The charge heterogeneity of soluble human galactosyltransferases isolated from milk, amniotic fluid and malignant ascites.
UDP-galactose: N-acetylglucosamine galactosyltransferase was isolated from pooled human milk, pooled amniotic fluid and from two different individual samples of malignant ascites. The purification procedure involving two successive affinity chromatography steps on N-acetylglucosamine--agarose and alpha-lactalbumin--agarose yielded an enzyme preparation homogeneous by size. Under non-denaturing conditions the ascites and amniotic fluid enzymes had identical electrophoretic mobility, but they moved faster than the milk enzyme. Isoelectric analysis in the presence and absence of urea resolved the milk enzyme into at least 13 different forms, nine of which had the same isoelectric points after refocusing. All enzyme forms showed similar activity when free N-acetylglucosamine, ovalbumin, sialic-acid-free ovine submaxillary mucin and glucose, in the presence of alpha-lactalbumin, were used as acceptor substrates. Comparative isoelectric focusing of the three galactosyltransferases revealed identical patterns of the amniotic and ascites enzymes, but only partial overlap with the milk enzyme, which was less negatively charged. Neuraminidase treatment of ascites and milk galactosyltransferases produced very similar focusing patterns. The possible structural basis for this charge heterogeneity is briefly discussed.
D000596:Amino Acids / Q000032:analysis; D000653:Amniotic Fluid / Q000201:enzymology*; D001201:Ascites / Q000201:enzymology*; D005260:Female; D005700:Galactosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008895:Milk, Human / Q000201:enzymology*; D009439:Neuraminidase; D010051:Ovarian Neoplasms / Q000201:enzymology*; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D013379:Substrate Specificity; D014508:Urea
D000596:Amino Acids; D014508:Urea; D005700:Galactosyltransferases; D009439:Neuraminidase