BIOCHEMICAL-PROPERTIES AND EXCRETION BEHAVIOR OF REPRESSIBLE ACID-PHOSPHATASES WITH ALTERED SUBUNIT COMPOSITION

Yeast repressible acid phosphatase (rAP) is the oligomeric extracellul ar enzyme encoded by the three structural genes PHO5 (p60), PHO10 (p58 ) and PHO11 (p56). We examined the ability of acid phosphatases formed by various subunit combinations to be excreted into the medium. Plasm ids with repressible acid phosphatase structural genes under control o f the yeast glyceraldehyde-phosphate dehydrogenase (GAP) promoter were constructed to obtain constitutive expression of acid phosphatase, an d yeast strains with disruptions in PHO5, PHO10 and PHO11, respectivel y, were used to generate mutants expressing single genes or specific g ene combinations. EndoF treatment of acid phosphatases, produced by th ese strains, followed by SDS-electrophoresis in combination with densi tometry techniques revealed that the ratio p60/(p56 + p58) among struc tural polypeptides in extracellular enzyme is constant and equals to 6 .0. A study of acid phosphatases formed by single type subunits was un dertaken. Expression products of PHO5, PHO10 and PHO11 genes were isol ated from the culture medium. The specific activities of the enzymes w ere found to be 33, 2 and 2 mM x mg(-1) x min(-1), respectively. The v alues of Mr estimated by HPLC chromatography for the enzymes encoded f or by the genes PHO5, PHO10 and PHO11 and SDS-polyacrilamide gel elect rophoresis data suggested an oligomeric organisation of the enzymes. I soelectric focusing in polyacrylamide gel with immobilised pH gradient followed by activity staining yielded numerous sharp bands of homopol ymeric acid phosphatases forms being different in their pi. The kineti c characterisation of the enzymes revealed differences in Km values, s ensitivity to temperature inactivation, inhibition by orthophosphate a nd the effect of pH on the enzyme activity.