Purification and further characterization of enteropeptidase from porcine duodenum.
Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.
Matsushima
M
M
Department of Internal Medicine, Faculty of Medicine, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392, Japan.
Ichinose
M
M
Yahagi
N
N
Tsukada-Kato
S
S
Miki
K
K
Omata
M
M
Kim
Y T
YT
Ito
H
H
Takahashi
T
T
Sakurai
Y
Y
Tsuchiya
Y
Y
Athauda
S B
SB
Inoue
H
H
Takahashi
K
K
D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D004386:Duodenum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013552:Swine
D002241:Carbohydrates; D004765:Enteropeptidase