Enzyme kinetic studies on human renin and its purified homologous substrate

Rosenthal, J.; Hp, Wolff; Weber, Petra; Dahlheim, H.

doi:10.1152/ajplegacy.1971.221.5.1292

Cited by 47 publications

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“…'• 10 The renin substrate preparation was dissolved in 0.15 M phosphate buffer of the desired pH, with the components Na 2 HPO 4 and NaH 2 PO 4 for the pH range between 4.5 and 9.0, or NaH 2 PO 4 and phosphoric acid for lower pH values. The substrate concentration (60,000 ng/ml) was in the saturation range for the small enzymatic activities measured.…”

Section: Determination Of Angiotensin-i-forming Enzyme (Aife) Activitymentioning

confidence: 99%

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Rosenthal¹,

Pfeifle²,

Michailov³

et al. 1984

Hypertension

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SUMMARY Investigations were performed on components of the renin-angiotensin system (RAS) in homogenate extracts of vascular tissue and aortic smooth muscle cells cultivated in vitro. Determinations of isoelectric points and pH optima indicated the existence in aortic homogenate extracts of two local angiotensin I (AI)-forming enzymes (AIFE) that were different from those of plasma, renal cortex, veins, and aortic smooth muscle cells. The pH optima for Al-converting enzyme (ACE) from vascular tissues, aortic smooth muscle cells, and plasma were in the same range (pH 8.0-8.5), and in agreement with those measured previously in other tissues. In contrast, in vitro studies with the ACE inhibitors MK-421 and MK-422 and measurement of isoelectric points suggested that aortic ACE was different from the plasma enzyme. AIFE and ACE activities were found to be elevated in spontaneously hypertensive rats (SHR). The biochemical characteristics of the enzymes investigated in the vascular tissue of SHR were not different from those of the normotensive controls. AI-and All-degrading enzymes were found both in aortic tissue and in aortic smooth muscle cells. One potent Al-degrading enzyme different from ACE was observed in aortic tissue. A high ratio of AI/AII immunoreactivities in arterial walls suggests the availability of renin substrate, and that Al-degrading enzymes are the rate-limiting enzymes for AH formation. were the first to demonstrate the existence of arterial and also venous wall renin by using systematically performed biochemical methods. In the last few years, there has been renewed interest in the vascular reninangiotensin system (RAS). Now, as before, it is thought to play a role in the regulation of blood pressure and in the development and maintenance of hypertension. The present investigation continues our previous studies in this field 4 ' 3 and was undertaken to recognize, quantify, and partly characterize those components of

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Section: Determination Of Angiotensin-i-forming Enzyme (Aife) Activitymentioning

confidence: 99%

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Rosenthal¹,

Pfeifle²,

Michailov³

et al. 1984

Hypertension

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“…Recently, various transgenic animal models of hypertension have been developed 15 to elucidate the mechanisms of hypertension. The transgenic rat TGR-(mRen-2)27, in which the mouse Ren-2 renin gene that codes for mouse salivary gland and kidney renin is transfected into the genome of the Sprague-Dawley rat, was the first genetic hypertensive rat model.…”

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confidence: 99%

Hypertension in the transgenic rat TGR(mRen-2)27 may be due to enhanced kinetics of the reaction between mouse renin and rat angiotensinogen.

et al. 1994

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The transgenic rat TGR(mRen-2)27, in which the Ren-2 mouse renin gene is transfected into the genome of the rat, develops severe hypertension with high adrenal renin and low kidney renin. These animals express both mouse and rat renin. To investigate the cause of hypertension in the TGR rat, we compared the kinetics of mouse renin acting on mouse and rat angiotensinogens. The optimum pH of the renin reaction in the Sprague-Dawley rat was 6.5, whereas the optimum pH of the reaction in the TGR rat was approximately 8.5. The optimum pH of the renin reaction in the DBA mouse was 6.0. Purified mouse Ren-2 renin acting on rat angiotensinogen showed a pH profile similar to that for the renin reaction in the TGR rat. The angiotensinogen concentration in pooled plasma from eight DBA mice was 104.5 ng angiotensin I/mL and was clearly lower than that in Sprague-Dawley rats (772.4±37.3 ng angiotensin I/mL, n=4). The reaction of purified mouse Ren-2 renin with rat angiotensinogen was 10 times faster than with mouse angiotensinogen. Plasma renin activity in DBA mice increased dramatically on addition of rat angiotensinogen (from T he renin-angiotensin system plays an important role in the development of hypertension. Recently, various transgenic animal models of hypertension have been developed 15 to elucidate the mechanisms of hypertension. The transgenic rat TGR-(mRen-2)27, in which the mouse Ren-2 renin gene that codes for mouse salivary gland and kidney renin is transfected into the genome of the Sprague-Dawley rat, was the first genetic hypertensive rat model. In this model an overexpression of the transgene is found in the adrenal glands, 2 and these rats show severe hypertension with remarkably high adrenal renin levels; however, the pathogenesis of the hypertension is not clear. We have reported that adrenal renin and circulating renin in TGR rats are probably derived primarily from the transfected mouse renin gene. 6 On the other hand, angiotensinogen in the TGR rat is solely rat angiotensinogen. It is known that mouse renin can generate angiotensin I (Ang I) by reacting with rat angiotensinogen.7 If the circulating renin in the TGR rat is mouse renin, then the kinetics of 253.4±66.7 to 225 000±48 000 ng angiotensin I/mL per hour). Intravenous injection of 2 or 10 £iL of DBA mouse plasma into the nephrectomized Sprague-Dawley rat increased the mean arterial pressure of the rat by 27.7±4.7 and 61.8±2.7 mm Hg, respectively, whereas injection of 200 JAL of Sprague-Dawley rat plasma did not change the mean arterial pressure of the rat. From these measurements and from the kinetic parameters measured by Poulsen and Jacobsen (/ Hypertens. 1986;4:175-180), we conclude that the molar concentration of mouse renin in the TGR rat is low compared with that of rat renin (1.5 versus 5.0 pmol/L) but that this low level of circulating mouse renin contributes significantly to plasma renin activity in the TGR rat. The elevation of renin activity and resulting hypertension in the TGR rat therefore can be attributed to the enhan...

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“…Homologous renin substrate was prepared from pooled plasma, obtained from normotensive women taking oral contraceptives, by ammonium sulfate precipitation between the molarity of 1.51 and 2.65 at pH 7.4. 20 The mean concentration of angiotensin I produced after incubation of substrate alone without enzyme (n = 10) was 1.2 ng/ml/hr ± 0.1 SE, and we attribute this to a renin contaminant. This "contaminant" was constant for all incubations and represents less than 2% of the concentrations of angiotensin generated with added renin.…”

Section: Methodsmentioning

confidence: 79%

Modification of the enzymatic activity of renin by acidification of plasma and by exposure of plasma to cold temperatures.

Welch¹,

Talwalkar²

1979

Hypertension

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SUMMARY Plasma renin activity (PRA) increases after acidification of plasma or exposure of plasma to cold temperatures. The purpose of the present study is to evaluate the hypothesis that these increases of PRA reflect inactivation of circulating renin inhibiting factors. In each of eight normotensive subjects, mean PRA increased (p < 0.01) after acidification of plasma by acid dialysis (98%), after acidification of plasma by addition of HCI (47%), or after exposure of plasma to -4°C for 5 days (155%). Renin substrate-free plasma was obtained by passing plasma over Sephadex G-50-40. Both untreated plasma and the substrate-free extract of plasma inhibited the enzymatic activity of renin (p < 0.01); little or no inhibition occurred after addition of acidified plasma or the protein-free extract of acidified plasma to renin-renin substrate. After addition of exogenous renin to cold-exposed plasma, the rate of angiotensin I generation was greater than that in control plasma (p < 0.05), although renin substrate did not differ. These observations suggest that denaturation of a circulating renin inhibitor may contribute to increased PRA after acidification or cold exposure of plasma. In a separate experiment, the addition of polyunsaturated free fatty acids (linoleic and arachidonic) to renin-renin substrate inhibited angiotensin production, although no inhibition occurred after dialysis of these fatty acids against an acid buffer (pH 3.3). Prolonged exposure of linoleic acid to cold temperatures did not affect its capacity to inhibit renin. Thus, loss of long chain, unSaturated free fatty acids may contribute to increased PRA after acid dialysis of plasma but not to increased PRA after exposure of plasma to cold temperatures. RENIN is a proteolytic enzyme that cleaves a circulating alpha-2-globulin substrate to form the decapeptide angiotensin I. Several reports suggest that plasma contains inhibitors of the reaction between renin and its substrate.19 Acetonesoluble neutral lipids extracted from plasma inhibit renin, 10 and we have recently demonstrated that physiologic concentrations of long chain, unsaturated fatty acids inhibit both the capacity of renin to. generate angiotensin in vitro and the pressor response to renin in vivo.11 Thus, in addition to renin and renin substrate concentrations, the measurement of plasma renin activity (PRA) may be affected by circulating renin inhibitors. We have used the term renin reactivity to refer to the capacity of added renin to generate angiotensin I either after its addition to crude

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Enzyme kinetic studies on human renin and its purified homologous substrate

Cited by 47 publications

References 0 publications

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Investigations of components of the renin-angiotensin system in rat vascular tissue.

Hypertension in the transgenic rat TGR(mRen-2)27 may be due to enhanced kinetics of the reaction between mouse renin and rat angiotensinogen.

Modification of the enzymatic activity of renin by acidification of plasma and by exposure of plasma to cold temperatures.

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