Purification and stabilization of galactosyltransferase from serum and lysolecithin extracted microsomes.

Abstract

Rat liver microsomes treated with increasing concentrations of lysolecithin released, after a brief lag, progressively increasing amounts of UDPgalactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. A second extraction of the microsomes with lysolecithin (8 mM) resulted in a total release of about 45% enzyme. The specific activity of the enzyme in the second extract was 12 times higher than that of the first extract. The galactosyl-transferase present in the extract was purified 417-fold by an affinity column chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. During purification, the column and elution buffers required 0.1% lysolecithin to keep the enzyme in active form. For purposes of comparison the soluble serum galactosyltransferase was also purified by identical techniques, which also required 0.1% lysolecithin in column and elution buffers to prevent the loss of enzyme activity. The pure serum and membrane galactosyltransferase contained no sialyltransferase and ran as a double band on polyacrylamide gels (molecular weight 63 000–64 000). The pure enzyme had an absolute requirement for Mn2+, not replaceable by Cu2+, Mg2+, Zn2+, and Co2+. The enzymes were active over a wide pH range, with optimum pH of 6.5. The apparent Km's for UDPgalactose for the serum and membrane enzymes were 12.05 and 11.8 μM, respectively. The specific activities of these two purified enzymes were also remarkably similar, 3.99 × 106 for serum and 3.84 × 106 for membrane enzyme. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose (DSG-fetuin). The enzyme activity with DSG-fetuin acceptor was inhibited to a lesser extent by α-lactalbumin.Effect of various additives on the stability of purified membrane and serum galactosyltransferase was studied at 0–5 °C and at −20 °C up to 60 days. At both temperatures, albumin was found to be the best stabilizer. Ammonium sulfate was a good stabilizer for the serum but not for the membrane enzyme. Glycerol showed some stabilizing effect for both enzymes. EDTA, p-methylbenzenesulfonyl fluoride, N-acetylglucosamine-Mn2+, and water did not offer any stabilization of the pure enzyme.