Proteolytic coefficient activity

Using four different peptides as substrates, Adams, et al.,21 found for 10 normal individuals the following ranges in activity (proteolytic coefficient x 104). 

Fig. 2. Effect of concentration of inhibitors on carboxypeptidase activity, following preincubation. Preincubation conditions Carboxypeptidase inhibitor, pH 7.5, 0°C., 60 minutes. Activity measurements 0.02 M CGP in veronal buffer containing 0.1 M NaCl, pH 7.5, 25°C. Activities were expressed as apparent proteolytic coefficients calculated from the strictly linear portions of first-order reaction plots.

The effects of increasing concentrations of 8-OHQ-5SA, OP, and aa D on the activity of carboxypeptidase at a constant substrate concentration of 0.02 M CGP are shown in Fig. 2. (Vallee and Neurath, 1955.) Activity of the inhibited reaction was expressed as per cent of the proteolytic coefficient observed at zero inhibitor concentration. The conditions of preincubation are indicated. Recent and unpublished data indicate the time course of the inhibitory effects of these agents OP in concentrations of 1 X 10" M causes 90 % inhibition of the reaction in 60 minutes. 80 % of the inhibition occurs in the first 15 minutes (Fig. 3), Addition of 1 X 10" M zinc ions to the enzyme thus inhibited restores enzymatic activity, demonstrating the reversibility of inhibition (unpublished results). Since inhibition did not occur when chelating agents were first incubated with zinc, cupric, or ferrous ions to form the respective metal chelate, it appeared that the sites of chelation of these compounds are responsible for the observed inhibition. Inhibition is therefore not caused by any structural similarity between the inhibitors and the substrate. 

The proteolytic coefficient is largest for compounds with N-terminal leucine and norleucine, but several other amino acids may be attacked at sufficient rates to make this enz3rme a generally useful reagent. Its activity is not restricted to small peptides, and it has been used in the stepwise degradation of proteins from the amino end, in the same manner that carboxypeptidase has been used in the analysis of the carboxyl ends of protein chains. 

Fig. 10. Effect of pH on the activation of crystalline paptun as measured by the proteolytic coefficient (Ci) for the hydrolysis of bmiaoyl>L>argininamide (153). Activators were used singly and in various combinations. Cys is cys teine and V is Versene, all used at 0.005 Af. The buffers were used at 0.02 M concentration acetate near pH 5, citrate at pH 6, phosphate at pH 7, and Tris near pH 8. Cysteine plus Versene gave the highest activity at all pH ralues and the Cl values were independent of the buffer used.

Mastitis is well known to decrease lactose content. This fact explains the relation between lactose content and determination of log SCC (4). This emphasizes the possibilities of detecting changes with lactose when analyzing milk spectra and proves its strong relation with SCC. Factors 5, 6, 8 and 10, which showed high correlation with regression coefficients, had the highest correlation with protein content. This is consistent with the fact that mastitis causes alteration of protein fractions in milk. Mastitic milk has more proteolytic activity than normal milk, due to increase of proteinase plasmin, which hydrolyzes the casein (25, 26). Harmon (6) and Urech et al. (25), have reported decreased ccs-casein and (3-casein content and elevated whey proteins and y-casein in the total protein of mastitic milk. 

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