In 1940, a method for determination of retin from its enzymatic activity was published by Leloier et al. Thereafter, especially since 1963, several improvements for the indirrect assay of renin were made by many investigators. However, we frequently noted disagreement among the results obtained by these methods. In this paper, we described experimental results concerning the reliability of the rat bioassay for determination of angiotensin (AT) content as well as purification of renin from hog kidneys, properties and inhibition of angiotensinase (ATase) activity, and some properties of renin activity.
Assay of AT was done by comparison of pressor response samples with standard AT(Hypertensin Ciba) solution when these solutions were injected to anesthetized rats intravenously. By this method, variance of the assay was ±7.2ng/ml (S. D.) when synthetic AT II were injected at a dose of 100ng/ml, ±4.7ng/ml at 50ng/ml and ±2.0ng/ml at 10ng/ml. The detectable lower limit was sometimes 1ng/ml, but usually 2-3ng/ml.
We used the renin solution extracted from hog kidneys by the method of Haas et al. (Arch. Biochem. Biophys. 1965). At a pH of 7.5, we could not detect ATase activity in this solution. At pH=5.0, though the activity was detected to a certain degree, addition of diisopropylfluorophosphate (DFP) decreased the activity considerably.
Destruction rate of synthetic AT II by ATase was very rapid early in the incubation. But it became slow with time. It seemed to us that the majer cause was reduction of AT content momentarily in the incubation mixture. ATase activity in plasma did not change obviously at the condition of pH 7.4, 37°C, for 90min. The AT destruction reaction seemed to be of the first order, when AT concentration was varied from 0.05-1μg/ml and the incubation carried on for a certain constant time. But successive change of AT concentration of each sample did not coinside with the first order reaction. We thought it was possible that the reaction had been prevented by a certain factor in plasma. The percentage of AT destraction increased with the rise of ATase content in incubation mixture, but the rate became disproportionate to the amount of ATase, when it was over a certain amount.
The largest ATase activity in plasma was shown near neutralty, and the activity was diminished considerably by acid treatment. Addition of ethylonediaminetetraacetate (EDTA) and DFP, at concentrations of 0.003 and 0.001mol/l respectively, inhibited the activity almost completely at a pH less than 5.5.
In our experimental condition, hog renin activity was the largest at pH=6. Although, in the initial stage of incubation, produced AT was proportional to the added amount of renin, the prolonged incubation lead to breaking such a relation. In order to complete that relation, the amount of AT produced should be less than 10 percent of angiotensinogen content. For this reason, it is the authors' opinion that the incubation to determine renin activity in plasma should be performedinapproximately 2 hours.
