Pseudorenin: A NEW ANGIOTENSIN-FORMING ENZYME

Abstract

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

Absence of renin-like activity in rat aorta and microvessels.

“…Our preliminary experiments (unpublished observations) as well as experiments reported by others 17 ' 18 demonstrated the presence of acid proteases in preparations of vascular enzymes. Since acid proteases can cleave angiotensin I from angiotensinogen, 17 ' I823U methods were used to: 1) remove acid proteases from preparations of vascular renin-like activity; 2) minimize the angiotensin I-generating activity of nonspecific acid proteases; and 3) quantitate any residual proteolytic activity.…”

“…When incubated with nephrectomized rat plasma, the acid proteases generated no AI. Although in previous studies 17 ' ' *• " • " and in our own unpublished experiments acid proteases can generate AI from angiotensinogen, in the current study the use of proteolytic inhibitors throughout isolation and assay procedures minimized the capacity of acid proteases to generate AI.…”

“…To maximize detection of vascular renin, activity was assayed near the pH optimum of renin. "• 19~22 Since nonspecific acid proteases can generate AI from angiotensinogen at near the optimal pH for renin activity, 17 ' 18 ' a all samples were incubated with proteolytic inhibitors and any residual activity was removed by chromatography over immobilized bovine hemoglobin. Activity of nonspecific acid proteases was monitored by radiochemical assay.…”

Specific antibody to hog renal renin and its application to the direct radioimmunoassay of renin in various organs.

“…Considerable debate exists in the literature concerning the identity of renin-like enzymes (based upon the ability to generate angiotensin I) in extra- (Day and Reid, 1976;Dorer et al, 1978;Ganten et al, 1976;Hackenthal et al, 1978;Skeggs et al, 1969). Pseudorenin and isorenin (Skeggs et al, 1969), an angiotensin-forming enzyme with an acidic pH optimum, are widely distributed in mammalian tissues.…”

“…Pseudorenin and isorenin (Skeggs et al, 1969), an angiotensin-forming enzyme with an acidic pH optimum, are widely distributed in mammalian tissues. Pseudorenin recently has been identified as cathepsin D (Dorer et al, 1978); the predominant portion of brain isorenin activity also has been identified as cathepsin D (Hackenthal et al, 1978).…”