Pooled effusions from patients with various cancers were used as an enzyme source for purification of both the cancer-associated galactosyltransferase (GT-II) and the normal isoenzyme (GT-I). The purification procedures involved taking the precipitate collected from a 30 to 70% ammonium sulfate fraction and subjecting them to chromatography on norleucine/Sepharose 4B with a linearly decreasing ammonium sulfate (1.25 M to 0.0 M) gradient. All of the fractions exhibiting galactosyltransferase activity were pooled, concentrated by (NH4)2SO4 precipitation, resuspended, and placed on a DEAE-cellulose column. A linearly increasing phosphate gradient was then used to separate isoenzyme GT-II from GT-I. The isoenzymes were further purified by sequential chromatography on α-lactalbumin/Sepharose 4B and N-acetylgucosamine/Sepharose 4B affinity columns achieving a final purification of 5400-fold for GT-II and 4300-fold for GT-I. The separated isoenzymes showed homogeneity by polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Each purified isoenzyme retained its respective mobility on polyacrylamide electrophoresis as initially demonstrated in the effusions and in serum. Kinetic studies showed that the two isoenzymes had identical pH optima (7.1), cation requirements, optimal (Mn+) of 10 mM, Km for UDP-galactose of 13.3 μM , and Km for ovalbumin of 0.2 mM. The isoenzymes differed by 1 order of magnitude in their affinity for a fetuin-derived acceptor with a Km of 0.2 mM for GT-I and Km of 2.0 for GT-II. The isoenzymes also differed in their relative sensitivity to α-lactalbumin; GT-II substrate specificity was only partially sensitive to the presence of the modifier protein. The molecular weight of GT-II was found to be greater than GT-I as determined by sodium dodecyl sulfate polyacrylamide electrophoresis and Bio-Gel P-150 chromatography (GT-I=54,000; GT-II=76,000). Both isoenzymes were found to be glycoproteins with 4% carbohydrate (by weight) for GT-I and 9% for GTII. GT-II contained twice as much hexosamine which was identified as glucosamine by gas-liquid chromatography. In addition, both enzymes were found to contain mannose and relatively small amounts of galactose. Peptide map analysis showed distinct compositional differences between GT-I and GT-II, althoght they also contained several peptides of identical mobility. The purified cancer-associated galactosyltransferase isoenzyme (GT-II) appears to be structurally and kinetically distinct from the normal isoenzyme (GT-I).
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1979|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology