Table 1.

Lipid galactosylation in postnuclear supernatants from exogenous UDP-galactose

	Enzyme activity (pmol/mg protein h)a
Transferases	CHOlec8	GalT-1 CHOlec8	UGT-CHOlec8	GalT-1 UGT-CHOlec8
GalT-1 + saponin	ND	9	ND	8b
GalT-1	ND	7	ND	28
GalT-2 + saponin	26	28	29	27
GalT-2	11	9	35	33

ND, not detectable: a fluorescent signal corresponding to less than 0.5 pmol/mg protein h.

^aPostnuclear supernatants prepared from CHOlec8 and UGT-CHOlec8 cells transiently transfected with GalT-1 and empty vector were analyzed for GalT-1 and GalT-2 activity on exogenous C₆-NBD-Cer and C₆-NBD-GlcCer, respectively, in the presence of UDP-galactose and in the presence or absence of saponin. The addition of UDP-glucose to the GalT-1 assay allowed the simultaneous measurement of the activity of the UDP-glucose: ceramideglucosyltransferase. C₆-NBD-GlcCer synthesis (on the cytosolic surface; Burger et al., 1996) was similar for all cell lines, 380 ± 15 pmol/mg protein h, corroborating that the same amounts of the lysates were used. Activities were determined in at least two independent experiments with similar results. A representative experiment is shown (n = 2, range is <20% of signal).

^bThe reduction in GalT-1 activity by saponin in GalT-1 UGT-CHOlec8 cells shows that saponin partially inhibited GalT-1 activity. When corrected for this inhibition, the apparent activity of GalT-1 CHOlec8 cells was enhanced four-fold by opening the membranes by saponin.