[Effect of the degree of phosphorylation of bovine pepsin A on its enzymatic activity].

Proteolytic and clotting activities of bovine pepsin A with respect to its degree of phosphorylation were studied on various substrates. The occurrence of phosphate group(s) on bovine pepsin A more or less strongly affects its enzymic properties according to the substrate and its environment. This is particularly obvious as far as kappa-casein is concerned. The specific flocculating activity of unphosphorylated (fA0) as well as dephosphorylated (treated with potato acid phosphatase) bovine pepsin A, determined on a 0.2% kappa-casein solution, is significantly higher than that observed with phosphorylated pepsins, especially after kappa-casein was treated with alpha-D.N-acetyl galactosaminyl oligosaccharidase, while specific milk clotting activity remains unchanged regardless to the level of phosphorylation of bovine pepsin A is. Using haemoglobin as substrate, unphosphorylated pepsin A exhibits the highest specific proteolytic activity and the less acidic pH optimum. Conversely, the amount of phosphate groups does not seem to have any effect on the peptidase activity assayed towards the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe. By treating whole bovine pepsin A with potato acid phosphatase during 24 h at 37 degrees C and pH 5.6, using a 1/100 E/S ratio, almost complete dephosphorylation can be reached. The stability of different bovine pepsin A preparations, more or less phosphorylated, treated or not with phosphatase was also investigated. At pH 2.2, phosphorylated bovine pepsin A is twice more stable at 37 degrees C than the dephosphorylated enzymes while dephosphorylated pepsin does not exhibit any degradation at pH 5.6, judging by isoelectric focusing patterns, or loss of activity. Such a result suggests that post-translational phosphorylation might play an essential physiological function by improving the stability and integrity of pepsin in the bovine abomasum, the pH of which is very acidic (between 1.0 and 2.0).