Purification and characterization of turtle pepsinogen and pepsin.
Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trionyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl-L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymosin.
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo.
A
A
Hirasawa
S B
SB
Athauda
K
K
Takahashi
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002845:Chromatography; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005753:Gastric Mucosa / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010435:Pepsinogens / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014426:Turtles / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D010435:Pepsinogens; D010434:Pepsin A