Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions.
1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37 degrees C for 1h released about 60% of the membrane-bound UDP-galactose-glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn(2+) that could not be replaced by Ca(2+), Mg(2+), Zn(2+) or Co(2+), and was active over a wide pH range (6-8) with a pH optimum of 6.5. The apparent K(m) for UDP-galactose was 10.8mum. The protein alpha-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. 4. The molecular weight of the membrane enzyme was 65000-70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2-5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-epsilon-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the epsilon-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.
Fraser
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IH
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1C 5S7, Canada.
Mookerjea
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