Two specific
N-acetylglucosaminyltransferases, alpha-1,3-
mannoside:beta-2-
N-acetylglucosaminyltransferase (
transferase I) and alpha-1,6-
mannoside:beta-2-
N-acetylglucosaminyltransferase (
transferase II), which catalyze the transfer of
N-acetylglucosamine (GlcNAc) from
uridine diphospho-GlcNAc to terminal branched alpha-mannosyl (Man) residues, were purified from liver
metastases of human
colon adenocarcinoma.
Transferase I was assayed with Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc beta 1, 4GlcNAc-Asn (Km 0.35 mM), and
transferase II was assayed with Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1,4GlcNAc beta 1,4-Glc-NAc-Asn (Km 1.0 mM), in which Asn is
asparagine. The Km of
transferase I for Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc-beta 1,4)-(Fuc alpha 1,6)GlcNAc-Asn was 1 mM. The specificity of the interaction of
transferase I with
ovalbumin,
ovomucoid, the modified heavy chain of porcine
immunoglobulin G and
glycopeptides prepared from these
glycoproteins was examined by kinetic and structural analysis. The best macromolecular substrates for
transferase I were
ovalbumin devoid of terminal GlcNAc and some
mannose, a solubilized preparation of the heavy chain of porcine
immunoglobulin G, devoid of
sialic acid,
galactose, and terminal GlcNAc, and untreated
ovomucoid. The apparent KmS were 45, 19, and 390 microM for
ovalbumin, the modified heavy chain of
immunoglobulin G, and untreated
ovomucoid, respectively. The apparent Km of the
enzyme for
uridine diphospho-GlcNAc was not significantly influenced by the nature of the
glycoprotein acceptor, and it varied between 14 and 20 microM for the different
glycoproteins. The structures of the
oligosaccharide chains in these
glycoproteins which acted as acceptors for the purified
enzyme were determined. A major
glycopeptide product with the structure Man alpha 1,3(Man alpha 1,6)Man alpha 1,6(14C-GlcNAc beta 1,2Man-alpha 1,3)Man beta 1,4GlcNAc-beta 1,4-GlcNAc-Asn was isolated from both
ovalbumin and
ovomucoid following incubation with
transferase I. The specificity of the
enzyme for terminal branched mannosyl residues attached to a beta-linked
mannose unit greatly restricts the action of this
transferase to this juncture in the synthesis of complex-type
oligosaccharide chains of N-
asparagine-linked
glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)