Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum.
1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.
D000117:Acetylglucosamine; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000302:isolation & purification*; D006602:Hexosyltransferases / Q000032:analysis; D007768:Lactalbumin / Q000494:pharmacology; D008345:Manganese; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology; D011755:Pyrophosphatases / Q000032:analysis; D051381:Rats; D012799:Sialyltransferases / Q000032:analysis; D014531:Uridine Diphosphate Galactose; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000302:isolation & purification*
D000596:Amino Acids; D014531:Uridine Diphosphate Galactose; D011092:Polyethylene Glycols; D008345:Manganese; D007768:Lactalbumin; D005700:Galactosyltransferases; D006602:Hexosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D012799:Sialyltransferases; D011755:Pyrophosphatases; D000117:Acetylglucosamine