Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus).
Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogeneous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS-polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2-3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.
D000375:Aging; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D010872:Caniformia / Q000378:metabolism*; D012085:Chymosin; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D046048:Seals, Earless / Q000378:metabolism*; D013270:Stomach / Q000254:growth & development; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D007527:Isoenzymes; D014508:Urea; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D012085:Chymosin