It has been shown by use of isolated, perfused rat kidneys that hypertensin II is a potent vasoconstrictor substance while hypertensin I is not. Hence it would appear that in intact animals the pressor activity of hypertensin I results from its rapid conversion to hypertensin II. An enzyme which effects this conversion has been procured from horse plasma in a semipurified form by means of ammonium sulfate fractionation and isoelectric precipitation. A method is described for estimating the activity of the enzyme. An example of the use of the preparation in converting purified hypertensin I to hypertensin II has been described.

The enzyme renin, which is found in extracts of kidney cortex, acts upon a protein substrate contained in the alpha-2 globulin fraction of the plasma (1) to produce the decapeptide hypertensin I. This peptide is further degraded by a plasma enzyme (2) to the powerfully vasoconstrictor octapeptide hypertensin II which appears to be the effector substance of the renin-hypertensin pressor system (3, 4).Since hypertensin has been found in the blood of many human beings with hypertensive cardiovascular disease (5) as well as in animals with experimental renal hypertension (6, 7) it is of great interest to discover a method of preventing its action in vivo. Owing to the recent purification and structural delineation of both hypertensin I and hypertensin II (8-12) this problem can now be rationally approached. Three separate methods appear feasible. First, it might be possible to prepare structural analogs of hypertensin II capable of interfering with the vasoconstrictor reaction. Second, the conversion of hypertensin I to hypertensin II might be prevented by compounds inhibiting the hypertensinconverting enzyme. Finally, the production of hypertensin I from renin substrate might be prevented by the inhibition of renin. Since renin is the initial and rate-limiting substance in the renin-hypertensin system it would seem that this last approach would be the most likely to succeed. This view is re-enforced by the observation that immunization with heterologous renin has been used successfully in the treatment of dogs with experimental renal hypertension (13).In order to prepare compounds capable of inhibiting renin it is necessary that the structure of its substrate be known. Inasmuch as renin substrate is a protein which has been only partially purified (14) the full determination of its structure is difficult. It appeared unlikely that the entire renin substrate molecule would be required for the action of renin since the specificity of the common proteolytic enzymes is based on one or at most two amino acid residues. An attempt was therefore made to partially degrade the renin substrate by the action of the enzyme trypsin. This effort has proven successful and has yielded a polypeptide renin substrate amenable to purification and structural determination.439

The pressor substance hypertensin, which is the product of the action of the renal enzyme renin upon its plasma substrate, has been shown to be present in the arterial blood of many patients with hypertensive cardiovascular disease (1) as well as in the arterial blood of animals with experimental renal hypertension (2--4). Hypertensin may, therefore, be concerned in causing the elevation of blood pressure in both of these conditions. In the course of attempts to purify this material it was found that certain preparations, when examined by means of the countercurrent distribution method, revealed not one but two pressor substances. Further study of this completely unexpected finding showed that the initial pressor substance, hypertensin I, formed as a result of the action of renin on its substrate, is quickly converted to a second substance with approximately equal pressor activity, hypertensin II, apparently by an enzyme in plasma, which is activated by chloride ion. I t is the purpose of this paper to describe the preparation of hypertensin I from suitable preparations of renin and substrate, to demonstrate its conversion to hypertensin I I by the action of the plasma enzyme, and to determine which ions are effective in activating this enzyme. MetkodsPreparation of Renin.--The renin was prepared from fresh hog kidneys according to the method described by Katz and Goldblatt (5) and modified by Dexter (6). Tiffs preparation contained a considerable amount of hypertensinase activity which was eliminated (reference 7, page 101) in the following fashion. Renin, prepared from 100 pounds of hog kidneys by ammonium sulfate precipitation, was dissolved in 1.5 liters of water. The solution was chilled to 0-5°C. and sufficient solid sodium chloride was added to insure complete saturation. 0.1 N HCI, saturated with NaC1, wa~nthen dripped into the solution slowly and with constant stirring until the pH dropped to 2.0. The solution was centrifuged at high speed at 0-5°C. The supernatant containing the hypertensinase was decanted and discarded. The precipitate was dissolved in 1 liter of cold 0.1 ~ Na,I-IPO4. The light brown solution was adjusted to pH 7.5, put into small diameter cellophane bags, and dialyzed exhaustively against cold distilled water. The resulting product, with a volume of about 1.25 liters, was found to be uniformly free of hypertensinase. Usually 1 volume of this enzyme preparation was adequate to catalyze the formation of hypertensin in 25 volumes of the substrate preparation described below in a period of 30 to 45 minutes when this mixture was incubated at 37°C. and pH 7.5.Preparation of Substrate.--Horse blood was collected in ~0 volume of 4 per cent sodium

Two dipeptides, phenylalanylarginine (Phe-Arg) and serylproline (Ser-Pro), are released sequentially from bradykinin by angiotensin-converting enzyme purified from hog lungs; chloride increases the rate of release of both dipeptides. Using an automated ninhydrin-reagent method, we studied the kinetics of bradykinin hydrolysis. The reaction proceeded in the absence of chloride; however, the addition of chloride increased the rate of hydrolysis by decreasing K m and increasing V m . The K m values for bradykinin were 3.9 x 10~6M in the absence of chloride and 0.85 x 10~6M in the presence of 0.01M NaCl (optimal concentration). Both of these K m values were well below the value of 30 x 10" 6 M determined for angiotensin I at its optimal chloride concentration of 0.1M. Hydrolysis of bradykinin had a pH optimum of 7 and was inhibited by low concentrations (10~6M) of ethylenediaminetetraacetic acid or the nonapeptide pyroglutamyl (Pyr)-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro. It is concluded that one enzyme, acting as a dipeptidyl carboxypeptidase, catalyzes both the conversion of angiotensin I to angiotensin II and the hydrolysis of bradykinin. KEY WORDSphenylalanylarginine enzyme kinetics dipeptidyl carboxypeptidase chloride activation serylproline hog lung Bothrops jararaca nonapeptide