Squires, E. James (1984) Isolation and characterization of gastric proteases from the Greenland cod (Gadus ogac). Doctoral (PhD) thesis, Memorial University of Newfoundland.
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The Greenland cod Gadis ogac is a sub-arctic species that thrives year round in the North Atlantic. It is postulated that the gastric proteases of this fish have several properties in common with the gastric proteases from mammalian species but also have several characteristics unique to fish species. Therefore, the gastric proteases and their zymogens were isolated from the stomach mucosa of the Greenland cod Gadus ogac and their properties compared to mammalian gastric proteases and the gastric proteases of other fish. Attempts were also made to purify porcine pepsin A and porcine gastricsin from a crude commercial pepsin preparation. The properties of the purified gastricsin fraction obtained differed substantially from the literature data on porcine gastricsin so that direct comparisons of many of the properties of gastricsin and the cod proteases could not be made. -- The zymogens of three gastric proteases were separated and purified by Sephadex G100 chromatography at pH 7, chromatofocusing and, after activation of the zymogens, Sephadex G75 chromatography at pH 2.5. The zymogens of protease 1, 2, and 3 had isoelectric points of > 7.5, 6.2 and 5.2 respectively. The zymogens of the Greenland cod gastric proteases were activated much more rapidly at low temperature than porcine pepsinogen. All three of the cod proteases had more alkaline pH optima with protein substrates than porcine pepsin, especially with methylated protein substrates. The pH optima of cod protease 2 and 3 and porcine pepsin with peptide substrates were all near pH 2 while the pH optimum of cod protease 1 with APDT was near pH 3. The specific activities of the individual cod proteases at 26 °C with protein substrates were generally lower than porcine pepsin. However, a mixture of the cod proteases had activity with the protein substrates that was greater than the sum of the activities of the individual proteases. Cod protease 2 and 3 were active on a number of peptide substrates that are good substrates for gastricsin while cod protease 1 was active only with APDT (N-acetyl-phenylalanine-diiodotyrosine) of all the peptide substrates investigated. The milk clotting activities of the cod proteases were much greater than that of porcine pepsin and the cod proteases had CU/PU ratios (the ratio of the clotting activity to hydrolytic activity with hemoglobin) that were 20-50 times higher than porcine pepsin. The individual cod proteases hydrolysed hemoglobin to a greater extent than porcine pepsin, indicating their wider substrate specificity. Porcine pepsin had a high Vmax and low Km' compared to cod protease 1 which had low to moderate Vmax and Km' with all substrates. Cod protease 2 had comparatively high Vmax and Km' with hemoglobin as substrate and moderate to low Vmax and Km’ with the other substrates. Cod protease 3 had comparatively low to moderate Vmax and Km' with hemoglobin and casein, high Vmax and Km' with methylated hemoglobin and low Vmax and high Km' with the peptide substrates. No significant differences were found in the activation energy for the hydrolysis of the various substrates by the different protease preparations. -- The cod proteases were less stable to heating and retained less activity at extremes of pH (less than 3 and greater than 6.5) than porcine pepsin. The activities of cod protease 1 and 2 with hemoglobin as the substrate were doubled in the presence of 25 mM NaCI while cod protease 3 and porcine pepsin were not stimulated by salt. The cod proteases did not cross react with antibodies raised against porcine pepsin. Antibodies were also obtained against the purified proteases T16 and T25 from psychrotrophic pseudomonads. Anti-T16 IgG precipitated cod protease 1 and porcine pepsin at similar IgG/enzyme ratios but did not affect cod proteases 2 and 3. Anti-T25 IgG precipitated all the proteases, cod protease 2 and porcine pepsin being precipitated at similar IgG/enzyme ratios. The subunit molecular weights of all the proteases were in the range of 35-37 kdal as estimated by SDS-PAGE and amino acid composition. The amino acid compositions of the cod proteases differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin and chymosin differ from each other. Cod protease 1 was the most different of the cod proteases from the mammalian proteases, while cod protease 3 was more like chymosin. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod protease 2 and 3 were intermediate between that of porcine pepsin A and chymosin.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Bibliography: leaves 160-171.|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Codfish; Digestion; Proteolytic enzymes|
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