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 th...
| Published in: | Canadian Journal of Biochemistry and Cell Biology |
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| Main Authors: | , |
| Format: | Article in Journal/Newspaper |
| Language: | English |
| Published: |
Canadian Science Publishing
1984
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| Subjects: | |
| Online Access: | http://dx.doi.org/10.1139/o84-091 http://www.nrcresearchpress.com/doi/pdf/10.1139/o84-091 |
| Summary: | 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 heterogenous 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. |
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