BIOSYNTHESIS IN-VITRO OF NEOLACTOTETRAOSYLCERAMIDE BY A GALACTOSYLTRANSFERASE FROM MOUSE T-LYMPHOMA - PURIFICATION AND KINETIC-STUDIES - SYNTHESIS OF NEOLACTO AND POLYLACTOSAMINE CORE

The galactosyltransferase, GalT-4, which catalyses the biosynthesis in vitro of neolactotetraosylceramide, nLcOse4Cer (Gal beta 1-4GlcNAc be ta 1-3Gal beta 1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcN Ac beta 1-3Gal beta 1-4Glc-Cer), and UDP-galactose has been purified 1 07 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using a ffinity columns. The purified enzyme is partially stabilized in the pr esence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after so dium dodecyl sulfate polyacrylamide gel electrophoresis of highly puri fied mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indi cating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polycl onal antibody against bovine lactose synthetase (UDP-Gal:Glc beta 4-ga lactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all o ther galactosyltransferases, GalT-4 has absolute requirements for diva lent cation (Mn2+). The K-m values for the substrate LcOse3Cer and don or UDP-galactose are 110 and 250 mu M, respectively. Substrate competi tion studies with LcOse3Cer and either asialo-agalacto-alpha 1-acid gl ycoprotein or N-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which show ed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc beta 1-1-3Gal beta 1-4Lc3), the precursor for polylactosamine antigen s. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental va lues were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an ab solute requirement for an N-acetylglucosamine residue in the substrate since the lyse-derivative (GlcNH(2) beta 1-3Gal beta 1-4Glc-sphingosi ne) of the acceptor glycolipid LcOse3Cer is completely inactive as sub strate while the K-m and V-max of the reacetylated substrate (GlcNAc b eta 1-3Gal beta 1-4Glc-acetylsphingosine) was comparable with LcOse3Ce r. Autoradiography of the radioactive product formed by purified P-179 8 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochroma tographed with authentic glycolipid. The monoclonal antibody IB-2, spe cific for nLcOse4Cer, also produced a positive immunostained band on T LC as well as giving a positive ELISA when tested with radioactive pro duct obtained using a highly purified enzyme from mouse P-1798 T-lymph oma.