Glycoprotein nature of yeast [Saccharomyces strain 1710] alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin

• Published in: The Journal of biological chemistry 1979-01, Vol.254 (23), p.11943-11952
• Main Authors: Onishi, H.R, Tkacz, J.S, Lampen, J.O
• Format: Article
• Language: English
• Subjects: Acid Phosphatase - biosynthesis; Alkaline Phosphatase - biosynthesis; Alkaline Phosphatase - isolation & purification; Carbohydrates - analysis; Glucosamine - analogs & derivatives; Glycoproteins - biosynthesis; Glycoproteins - isolation & purification; Molecular Weight; plant biochemistry; plant physiology; Saccharomyces - drug effects; Saccharomyces - enzymology; Tunicamycin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)86408-6

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.