Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences.
Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005075:Biological Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005399:Fishes / Q000378:metabolism*; D005753:Gastric Mucosa / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000302:isolation & purification / Q000378:metabolism*; D010435:Pepsinogens / Q000302:isolation & purification / Q000378:metabolism*; D014413:Tuna / Q000378:metabolism*; D014426:Turtles
D000596:Amino Acids; D010435:Pepsinogens; D010434:Pepsin A