title: Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.
abstract: 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.
mesh_terms: 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*
chemical_list: D002241:Carbohydrates; D006023:Glycoproteins; D014415:Tunicamycin; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D005944:Glucosamine