Fluorimetric assay of renin

doi:10.1016/0005-2744(78)90051-7

Biochimica et Biophysica Acta (BBA) - Enzymology

Volume 523, Issue 2, 12 April 1978, Pages 485-493

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Abstract

A simple fluorimetric assay was set up to test renin within 2 h. $N- acetyltetradecapeptide$ was synthesized and used as substrate. It was demonstrated that $N- acetyl-angiotensin$ I and Leu-Val-Tyr-Ser were the two peptides obtained after hydrolysis by renin. Fluorescamine reacted with the free NH₂ of the tetrapeptide generated to induce a fluorimetric reaction detected at 395–495 nm. The Michaelis constant of the reaction was 1.87 · 10⁻⁵ M. With this method as little as one milliGoldblatt Unit (mG.U.) of hog renin could be detected and the generation of tetrapeptide was linear with respect to the renin concentration up to 20 mG.U. The fluorimetric assay was applied to the detection of renin during its purification and to the characterization of renin inhibitors.

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Cathepsin E (EC 3.4.23.34), an intracellular aspartic proteinase, was purified from monkey intestine by simple procedures that included affinity chromatography and fast protein liquid chromatography. Cathepsin E was very active at weakly acidic pH in the processing of chemically synthesized precursors such as the precursor to neurotensin/neuromedin, proopiomelanocortin, the precursor to xenopsin, and angiotensinogen. The processing sites were adjacent to a dibasic motif in the former two precursors and at hydrophobic recognition sites in the latter two. The common structural features that specified the processing sites were found in the carboxyl-terminal sequences of the active peptide moieties of these precursors; namely, the sequence Pro-Xaa-X'aa-hydrophobic amino acid was found at positions P4 through P1. Pro at the P4 position is thought to be important for directing the processing sites of the various precursor molecules to the active site of cathepsin E. Although the positions of Xaa and X′aa were occupied by various amino acids, including hydrophobic and aromatic amino acids, some of these had a negative effect, as typically observed when Glu/Arg and Pro were present at the P3 and P2 positions, respectively. Cathepsin D was much less active or was almost inactive in the processing of the precursors to neurotensin and related peptides as a result of the inability of the Pro-directed conformation of the precursor molecules to gain access to the active site of cathepsin D. Thus, the consensus sequence of precursors, Pro-Xaa-X′aa-hydrophobic amino acid, might not only generate the best conformation for cleavage by cathepsin E but might be responsible for the difference in specificities between cathepsins E and D.
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1992, Analytical Biochemistry
Six intramolecularly quenched fluorogenic peptides related to the sequences Phe⁸ to His¹³, His⁶ to His¹³, and Tyr⁴ to His¹³ of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC₅₀ = 7.8 × 10⁻⁹m), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707–2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 μGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.
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1991, Advances in Pharmacology
This chapter reviews the advances and challenges encountered in the process of discovering and developing renin inhibitors. Renin inhibitors have emerged as agents capable of reducing blood pressure in man and experimental animals when administered intravenously. These compounds are more specific than angiotensin-converting enzyme (ACE) inhibitors, but they exert their actions through vasodilation—for example, peripheral, renal. The earlier renin inhibitors are high-molecular-weight peptides. However, structural modification and physicochemical optimization have led to smaller dipeptide inhibitors, which represent the most promising leads for drug candidacy to date. The dipeptide-renin inhibitors lack good oral bioavailability caused by either limited gastrointestinal absorption or high hepatic extraction, or both. These insufficiencies provide the challenge for the improvement of the dipeptide-renin inhibitors series and the development of nonpeptide-rennin inhibitors. Thus, the evolution of renin inhibitors continues. If solutions to poor gastrointestinal absorption and rapid hepatic extraction are found, then rennin inhibitors play a significant role in the treatment and control of hypertension and renin-angiotensin-aldosterone system (RAAS)-related diseases.
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The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH₂ containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC₅₀ of ∼ 1 μM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.

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Fluorimetric assay of renin

Abstract

Arch. Biochem. Biophys.

Lancet

Anal. Biochem.

Clin. Chim. Acta

FEBS Lett.

Can. Med. Assoc. J.

J. Clin. Endocrinol. Metab.

Clin. Chim. Acta

Am. J. Med.

Biochem. Med.

J. Exp. Med.

Am. J. Physiol.

Science