Solubilization and partial characterization of UDP-N-acetylgalactosamine: globoside alpha-N-acetylgalactosaminyltransferase from dog spleen microsomes.
UDP-N-acetylgalactosamine:globoside alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn2+; this was not substituted by Ca2+ or Mg2+. Apparent Km values for UDP-N-acetylgalactosamine and globoside were 1-10(-5) and 5-10(-4) M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparaton, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Cholormercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002554:Cerebrosides; D004285:Dogs; D004793:Enzyme Reactivators; D005700:Galactosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D017350:N-Acetylgalactosaminyltransferases; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility; D013154:Spleen / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014500:Uracil Nucleotides / Q000494:pharmacology; D014536:Uridine Diphosphate N-Acetylgalactosamine
D002554:Cerebrosides; D004793:Enzyme Reactivators; D010743:Phospholipids; D013438:Sulfhydryl Compounds; D014500:Uracil Nucleotides; D008345:Manganese; D014536:Uridine Diphosphate N-Acetylgalactosamine; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C023216:globoside alpha-N-acetylgalactosaminyltransferase; D008274:Magnesium; D002118:Calcium