A Kinetic Analysis of the Renin-Angiotonin Pressor System and the Standardization of the Enzymes Renin and Angiotonase

Abstract

Sufficient is now known of the renal vasopressor system and its relationship to the mechanism of hypertension to justify a more exacting analysis of it. Shortly after the discovery of angiotonin it was noted that when renin and plasma were allowed to interact, angiotonin at first increased and then slowly began to decrease (3). We had no evidence at that time as to the nature of the decrease, and it was merely suggested that angiotonin was an intermediate rather than an end product in these closely associated reactions. This has proved to be true, and the catalyst causing the further breakdown was shown by Fasciolo, Leloir, and Mufioz (20,4) to be an enzyme denominated hypertensinase by them. In line with the nomenclature employed by us (22, 23) this becomes "angiotonase."Good evidence had suggested that the reaction between renin and reninsubstrate is enzymatic, but proof that this is so came from a recent kinetic analysis of the problem demonstrating the reaction to be of the first order (1). Since both physiological and chemical investigation of renin depends on the ability to assay it quantitatively, we have sought such a method.The methods of bioassay of renin have recently been summarized by Goldblatt, Katz, Lewis, and Richardson (11) in a paper in which they described a method of assaying renin which does not differ appreciably from some previously reported. They overlooked an important contribution by Leloir et al. (5) in which there is proposed a renin unit based upon the amount of pressor substance (anagiotonin) formed. The method of Goldblatt, Katz, Lewis, and Richardson and previous methods referred to by them, are based upon the determination of the rise in arterial pressure elicited by intravenous injection of given amounts of renin solution. Since renin itself is not a pressor substance, it should not be tested for its pressor but for its enzymatic action; that is, in terms of renin-substrate decomposed or angiotonin formed. Similar arguments apply to destruction of angiotonin by angiotonase. A satisfactory method for the assay of angiotonase has been described by Page and Helmer (21).Since kidney extracts usually contain both renin and angiotonase the assay of renin becomes difficult because angiotonase interferes with the determination

The Renal Humoral Pressor Mechanism in Man. I. Preparation and Assay of Human Renin, Human Hypertensinogen, and Hypertensin 12

“…Hg (19) (Figure 1). We have never observed the linear relationship described by some workers (23) 3: 0.03 ml. (0.5 cat unit) of hypertensinase-free human renin incubated with hypertensinase-free beef plasma as in tube 1 without preliminary treatment with salt, low pH, and dialysis, i.e., by the direct method of Leloir and co-workers (2), using hypertensinase-free solutions.…”

The Action of Crystalline Proteolytic Enzymes on Angiotonin

“…I Hence the course of the hydrolysis was followed by bioassay of angiotonin using a pithed cat as test animal. First order constants were determined as described for the assay of angiotonase (16). Dividing this constant by the concentration of the enzyme expressed in milligrams proteins nitrogen per cubic centimeter test solution, gave the proteolytic coefficient CA.…”

Isolation of a Phospholipid Renin Inhibitor from Kidney*

“…The entire procedure, except where noted, was carried out at 3-4°. Dog renin was prepared using a modification of the procedure of Plentl and Page (1943) followed by ammonium sulfate fractionation. An acetone powder was prepared by mincing 1710 g of dog kidney with 3.8 1. of acetone.…”