Purification and partial amino acid sequence of fuctinin, an endogenous inhibitor of fucosyltransferase activities.
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 functinin-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.
Ruggiero-Lopez
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D
Department of Biochemistry, Institut National de la Santé et de la Recherche Médicale, Oullins, France.
Manioc
C
C
Geourjon
C
C
Louisot
P
P
Martin
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors*; D006031:Glycosylation; D007413:Intestinal Mucosa / Q000737:chemistry*; D007421:Intestine, Small / Q000737:chemistry*; D007526:Isoelectric Point; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D011506:Proteins; C089262:fuctinin; D005647:Fucosyltransferases