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SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX
Author(s) -
Bosmann H. B.
Publication year - 1972
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1972.tb01391.x
Subject(s) - transferase , fetuin , chemistry , biochemistry , glycoprotein , enzyme , orosomucoid , mucin , neuraminic acid
— Four glycoprotein:glycosyl transferases (a fetuin: N ‐acetylglucosaminyl transferase; a bovine submaxillary mucin: N ‐acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34‐, 45‐, 37‐ and 47‐fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G‐100 and G‐150 of 0 , 1% (w/v) Triton X‐100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N ‐acetylglucosaminyl transferase functioned only with fetuin minus N ‐acetylneuraminic acid, galactose and N ‐acetylglucosamine; the bovine submaxillary mucin: N ‐ acetylgalactosaminyl transferase with bovine submaxillary much minus N ‐acetylneuraminic acid and N ‐acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N ‐acetylneuraminic acid and galactose or fetuin minus N ‐acetylneuraminic acid and galactose. Each transferase required a specific (XDP)‐monosaccharide for transfer. The transferases were entirely dependent on either Mn 2+ or Mg 2+ for activation and Fe 2+ and Hg 2+ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N ‐acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N ‐acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K m (μM) and V mex (pmoles/h/mg of protein) for the transferases were: fetuin: N ‐acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N ‐acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.