Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.
The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.
D000135:Acid Phosphatase / Q000096:biosynthesis; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000302:isolation & purification; D002241:Carbohydrates / Q000032:analysis; D005944:Glucosamine / Q000031:analogs & derivatives*; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D008970:Molecular Weight; D012440:Saccharomyces / Q000187:drug effects / Q000201:enzymology*; D014415:Tunicamycin / Q000494:pharmacology*
D002241:Carbohydrates; D006023:Glycoproteins; D014415:Tunicamycin; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D005944:Glucosamine