as a novel type of ion pump [25,26,63,[84][85][86][87]. The ATPase gene from S. eerevisiae and N. erassa has recently been cloned and sequenced [68,91,92] and shows a great deal of sequence homology.…”

Distribution of Plasma-membrane Fragments during Zonal Centrifugations of Homogenates from Aerobic Saccharomyces cerevisiae

“…Content oj' marker components in whole homogenates The contents of the marker components in the whole homogenates [have been reported (Nurminen et al, 1976, Results are expressed as percentages of the total amounts recovered in the density range 1-00 to 1-29 g ~r n -~.…”

“…Fresh cells were disrupted in a Mini-mill disintegrator, high-speed zonal centrifugations were performed in a B XV zonal rotor, and several of the membrane fractions obtained by zonal centrifugation on buffered sucrose were further fractionated on gradients of iso-osmotic Urografin as described previously (Nurminen et al, 1976).…”