Trypsin activation volumes

Figure 4 shows the temperature profiles for amidase activity of the protease from M. jannaschii at 10,250, and 500 atm. The optimum temperature for activity of 116° at low pressure (10 atm) is one of the highest optimum temperatures for a proteolytic enzyme reported in the literature. Figure 4 also shows that pressure substantially enhances the activity of the protease at each temperature for example, application of 500 atm increases the maximum reaction rate about twofold and the rate at 130°C fivefold. The 400% enhancement of amidase activity at 130° translates to an overall activation volume, A V, of — 106 ml mol , as determined by the Johnson-Eyring equation [Eq. (2)]. The most negative activation volumes previously reported for serine proteases were —36 ml mol for the digestion of casein by trypsin and —33 ml mol for the hydrolysis of Suc-Ala-Ala-pNA by a-chymotrypsin. Michels and Clark also found that the protease is stabilized... 

Total volume of pancreatic juice, amount or concentration of bicarbonate, and activities of pancreatic enzymes are measured in duodenal contents. The enzyme most commonly measured is trypsin, but amylase, lipase, chy-motrypsin, and elastase may also be evaluated. The Lundh test consists of administering a standardized meal consisting of 5% protein, 6% fat, 15% carbohydrate, and 74% nonnutrient fiber. Advantages of the Lundh meal are that it provides a physiological stimulus to the pancreas and is simple to administer. However, administration of the meal prevents determination of the total enzyme and bicarbonate or secretory volume. Moreover, it provides inadequate or no stimu-... 

X 10 moles trypsin per liter fluid volume. To demonstrate the feasibility of using the Ford method to determine the active-site of our immobilized enzyme systems, trypsin CVB-PHEMA-PABS-carbamate was treated in a circulation reactor with NPGB and the titration is Illustrated in Figure 4. The amount of p-nitro-phenol produced by the burst is equal to the amount of the active immobilized trypsin which, for this particular system, turns out to be 31% of the total bound enzyme. Active-site titrations of soluble trypsin were performed according to Chase and Shaw (16), and the active molecules for free trypsin was found to be 70% of the total protein involved. Consequently, the retention of active molecules for the immobilized enzyme was calculated 45%. The specific activity is 17% (Table III) for the same system so the efficiency of the system, based on the actually available active sites, was 38%. Thus, 62% of the initially active trypsin bound has lost its activity upon binding. 

Herbicide Binding Assays. Control and trypsin-treated chloroplast thylakoids were suspended in PSNM buffer. Buffer (1 ml volume) containing 50 Chi was incubated 3 min with l C-atrazine (specific activity 27.2 pCi/mg). Chloroplasts were pelleted and an aliquot of the supernatant was removed for determination of the amount of unbound atrazine. Details of this procedure are described elsewhere (6, 9). Radiolabeled atrazine was a gift of Dr. H. LeBaron, CTBA-GEIGY, N. Carolina. 

Russet Burbank potatoes were quartered and placed in a water bath at 80 C for 10 min. The tubers were rinsed with cold water and blended to a paste. Absolute ethanol was blended in slowly with the paste until it was 80% ethanol by volume. The mixture was filtered through Whatman No. 1 filter paper with the aid of a vacuum. The ethanol in the filtrate was removed by vacuum evaporation and the resulting solution was dialyzed in a 2,000 MW cut off dialysis bag against several changes of distilled water. The remaining extract in the dialysis bag was lyophilized. The dry material contained one major polypeptide, CPI, and some minor components. On a protein basis the material was over 95% CPI. Purity of this crude CPI fraction was also demonstrated by electrophoresis by the method of Panyim and Chalkley (8). No Inhibitor I nor Inhibitor II could be detected by immunological techniques, however a small quantity of trypsin inhibitor activity (less than 0.5%) was present, presumably from contamination by the polypeptide trypsin inhibitor that is present in potato tubers (4). This CPI preparation was utilized for the chick feeding experiments. 

Fig. 1. Time-course study of the stimulation of phospholipase activity in the 100,000 g supernatant fraction from potato leaves. Control (0) = 1 ml2 00,000 g supernatant fraction containing 3 mM Mg and 0.3 mM ATP, control plus 50 mM NaF (A), control plus 50 mM NaF and the catalytic subunit of cyclic AMP-depen-dent protein kinase (500 u) ( ), control plus 50 mM NaF and calmodulin (10,000 u) (X), control plus 25 grams trypsin ( ). All reported concentrations are those which occur in the final volume of 1.1 ml.

---