Hypertensinase

Braun-Menendez and co-workers (22) observed that the amount of hypertensin produced when serum globulins were incubated with kidney extracts reached a maximum after about ten to fifteen minutes. When the incubation was continued beyond this period, a gradual disappearance of the pressor principle was noticed (Fig. 2). Page and Helmer (128), who made similar observations, stated that renin destroys hypertensin. The Argentine workers, however, aware of the fact that kidney extracts contain other enzymes beside renin, showed that not renin but a new enzyme. 

Hypertensinase occurs in a large variety of tissues. Its distribution in various organs was studied by Fasciolo and co-workers (44) and is sum- 

By the inactivation of hypertensinase in renin substrate and renin solutions before they were mixed it thus became possible to obtain maximal yields of hypertensin unaffected by prolonged incubation time. An alternative way to prevent the action of hypertensinase in mixtures of renin and renin substrate was described by Sapirstein, Reed, and Southard (154). It was found that the action of hypertensinase is practically arrested if the incubation experiments are carried out at 0°C. while renin continues to form hypertensin, though at a diminislied rate. 

Little is known about the chemical nature of hypertensinase. The enzyme was not precipitated by dialysis. In fractionation experiments it was found in the fraction precipitated between 30 and 60% saturation with ammonium sulfate. In a comparative study of hypertensinase and proteinase activity of blood plasma (30,31), there was found in various plasma fractions a parallel between hypertensinase activity and hydrolyzing effect on Z-leucylglycine. These findings were interpreted as supporting evidence for the hypothesis that plasma hypertensinase active at pH 7.3 to 7.8 is an aminopeptidase. 

Helmer and co-workers (81), who compared hypertensinase preparations from muscle, liver, and intestinal mucosa with that contained in kidney extracts, found that the greatest activity in kidney preparations was reached at pH 4 while the other extracts showed maximum activity at pH 7. Exposure of kidney extracts to pH 3.5 to 3.9 inactivated only the hypertensinase which had an optimal activity at pH 7 but did not destroy the acid-active hypertensinase (80,81). The existence of an acid-resistant hypertensinase in kidney extracts was also demonstrated by Schales, Stead, and Warren (158), who found 10% of the original hypertensinase activity left (tested at pH 7.4), after incubating extracts for nine hours at 37°C. and pH 3.7. These findings restrict to some extent the statement already made that no loss of hypertensin occurs if kidney extracts and renin substrate are exposed to prolonged incubation after exposure to pH 3.9 for a short period. 

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Under the proper stimulus renin is released into the circulating blood where it can be identified, especially in that from the renal vein. It acts rapidly upon its specific substrate, splitting the protein into peptides, one or more of which have been called hypertensin or angiotonin. Hypertensin has been concentrated but not obtained in pure form. It is the effector substance of renin, constricting arterioles and raising blood pressure. The action of hypertensin is abolished by hypertensinase, an enzyme found in blood and renal extracts. Fortunately, the latter is destroyed by heat and alkalinity. 

Hypertensin is soluble in alcohol, glacial acetic acid, phenol, and water, and insoluble in ether (61). Because it is inactivated by tyrosinase it probably contains a catechol or phenol group, and by amine oxidase, an amine group on an a-carbon atom (Figure 2). Hypertensin is inactivated by certain phenolic, catecholic, and amine oxidases, by pepsin, trypsin, chymotrypsin, and carboxypeptidase, and by hypertensinase found in plasma. The nature of hypertensinase is unknown, but it is probably not an oxidative enzyme. Because it is heat-labile, hypertensinase can be removed from blood and renin preparations by heating hypertensin itself is heat-stable. Lack of pure preparations of hypertensin has delayed its further chemical identification. 

Principally because amine oxidase was so abundantly present in the body and could be shown to deaminate certain pressor amines in vitro, it has been thought by some to play a role in the in vivo inactivation of adrenaline and in the etiology of hypertension. The administration of crude amine oxidase has been reported to have decreased the blood pressure of normal and hypertensive rats (143), but this has not provoked substantiation. Croxatto and Croxatto (53, 54) have shown that renal hypertensinase and amine oxidase were different enzymes. Also, Bing, Zucker, and Perkins (34) found angiotonin and amine oxidase to be fundamentally different. Thus the hypertensin and the phenylethyl (pressor) amine approaches to hypertension seem quite dissimilar on these grounds. 

Numerous enzymes also destroy the hypertensive action, such as tyrosinase, intestinal aminopolypeptidases. Plentl and Page (471) drew various conclusions concerning the structure of the peptide, from data on the controlled hydrolysis by four crystalline enzymes, namely pepsin, chymotrypsin, trypsin, carboxypeptidase, all of which destroy the physiological activity. Thrombin (149) and thromboplastin (150), but not prothrombin, also destroy the activity. An enzyme present in plasma, hypertensinase, has been described which destroys the hypertensin action in the animal. This enzyme acts under anaerobic as well as aerobic conditions, and the presence of octyl alcohol and cyanide do not affect its behavior its pH optimum is between 7.8 and 8.5. Hypertensinase is also present in red blood cells, with a pH optimum differing from the plasma enzyme. These enzymes, whose specificity and identity are not yet well established, are probably aminopolypeptidases (151). 

A. Treatment with Renal Extracts Containing Hypertensinase. 538... 

Fig. 1. pH-activity curve of renin and hypertensinase (117). Each curve corresponds to one experiment. 

One unit of hypertensinase was defined as the amount which destroys 0.5 unit of hsrpertensin in 4 hours at 37 °C. and pH 7.4 with a total volume of 10 ml. and an initial amount of 1 unit of hypertensin present (44). One unit of hypertensin was defined as the amount which gives a blood pressure increase of 20-30 mm. Hg in a 10-kg. chloral-injected dog (22,117). 

The hypertensinase with an optimal activity at pH 4 was destroyed when the acidity was increased to pH 2.0 but under these conditions large losses of renin were observed (137). The al)ility of hypertensinase from red cells to destroy hypertensin in vivo is illustrated in Figure 3. Sapirstein, Reed, and Page (153) hemolyzed about 2% of the circulating erythrocytes in dog and noted a definite decrease in magnitude and duration of the... 

Friedman (47) found less hypertensinase in the renal vein plasma of acutely hypertensive dogs than in the plasma of normal dogs. Later inves-... 

A. TREATMENT WITH RENAL EXTRACTS CONTAINING HYPERTENSINASE... 

Fig. 4. Effect of intramuscular injection of kidney extract on the blood pressure and body temperature of a 33-year-old patient with malignant hypertension (158). White squares unmodified renal extract given. Black squares hypertensinase-poor extract given. The local reactions are graded from 1-t- to 4-t-, depending on their severity. R indicates a shock-like reaction.

Schales, Stead, and Warren (158) confirmed the blood-pressure-lowering effect of certain kidney extracts on hypertensive patients and found considerable hypertensinase activity in their preparations. The daily dose of extract (10 ml.) was obtained from 750 to 1000 g. of hog kidney. It contained an amount of hypertensinase capable of destroying at pH 7.4 in two hours as much hypertensin as is obtainable from 10 to 30 liters of beef serum, using an excess of renin under optimal conditions. These authors observed,... 

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