Isolation and characterization of cancer-associated galactosyltransferase isoenzyme.
We isolated galactosyltransferase (EC 2.4.1.22) from pleural effusions of a lung cancer patient and a patient with cirrhosis by precipitation with ammonium sulfate, followed by gel filtration on Sepharose 6B, and affinity chromatography on columns of alpha-lactalbumin-agarose and protein A-Sepharose. By this procedure the enzyme from both sources was purified 40 000-fold with approximate yields of 37% and 60%, respectively, and did not contain immunoglobulin. Electrophoresis on polyacrylamide gel of the enzyme from the cancer patient (slower moving) and from the non-cancer patient (faster moving) gave one sharp band for each. Their respective relative molecular masses, 74 131 and 107 151, were estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel filtration, respectively. The isoenzymes were active between pH 5 and 8, most active at 7, and showed no activity below pH 4 or above pH 9. Activity was greatest at temperatures between 37 and 40 degrees C. At 30 degrees C or 50 degrees C the activity was more than halved, and was lost completely above 60 degrees C. The isoenzymes had an absolute requirement for Mn2+. Omitting the surfactant Triton X-100 from the buffer resulted in considerable loss in activity of both isoenzymes. Glucose can be used as an acceptor for these isoenzymes if alpha-lactalbumin is present in the assay mixture. These isoenzymes had different Km values for UDP-galactose, N-acetylglucosamine, and Mn2+.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000302:isolation & purification*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D007768:Lactalbumin / Q000494:pharmacology; D008103:Liver Cirrhosis / Q000201:enzymology*; D008175:Lung Neoplasms / Q000201:enzymology*; D008970:Molecular Weight; D010996:Pleural Effusion / Q000201:enzymology*
D007527:Isoenzymes; D007768:Lactalbumin; D005700:Galactosyltransferases