Purification and characterization of alkaline phosphatase, a lysosomal glycoprotein from Saccharomyces cerevisiae

Abstract

The function of the oligosaccharide moiety of lysosomal glycooroteins is presently unclear. A lack of information concerning both the structure of these oligosaccharides and the effects of deglycosy- 'ation of lysosomal glycoproteins has prevented the elucidation of the function of the carbohydrate moiety. In order to obtain information concerning the structure of these oliqosaccharides, the lysosomal glycoprotein, repressible alkaline phosphatase was purified from the yeast Saccharomyces cerevisiae. The repressible form of alkaline onosphatase was specifically purified by antibody affinity chromatography. The purified enzyme migrated as a single band when electrophoresed at several different pH values and in the presence of SDS. "he native form of repressible alkaline ohosphatase appeared to be a dimer with a molecular weight of approximately 120,000 daltons. The subunit molecular weight was 62,000 daltons for the glycosylated enzyme and 53,000 for the deglycosylated form. The enzyme was inactivated by EDTA suggesting alkaline phosphatase is a metallo-enzyme. A wide variety of phosphorylated compounds were hydrolyzed by the enzyme at pH 8.5, but polyphosphates and phosphorylated compounds from yeast vacuoles served poorly as substrates. The enzyme contained 2 N-asparagine linked oligosaccharides per subunit. The oligosaccharides were homogeneous in size and composed of 8 mannose residues and 2 N-acetyl glucosamine residues. Based upon Smith degradation, acetolysis and sequential exoglycosidase digestion the following structure is proposed...