Abstract
UDP-N-acetyl-D-gaiactosamine: κ-casein polypeptide N-acetylgalactosaminyltransferase was purified from a crude Golgi apparatus of lactating bovine mammary gland after solubilization with Triton X-100. Through chromatography on DEAE-Sephadex A-50, apomucin-Sepharose 4B, FPLC mono S, and Sephacryl S-200, and then electrofocusing, the enzyme was purified up to 7500-fold from the homogenate.
The molecular weight of the enzyme was estimated at 200, 000 from gel filtration. The pI value of the enzyme was 6.4 on electrofocusing. The purified enzyme transferred GalNAc from UDP-GalNAc, not to the carbohydrate chains but to the polypeptide chains of the substrates, κ-casein and mucin. The enzyme required Mn2+, DTT, and Triton X-100 for maximal activity. The Km value for UDP-GalNAc was 16.2μM. Km values for κ-subcomponents 1 and 7, and apomucin were 1.15, 5.10, and 0.192mg/ml, and Vmax values were 254, 259, and 581 nmol/hr/mg, respectively. Thermal stability and the effects of pH, milk components, lectins, and nucleotides were examined.
αsl-Casein strongly inhibited GalNAc transfer to κ-casein. The inhibitory effect of αsl-casein was canceled by the addition of Ca2+, which causes casein micelle formation. This means that the glycosylation of κ-casein occurs after casein micelle formation triggered by the accumulation of Ca2+ in vivo.
