Characterization of a Galactosyltransferase in Plasma Membrane- enriched Fractions from Balb/c 3Tl2 Cells*

Plasma membrane-enriched fractions were obtained from the Balb/c 3T12 cell line by a conventional technique. The specific activities of two plasma membrane markers, 5’-nucleotidase and (Na+-K+)-ATPase were enriched loto l&fold and 6to lo-fold, respectively, over homogenates in these preparations. Galactosyltransferase activity toward N-acetylglucosamine and asialo-agalacto-fetuin was associated with these plasma membrane markers with an enrichment in specific activity of 1.5to a-fold over homogenates. The galactosyltransferase activity toward GlcNAc was partly characterized with respect to UDP-galactose and GlcNAc, yielding K,,, values of 12.7 PM and 4.6 mM, respectively. cy-Lactalbumin, the modifier protein of the lactose synthetase system, was shown to inhibit N-acetyllactosamine synthesis in the plasma membrane fractions by greater than 85%, while stimulating lactose synthesis by 60-fold. Dialdehyde-UDP, reported by Powell and Brew to bind to and inactivate bovine colostrum galactosyltransferase (Powell, J. T., and Brew, K. (1976) Biochemistry 15, 3499-3505), inactivated the 3T12 plasma membrane-enriched galactosyltransferase activity by greater than 90%. When dialdehyde[3H]UDP was incubated with living, intact cells in culture, there was a 3.5-fold enrichment of bound radioactivity in the subsequently isolated plasma membrane-enriched fractions compared to the more dense membrane fractions. When cell homogenates were incubated with dialdehyde-[3H]UDP and plasma membranes subsequently isolated, there was no preferential enrichment for bound radioactivity in any membrane fraction. Also, when intact cells in monolayer were treated with unlabeled dialdehyde-UDP, there was a significant loss of galactosyltransferase activity in the plasma membrane-enriched fractions compared to denser membrane fractions. We conclude that plasma membrane-enriched fractions with enriched galactosyltransferase activity can be isolated from these cells and that dialdehyde-UDP could be a useful tool for studying the function and localization of the membrane-bound galactosyltransferase.