title: Pepsinogens and pepsins from largemouth bass, Micropterus salmoides: purification and characterization with special reference to high proteolytic activities of bass enzymes.
abstract: Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin.
authors.affiliation: Division of Biomolecular Science, Graduate School of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan; Department of Health and Nutrition, Nagoya Bunri University, Inazawa 492-8213, Japan. Electronic address: y.miura@nsc.nagoya-cu.ac.jp.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Miura
authors.affiliation: Department of Health and Nutrition, Nagoya Bunri University, Inazawa 492-8213, Japan. Electronic address: kageyama.takashi@nagoya-bunri.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kageyama
authors.affiliation: Division of Biomolecular Science, Graduate School of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan. Electronic address: moriyama@nsc.nagoya-cu.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Moriyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001492:Bass / Q000235:genetics*; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry* / Q000302:isolation & purification; D010435:Pepsinogens / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D013270:Stomach / Q000737:chemistry / Q000201:enzymology*
chemical_list: D010435:Pepsinogens; D010434:Pepsin A
keywords: Aspartic proteinase; Gastric digestion; Largemouth bass; Pepsin; Pepsinogen