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On the isolation enzymes and lipid composition of the plasma membrane and cell wall of bakers yeast saccharomyces cerevisiae



On the isolation enzymes and lipid composition of the plasma membrane and cell wall of bakers yeast saccharomyces cerevisiae



Reports Monographs Non Serials : 46



Cell envelopes containing the cell walls and fragments of plasma membrane were isolated from baker's yeast (S. cerevisiae). After enzymatic removal of most of the carbohydrates, the plasma membrane fraction was obtained by differential centrifugation. It contained mainly proteins and lipids, and an appreciable amount of carbohydrate. The cell walls were prepared from the cell envelopes by repeated washings by centrifugation. EM and the absence of known intracellular enzymes revealed that the cell envelopes were free from cytoplasmic material. Saccharase, acid phosphatase, inorganic pyrophosphatase and ATPase active at pH 3.5 were released into the medium during enzymatic digestion, which indicates that they are located in the cell wall or in the periplasmic space. The Mg2+ dependent ATPase and lipase apparent in the cell envelopes are located in the plasma membrane only, whereas a part of lysophospholipase is present also in the cell wall. An adenylate cyclase preferentially activated by Mn2+ was demonstrated in the cell envelopes. Most of the envelope lipids were in the plasma membrane. The main phosphoglycerides present in the plasma membrane were phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. The major envelope fatty acids in aerobic S. cerevisiae were C16:1 and C18:1 acids. The principal fatty acids with more than 18 carbons were 2 hydroxy-C26:0 and C26:0 acids. They largely originated from a glycosphingolipid containing inositol, phosphorus and mannose, present in both the cell wall and the plasma membrane. The minor glycoplipids detected included ceramides, sterol glycosides, sulpholipids, cerebrosides and acylglucoses. Sphingolipids dominated in the envelope glycoplipids. Their main long-chain base was C18-phytosphingosine. Neutral lipds constituted a large part of the envelope lipids. The cell-wall lipids largely consisted of neutral lipids, principally triacylglycerols and lower acylglycerols and small amounts of esterified sterols. The plasma membrane contained a considerable amount of triacylglycerols and most of the envelope sterols. Compared to the plasma membrane of nearly anaerobic S. carlsbergensis, the plasma membrane of aerobic S. cerevisiae contained more C16:1, C18:1 and other unsaturated fatty acids, more sterol and less squalene. The main sterol in the aerobic membrane was ergosterol. It was in the free form, whereas zymosterol, 24(28) dehydroergosterol, epi- or fecosterol and lanosterol were mostly esterified. The anaerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol, and was richer in saturated fatty acids having a greater variation in chain length. For the larger scale isolation of plasma membranes from glucose repressed S. cerevisiae, the fractionation of homogenates by zonal centrifugation in density gradients of sucrose and iso-osmotic Ficoll was studied. The distribution of several enzymes and other marker components, and the composition and morphology of the fractions were investigated. In sucrose, a minor peak of phospholipid and sterol coincident with a peak of oligomycin insensitive Mg2+-dependent ATPase at around the density 1.25 contained heavy fragments of plasma membrane of much higher carbohydrate content, especialy mannose, than did the lighter vesicles of plasma membrane. Apparently the plasma-membrane fragments can differ in density depending on their content of heavy mannanprotein particles.

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Accession: 006028133

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