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

No abstract

Angiotensin-converting enzyme was purified 1500-fold from a homogenate of hog lungs. The purification procedure included fractionation with ammonium sulfate, inactivation of contaminating enzymes at pH 4.7, batch treatment with CM-cellulose, and chromatography on columns of DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was assayed with hippurylglycylglycine and, after pH 4.7 was established, with both hippurylglycylglycine and angiotensin I as substrates. The ratio of activity toward the two substrates remained constant throughout the later stages of purification. The final enzyme preparation, on analytical disc gel electrophoresis, showed one major protein band with the expected ratio of activities toward both substrates. The molecular weight was estimated to be approximately 300,000 by gel filtration. The pH optimums with hippurylglycylglycine and angiotensin I as substrates were 8.4 and 7.5, respectively, and chloride ion was required for optimal activity of the enzyme with both substrates. Hydrolysis of both substrates was inhibited by ethylenediaminetetraacetic acid (EDTA) or the pentapeptide pyroglutamyllysyltryptophanylalanylproline. It would, therefore, appear that a single enzyme, purified in the described manner, hydrolyzes both angiotensin I and hippurylglycylglycine.

A new enzyme, pseudorenin, has been discovered which resembles renin in its ability to form angiotensin I from the synthetic tetradecapeptide renin substrate and from purified hog renin substrate A. Its maximal activity occurs at a much lower pH value than does that of renin. The two enzymes may be easily separated by DEAE-cellulose chromatography. Unlike renin, pseudorenin does not attack substrate A in the presence of serum, nor does it produce angiotensin I from renin substrate as it exists in serum. In contrast to renin, which occurs primarily in the kidney, pseudorenin has been found in every one of the 13 different tissues which have been tested, and also in the plasma. The natural substrate for the new enzyme as well as its physiological function are not known. ADDITIONAL KEY WORDS pseudorenin renin• The enzyme renin is the primary activator of the renin-angiotensin pressor system. It is found in the kidney, from which it may be secreted into the bloodstream, where it hydrolyzes a protein substrate releasing the decapeptide angiotensin I. A converting enzyme cleaves this latter peptide, removing the dipeptide His-Leu from its C-terminal, thus producing angiotensin II, a powerful vasoconstrictive compound which is the effector substance of the system (1). The protein substrate for renin was purified from hog plasma (2). Five major forms were found: A, B 1; B 2 , Ci, and C 2 . All were glycoproteins with molecular weights of about 57,000. The renin substrate from horse plasma was degraded with trypsin to yield a peptide fragment which yielded angiotensin I on further treatment with renin. The fragment, a tetradecapeptide, was isolated and its structure determined and confirmed by synthesis (3, 4). The kinetics of the reaction of hog Received July 15, 1969. Accepted for publication August 26, 1969. renin with this tetradecapeptide and with a number of related synthetic peptides were recently described (5).A new enzyme has now been found which produces angiotensin I from the tetradecapeptide renin substrate and from purified hog renin substrate A. It differs from renin in that it does not produce angiotensin I from the substrate occurring naturally in plasma, nor does it attack the purified substrate A in the presence of plasma. It is chromatographically distinct from renin, and has optimum enzymatic activity at much lower pH values. Its activity has been found in every tissue thus far tested. This paper contains a description of the new enzyme, which has been named pseudorenin. ExperimentalMethods of Assay.-Samples to be assayed for pseudorenin were diluted to 1 ml with icecold saline in siliconized Pyrex tubes. One ml of a cold solution containing 1 nmole of tetradecapeptide substrate was then added. The substrate solution was prepared in 0.05M sodium citrate buffer, pH 4.0, containing 0.1M NaCl. The tubes were then incubated at 37.5°f or 15 minutes. The incubation, as well as all