J.L. Prado

Kinin-converting aminopeptidase from human serum

Abstract A kinin-converting aminopeptidase from human serum (HuPA) which converts kallidin (lysylbradykinin, LBK) to bradykinin (BK) was purified about 900-fold by a three-step procedure involving ammonium sulfate fractionation, continuous loading Sephadex gel electrophoresis, and gel filtration on Sephadex G-200; the final yield was about 7 per cent. A bioassay was developed in which BK formed in the presence of excess LBK or methionyllysylbradykinin (MLBK), pH 7.8, 37°, was separated on carboxymethylcellulose columns and assayed on the isolated guinea pig ileum. L -Aminoacyl-β-naphthylamidase activities of HuPA measured on L -lysine, L -arginine- and L -leucylnaphthylamides (Lys-NA, Arg-NA and Leu-NA) were parallel to the kinin-converting activity in all purification steps. The activity of the purest preparation on LBK and MLBK, measured at the approximate enzyme:substrate molar ratio 1:1500, was respectively 267 and 174 nmoles BK/min/mg of protein. Considering the activity on Met-NA as 100, the activities on other naphthylamides were: Leu-NA, 65; Arg-NA, 46; Lys-NA, 29; Asp-NA, 0; Glu-NA, 0. A few tri- and dipeptides, angiotensin II and its amide, polylysine and polyarginine were poorer substrates. The following substances were inhibitors: 1,10-phenanthroline, puromycin, EDTA and 2,3-dimercapto-l-propanol. 1,10-Phenanthroline or EDTA inhibition could be partially reversed by Co 2+ , Mn 2+ and Zn 2+ . The molecular weight was estimated as 95,000 on Sephadex gel filtration. On agarose gel microelectrophoresis, the enzyme migrated as α 1 -globulin before the electrophoresis step and had a migration intermediate between α 2 - and β -globulin following this step. In this microelectrophoresis, only one band of arylamidase activity was detected on Lys-, Arg- and Leu-NA. The purest preparation was still contaminated with albumin but was free of kininase activity.

Pharmacologically-active polypeptide formed from blood globulin by a cysteine-activated protease from Clostridium histolyticum

Abstract A cysteine-activated protease secreted by Clostridium hystolyticum has been shown to release from fraction IV- 4 of bovine plasma a slow-reacting substance, which contracts the isolated guinea-pig ileum, rat uterus, and hen rectal caecum, and produces a transitory drop in rat blood pressure. Parallel pharmacological assays, the action of trypsin, chymotrypsin and pepsin, paper chromatography and paper electrophoresis, revealed a close similarity between the active principle described and bradykinin.

Partial purification of a plasma-kinin-forming enzyme from horse urine

Durch Chromatographie mit Diäthylamino-äthyl-cellulose nach Ammoniumsulfat-Fraktionierung wurde ein Plasma-Kinin-freisetzendes Ferment aus Pferdeharn teilweise (80×) gereinigt. Das so gereinigte Präparat war gegen Pferde-, Menschen-, Rinder (Fraktion IV-4)- und Ratten-Blutplasma, nicht aber gegen Schweine-(Fraktion IV-4)-Globuline wirksam. Durch Sojabohnen-Trypsininhibitor oder Ovomucoid erfolgt keine Hemmung. Präparate von verschiedenem Reinheitsgrad zeigten entsprechendep-Toluenesulfonyl-l-arginin-methylester-spaltende und Kinin-freisetzende Wirksamkeit.

Kinin-converting aminopeptidase from human urine partial purification and properties

Abstract 1. 1. An aminopeptidase from human urine, which hydrolyses dipeptides and β-naphthylamides of neutral and basic amino acids and which converts the peptides lysylbradykinin and methionyllysyl-bradykinin into bradykinin, was highly purified by a four-step procedure. 2. 2. The enzyme (mol. wt 100,000) has several similarities with kinin-converting aminopeptidases found in human serum and liver, and is inhibited by 1,10-phenanthroline and puromycin.

Kallidin (lysylbradykinin), the kinin formed from horse plasma by horse urinary kallikrein

Abstract Horse urinary kallikrein when incubated with horse plasma formed kallidin (lysylbradykinin) from the kininogens in the plasma. Horse plasma, like human plasma, was found to contain an aminopeptidase capable of converting kallidin to bradykinin. No evidence, however, could be found that the plasma contained an aminopeptidase capable of converting Met-Lys-bradykinin to kallidin, thus eliminating the possibility that the kallikrein had released Met-Lys-bradykinin which was converted to kallidin during the 1–5 min incubations. The method used for identification of the kinins is rapid, gives a good recovery and requires small amounts of plasma and enzyme.

Inactivation of kinins by chymotrypsin

Abstract The inactivation rate of synthetic bradykinin (BK) by different samples of crystallized α-chymotrypsin at pH 7.0, 30° and a 1:10 enzyme/substrate molar ratio was reduced in the presence of 1,10-phenanthroline. Selective inaetivation of the samples by preincubation with l -(1-tosylamido-2-phenyl) ethyl-chloromethylketone (TPCK) in 1:10 enzyme/TPCK ratio reduces the inactivation rate about 60 per cent. This residual BK-inactivating action is probably due to a carboxypeptidase-B-type enzyme present in the chymotrypsin commercially available. In a 1:200 enzyme/substrate ratio and in the presence of 1,10-phenanthroline, a few min of incubation at pH 7.0, 30°, is sufficient to inactivate bradykinin at a constant rate. This inaetivation is due to the cleavage of the Phe-Arg bond by chymotrypsin, releasing free arginine. The rate of inactivation of BK by chymotrypsin in the presence of 3.3 × 10 −3 M 1,10-phenanthroline was 43.9 nmoles/min/mg of enzyme, and the inactivation rates of the longer kinins. kallidin (lysylbradykinin) and Met-Lys-bradykinin. were 60.1 and 138.18 nmoles/min/mg of enzyme respectively.

Methionine aminopeptidase associated with liver mitochondria and microsomes

Abstract 1. 1. A new, unstable, particle-bound (particularly mitochondria and microsomes) aminopeptidase, mol. wt 74,000, was partially purified from rat liver. 2. 2. Among five aminoacyl-β-naphthylamides assayed, MetNA was the best substrate. The aminopeptidase releases NH 2 -terminal methionine from methionyllysylbradykinin. 3. 3. Titration with p -hydroxymercuribenzoate indicated that this methionine aminopeptidase contains ca five SH groups per mol.

Two arylamidases from human liver and their kinin-converting activity.

Abstract 1. 1. Two arylamidases, called B and N, were found in human liver homogenates. 2. 2. Arylamidase N, purified 300-fold, MW 200,000, converts lysylbradykinin into bradykinin at a rate higher than 1.53 μniole/min per mg protein, and does not depend on chloride ions. 3. 3. Arylamidase B, unstable, is a chloride dependent arylamidase and has no kinin-converting activity. 4. 4. Comparative hydrolysis rates of some aminoacyl-β-naphthylamides and other properties also show that they are different enzymes.

Assay of Renin and Hypertensin with the Isolated Guinea-Pig Ileum

Renin and hypertensin may be easily assayed with the isolated guinea-pig ileum, which is as sensitive as the vascular toad preparation. Hypertensin itself is the substance which acts on the smooth muscle of the ileum.

Renin Content of Systemic Blood of Rats with Desoxycorticosterone and Metacorticoid Hypertension

No increased renin concentration was detected in the systemic blood of rats with desoxycorti-costerone and metacorticoid hypertension, using the guinea-pig; ileum method of renin estimation. Re-establishment of the circulation through totally ischemic rat kidney is followed by the liberation of large amounts of renin.