The
galactosyltransferase,
GalT-4, which catalyses the biosynthesis in vitro of
neolactotetraosylceramide, nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) from
lactotriaosylceramide, LcOse3Cer (Glc NAc beta 1-3Gal beta 1-4Glc-Cer), and
UDP-galactose has been purified 107 500-fold from a
mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified
enzyme is partially stabilized in the presence of
phospholipid liposomes. Two closely migrating
protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after
sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse
GalT-4. These two
protein bands, when subjected to limited proteolysis, resulted in three
peptides with identical mobilities indicating amino acid sequence identity between the
proteins. Both
protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine
lactose synthetase (
UDP-Gal:Glc beta 4-
galactosyltransferase) following Western blot analysis on
nitrocellulose paper. The
enzyme has a pH optimum between 6.5 and 7.0 and like all other
galactosyltransferases,
GalT-4 has absolute requirements for divalent
cation (Mn2+). The K(m) values for the substrate LcOse3Cer and donor
UDP-galactose are 110 and 250 microM, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-
alpha 1-acid glycoprotein or
N-acetylglucosamine revealed that these reactions might be catalysed by the same
protein. The only other
glycolipid which showed acceptor activity toward the purified
GalT-4 was iLcOse5Cer (GlcNAc beta 1-1-3Gal beta 1-4Lc3), the precursor for
polylactosamine antigens. 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 values were closer to the theoretical values calculated for two
enzymes. Interestingly however, it seems that the
GalT-4 from P-1798 has an absolute requirement for an
N-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH2 beta 1-3Gal beta 1-4Glc-
sphingosine) of the acceptor
glycolipid LcOse3Cer is completely inactive as substrate while the K(m) and Vmax of the reacetylated substrate (GlcNAc beta 1-3Gal beta 1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798
GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic
glycolipid. The
monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified
enzyme from mouse P-1798 T-
lymphoma.