Comparison of angiotensinogen and tetradecapeptide as substrates for human renin

Stammers, D.K.; Dann, J. G.; Harris, Christopher; Smith, Dana R.

doi:10.1016/0003-9861(87)90362-6

Cited by 20 publications

(9 citation statements)

References 29 publications

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“…In this case, Ang-(1-12) apparently lacks the minimum number of residues on the carboxy terminus recognized and cleaved by renin. Indeed, the Ang-(1-14) substrate is considered to contain the minimum sequence for renin to cleave the His-Leu in the formation of Ang I [11, 26]. Although our results do not support a role for renin to process Ang-(1-12), enzymatic pathway(s) for generation of the peptide in the circulation is not known.…”

Section: Discussionmentioning

confidence: 59%

“…The substrate properties of the 14 amino acid peptide may potentially be quite different than the >350 residue protein and use of the precursor protein is the preferred approach to demonstrate a putative role of an enzyme in the processing of angiotensins [11, 26]. Renin specifically cleaves the Leu 10 -Leu 11 bond of rat angiotensinogen to form Ang I, while the hydrolysis between the two aromatic residues Tyr 12 -Tyr 13 is required for the direct generation of Ang-(1-12) [11,26]. The processing pathway we modeled in the circulation apparently favors the initial renin-dependent formation of Ang I rather than the formation of Ang I from Ang-(1-12) by ACE.…”

Section: Discussionmentioning

confidence: 99%

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Divergent pathways for the angiotensin-(1–12) metabolism in the rat circulation and kidney

Westwood¹,

Chappell²

2012

Peptides

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Evidence of endogenous angiotensin–(1-12) [Ang–(1-12)] may necessitate revision of the accepted view that Ang I is the immediate peptide product derived from the precursor protein angiotensinogen. As the processing of this peptide have not been fully elucidated, we characterized Ang-(1–12) metabolism in the serum and kidney of the mRen2.Lewis rat, a model of high circulating renin and ACE expression. A sensitive HPLC-based method to detect the metabolism ex vivo of low concentrations of 125I-labeled Ang-(1–12) was utilized. Ang-(1–12) processing to serum did not reveal the participation of renin; however, serum ACE readily converted Ang-(1–12) to Ang I with subsequent metabolism to Ang II. Ang I and Ang II forming activities for serum ACE were 102 ± 4 and 104 ± 3 fmol/ml/min serum (n = 3), respectively and both products were abolished by the potent ACE inhibitor lisinopril. The metabolism of Ang-(1–12) in renal cortical membranes also revealed the formation of Ang I; however, the main products were Ang-(1–7) and Ang-(1– 4) at 129 ± 9 and 310 ± 12 fmol/mg/min protein (n = 4), respectively. Neprilysin inhibition abolished these products and substantially reduced the overall metabolism of Ang-(1–12). Incubation of Ang-(1–12) with either human or mouse neprilysin revealed identical products. We conclude that endogenous Ang-(1–12) may contribute to the expression of biologically active angiotensins through a renin-independent pathway. The preferred route for Ang-(1–12) metabolism likely reflects the relative tissue content of ACE and neprilysin.

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Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Divergent pathways for the angiotensin-(1–12) metabolism in the rat circulation and kidney

Westwood¹,

Chappell²

2012

Peptides

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“…The K m values for hTDP were found to be 13.3 + − 6.2 µM [31] and 20.7 + − 7 µM [32]. High K m values have been also found when the asparagine residue was replaced by serine (K m = 8.4 µM) [11] or threonine (K m = 27.0 µM) [32], whereas the K m values reported for angiotensinogen are low, varying from 0.4 to 5.7 µM [33]. The modification of peptide substrates for proteases with bulky hydrophobic groups can alter their kinetic behaviour.…”

Section: Effect Of Substrate Length On Kinetic Parameters and Quenchimentioning

confidence: 99%

Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

Paschalidou

Neumann

Gerhartz

et al. 2004

Biochemical Journal

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The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3' (substrate 3) resulted in an acceptable k(cat)/K(m) (41000 M(-1).s(-1)) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k(cat)/K(m) value of 350000 M(-1).s(-1) and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k(cat). The pH dependence of k(cat)/K(m) for renin and substrate 9 showed that the optimal pH is at pH 6-7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK(a) of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg [where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid] (k(cat)/K(m)=268000 M(-1) x s(-1), quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.

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“…A second possibility is a difference in the reaction kinetics between renin and AGT in comparison to the tetradecapeptide. It has been demonstrated that human renin cleaves AGT more slowly in vitro than the synthetic human tetradecapeptide substrate (Cumin et al 1987; Stammers et al 1987). If mouse renin reacts more readily in vivo with the tetradecapeptide than with AGT, this could explain the rather rapid pressor response observed in the present experiment.…”

Section: Discussionmentioning

confidence: 99%

Influence of elevated renin substrate on angiotensin II and arterial blood pressure in conscious mice

Cholewa

Mattson

2005

Experimental Physiology

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The present experiments were performed to determine the influence of intravenous administration of renin substrate on plasma angiotensin II levels and mean arterial blood pressure in conscious C57BL/6J mice. Mice with chronic indwelling femoral arterial and venous catheters were acutely or chronically administered intravenous doses of a synthetic peptide corresponding to the 14 amino acids on the N-terminal of angiotensinogen. A dosedependent increase in arterial blood pressure was observed as the intravenous bolus dose of the renin substrate was increased from 0.18 to 180 nmol kg −1 with a maximal increase in pressure of 40 ± 3 mmHg achieved following administration of the 18 nmol kg −1 bolus (n = 11). Additional experiments demonstrated that a sustained intravenous infusion of the renin substrate led to a long-term increase in arterial blood pressure. The continuous infusion of renin substrate at 0.05 nmol kg −1 min −1 for 3 days did not alter arterial blood pressure from the control level of 119 ± 5 mmHg (n = 5); however, arterial blood pressure significantly increased to 129 ± 6 mmHg with an infusion rate of 0.5 nmol kg −1 min −1 and further increased to 141 ± 3 mmHg when the renin substrate infusion was increased to 5.0 nmol kg −1 min −1 . Finally, the infusion of renin substrate at 5.0 nmol kg −1 min −1 resulted in a significant increase in plasma angiotensin II concentration from 34 ± 6 pg ml −1 in vehicle-infused mice to 288 ± 109 pg ml −1 . These results demonstrate that modulation of the circulating level of angiotensinogen can alter the plasma angiotensin II level and arterial blood pressure in normal animals.

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Comparison of angiotensinogen and tetradecapeptide as substrates for human renin

Cited by 20 publications

References 29 publications

Divergent pathways for the angiotensin-(1–12) metabolism in the rat circulation and kidney

Divergent pathways for the angiotensin-(1–12) metabolism in the rat circulation and kidney

Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

Influence of elevated renin substrate on angiotensin II and arterial blood pressure in conscious mice

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