Extraction, Purification, and Assay of Human Renin Free of Angiotensinase

Haas, Erwin; Goldblatt, Harry; Gipson, Edwin C.; Lewis, L

doi:10.1161/01.res.19.4.739

Cited by 170 publications

(40 citation statements)

References 15 publications

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“…Ltd), cyproheptadine hydrochloride (Merck, Sharpe & Dohme Ltd), mepyramine maleate (May & Baker Ltd), val-5-angiotensin II amide (Ciba Laboratories Ltd). Renin was prepared from rat kidneys by the method of Haas, Goldblatt, Gipson & Lewis (1966).…”

Section: Drugsmentioning

confidence: 99%

Central components of the renin‐angiotensin system

Hayden

Targett

1971

British J Pharmacology

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Summary1. Re-establishment of renal blood flow after total renal ischaemia of 4 h duration causes a pressor response in pentobarbitone anaesthetized rats and cats. 2. The pressor response is related to the release of renin from the ischaemic kidney. 3. Spinal section at any cervical level or stellate ganglionectomy abolishes the pressor response but spinal section caudad to the thorax or vagotomy and treatment with hexamethonium have no effect. 4. Spinal section and stellate ganglionectomy interfere with the release of renin and with the pressor response to angiotensin.

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Section: Drugsmentioning

confidence: 99%

Central components of the renin‐angiotensin system

Hayden

Targett

1971

British J Pharmacology

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“…Human renin used in the RIBA was extracted from cadaver kidneys 5 and purified by immunoaffinity chromatography with R 3-36-16, a monoclonal antibody directed against human renin. 6 The specific activity of the purified enzyme was 150-200 Goldblatt units (GU)/mg protein.…”

Section: Methodsmentioning

confidence: 99%

Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma.

et al. 1989

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A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r=0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 fiM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with T he renin-angiotensin system plays a major role in the regulation of blood pressure and in the maintenance of sodium and volume homeostasis. The enzymatic cleavage of angiotensinogen by human renin (EC 3.4.23.15) generates a decapeptide, angiotensin I, which is immediately hydrolyzed by a carboxydipeptidyl hydrolase, the angiotensin converting enzyme (EC 3.4.15.1), to the octapeptide angiotensin II, a potent vasoconstrictor. Renin has only one known substrate, angiotensinogen, and therefore provides an interesting target for the design of specific blockers of the renin-angiotensin system. During recent years, several specific and potent inhibitors of primate renin have been synthesized and characterized in vitro and in vivo. The in vitro studies have demonstrated the potency and specificity of the compounds against renin from different species and other aspartic proteinases, 1 and the in vivo studies have demonstrated the effects of renin inhibitors on blood

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“…Rat renin was prepared essentially following procedure A described by Haas and Goldblatt [25]. The preparation method used included an incubation of the partially purified enzyme at pH 9.5 in the presence of 0.1 M EDTA to inactivate angiotensinase activities [25,26].…”

Section: Preparation Of Rat Reninmentioning

confidence: 99%

“…The preparation method used included an incubation of the partially purified enzyme at pH 9.5 in the presence of 0.1 M EDTA to inactivate angiotensinase activities [25,26]. After this treatment, the enzyme was precipitated by addition of (NH4)2S04 to a final concentration of 2.3 M, redissolved in 50 mM Tris/HCl, pH 7.4, and extensively dialysed against the same buffer.…”

Section: Preparation Of Rat Reninmentioning

confidence: 99%

Purification and Characterization of Two Forms of Rat Plasma Proangiotensin

Voigt¹,

Wittmann‐Liebold²,

Köster³

1982

European Journal of Biochemistry

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Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose at pH 6.5, DEAE-Sepharose at pH 8.9, Sephadex G-I SO, hydroxyapatite and hexyl-agarose. Both forms were finally separated by affinity chromatography on concanavalin-A -Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55 000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino-terminal amino acid sequence which was found to be the same (HzN-AspArg-Val) as for angiotensin I and 11. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be rC, = 5.0 pM proangiotensin and V = 270 nmol of angiotensin I lip' unit renin-' for the concanavalin-A-binding form and K,,, = 5.6 pM proangiotensin and V = 250 nmol angiotensin I h -l unit renin-' for the prohormone which did not bind to concanavalin-A-Sepharose. The form of proangiotensin not bound to concanavalin-A-Sepharose was found to be more thermally labile (1, of 59.0 "C) than the form binding to concanavalin A ( t , of 61.5 'C, where t , temperature at which 50 reactivity is lost).Proangiotensin, the prohormone of the angiotensin peptides (for a review see [l]), is the protein substrate for renin, an acid protease synthesized by the juxtaglonierular cells of the kidney and secreted into the blood circulation via the renal vein [2]. Renin cleaves a leucine-leucine sequence of proangiotensin ( Fig. 1) to release the decapeptide angiotensin I [3], which is then converted to the octapeptide angiotensin 11 by converting enzyme (kininase IT) which cleaves the decapeptide to release the two COOH-terminal amino acids, histidine-9 and leucine-I0 [4,5]. Angiotensin 11, the most potent endogenous pressor hormone isolated to date, appears to be involved in blood pressure homeostasis and indirectly, via the mediation of aldosterone release by the adrenal gland, in the regulation of sodium excretion in the kidney.Proangiotensin is produced by and secreted from the liver 16-91, Its biosynthesis is regulated by sex hormones [7, and particularly by the corticosteroids [9,12-151. Induction of proangiotensin by corticosteroids can be reversed by inhibitors of translation and RNA biosynthesis [12]. To gain more insight into the induction process immunochemical techniques have to be employed. For the preparation of antibodies against rat plasma proangiotensin it was necessary to isolate sufficient amounts of this protein. Isolation of human proangiotensin has been described by several groups 116-221 and partial puri...

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Extraction, Purification, and Assay of Human Renin Free of Angiotensinase

Cited by 170 publications

References 15 publications

Central components of the renin‐angiotensin system

Central components of the renin‐angiotensin system

Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma.

Purification and Characterization of Two Forms of Rat Plasma Proangiotensin

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