Division of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20892.
A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.