Open AccessPurification and Partial Amino Acid Sequence of Fuctinin, an Endogenous Inhibitor of Fucosyltransferase Activities
Author(s) -
RuggieroLopez Daniel,
Manioc Claudine,
Geourjon Christophe,
Louisot Pierre,
Martin Ambroise
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb19993.x
Subject(s) - biochemistry , gel electrophoresis , isoelectric point , peptide sequence , biology , glycosylation , peptide , molecular mass , isoelectric focusing , amino acid , microbiology and biotechnology , chemistry , enzyme , gene
A powerful endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, was purified to homogeneity from rat small‐intestinal mucosa. The purification scheme involved DEAE–cellulose ion‐exchange chromatography, ammonium sulfate fractionation, hexyl–agarose hydrophobic chromatography and size‐exclusion HPLC. Active native fuctinin has an isoelectric point of 4.55 and apparent molecular mass approximately 66 kDa, whereas a single protein band with a molecular mass of approximately 24 kDa was obtained by denaturing polyacrylamide gel electrophoresis, suggesting that fuctinin is an oligomeric protein. Two‐dimensional polyacrylamide gel electrophoresis displayed eight spots in this single band. Comparisons of the N‐terminal amino acid sequences of each spot support the idea of the existence of three related polypeptides and suggest a proteolytic N‐terminal cleavage despite the use of an efficient protease inhibitor throughout the purification. In spite of the presence of an N‐glycosylation site, fuctinin is not glycosylated. One of the three polypeptides, peptide 3, possesses two consensus sequences for phosphorylation and a consensus sequence for myristoylation. The sequences of fuctinin‐related peptides, especially peptide 3, exhibit high similarity to the N‐terminal domain of the Set protein and a putative human leukocyte antigen‐associated protein. The possible implications of these results are discussed.