Springer Online Journal Archives 1860-2000
Abstract Almost all the vacuoles (about 95%) remained intact after “polybase-induced lysis” of the yeast protoplasts. These vacuoles could be sedimentated together with other cell organelles which were equally well preserved, leaving as a supernatant a cytosol fraction which was essentially uncontaminated by the contents of disrupted vacuoles. After density gradient centrifugation more than half of the vacuoles were recovered in a fraction which was highly purified as judged from the measurement of several marker enzymes and from light and electron microscopic observations. Polyphosphate, which has been shown to be located exclusively in the vacuolar sap of protoplasts, was used as a vacuolar marker to determine the yields of vacuoles in the different fractions obtained from the density gradients. It was also used to assess the overall distribution of lytic enzymes in the cytosol and in the vacuome. The results indicate that the following enzyme activities are mostly, if not exclusively (〉90%), located in the vacuome, probably all in the typical large vacuoles present in the protoplasts: exo-and endopolyphosphatase, proteases A and B, carboxypeptidase Y, an aminopeptidase, RNase, α-mannosidase, and phosphatases which hydrolyze a number of different substrates. The polyphosphatases are thus in the same compartment as the polyphosphate. The activities of some other hydrolases, notably of a Mg2+ dependent, Oligomycin and NaN3 insensitive ATPase and alkaline phosphatase, were partially associated with the vacuoles. The activities of pyrophosphatase, tripolyphosphatase, α-glucosidase, and aminopeptidase active in the presence of EDTA, were located almost exclusively in the soluble, cytosolic fraction.
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