DISCUSSION: Bovine Colostrum and Murine Lymphoma Cells
UDP-Ga1NAc:polypeptide N-acetylgalactosaminyltransferase purified to homogeneity from bovine colostrum is a monomer of approximately 70,000 daltons as determined by both SDS-PAGE and gel filtration chromatography. An antibody raised against the purified 70,000-dalton enzyme precipitates the enzyme activity as well as the radiolabeled protein. The difference in the precipitation capacity of the antibody between the two experiments (-0.7 ng of enzyme precipitated/pl when calculated on the basis of activity versus -1.8 ng/pl when calculated on the basis of the iodinated enzyme protein) is probably due to the presence of inactive enzyme. This would be expected since the enzyme loses activity upon storage. Isolation of the intracellular form of this enzyme from mouse lymphoma BW5147 cells yielded a preparation which, although not homogenous, eluted at a position corresponding to a molecular mass of 71,500 on gel filtration chromatography and contained a band of approximately 70,000 daltons on SDS-PAGE (Figs. 4B and 5B). Blotting of this preparation with anti-bovine colostrum N-acetylgalactosaminyltransferase antibody resulted in a band of approx-imately 71,000 daltons.
If one assumes that the soluble transferase is derived from a membrane-bound intracellular form by proteolytic cleavage, these results indicate either that the membrane-anchoring portion of the intracellular transferase is relatively small or that this part of the enzyme is very sensitive to proteases and is readily cleaved during the purification procedure. The former possibility seems more likely since even when a crude membrane extract prepared in the presence of protease inhibitors is blotted, no species of higher molecular mass could be detected. The fact that antibody raised against the soluble bovine enzyme cross-reacts with the membrane-derived murine enzyme lends further support to the conclusion that the soluble enzyme is a cleavage product. In the purification of N-acetylgalactosaminyltransferase from ascites hepatoma AH66 cells, Sugiura et al. (9) reported an apparent M, of 54,000-56,000. The specific activity of this enzyme was 390 units/mg as compared to 1,860 units/mg for the bovine colostrum enzyme. However, the assays of the ascites hepatoma enzyme were done at UDP-GalNAc concentrations below the K,.
The two enzymes differ in several other respects. While both enzymes are specific for UDP-GalNAc, the ascites hepatoma enzyme has a K, of 42 pM for this nucleotide sugar whereas the apparent K, of the bovine colostrum enzyme is 8 p~. Another major difference is in the cation requirement. Both enzymes prefer Mn2+, but the bovine colostrum enzyme utilizes Co2+ almost as well as Mn2+ whereas Ca2+ is poorly utilized. By contrast, the ascites hepatoma enzyme uses Co2+ and Ca2+ equally well, each being 50% as effective as Mn2+. The reasons for these differences in the properties of the two enzymes is not clear. One possiblity is that the two enzymes represent different proteins. Alternatively the ascites hepatoma enzyme may be a proteolytic fragment of the intracellular enzyme resulting in some alterations in its properties. Further studies will be required to resolve this discrepancy. An intriguing finding is the presence of complex-type Asnlinked oligosaccharides on the bovine enzyme.