FIGURE 4.

Functional relevance of the enzyme complexes. COS7 cells were transfected with the indicated enzyme constructs and stained for sialic acid content (A) and T-antigen expression (B) as described under “Experimental Procedures.” Prior to T-antigen measurements, N-glycans were removed by peptide N-glycosidase F. Quantification was done by using flow cytometry. All measurements were made in triplicate and are expressed as fold changes relative to endogenous level (mean ± S.D.; n = 3). Shown are size-exclusion chromatography and in vitro activities of the fractions in cells expressing GalT-I alone (C), ST6Gal-I alone (D), or both GalT-I and ST6Gal-I (E). COS7 cells transfected with the appropriate plasmids were solubilized, fractionated, and subjected to enzyme activity measurements as described under “Experimental Procedures.” Enzyme activities are expressed as μmol/min/μg protein. Solid squares denote GalT-I activity values in fractions, and open circles denote ST6Gal-I activity. Arrows denote the standard proteins used to calibrate the column (Bio-gel A0.5m, Bio-Rad). Shown are GalT-I (F) and ST6Gal-I (G) activities of total cell lysates. Activities were measured as described above and are expressed as the mean (μmol/min/μg protein ± S.D.) from three independent measurements. **, p < 0.01; ***, p < 0.001.