Purification of human renin

Human renin was purified 2,800-fold from a partially purified preparation to an electrophoretically homogeneous state by a series of three different types of affinity chromatography and two additional conventional chromatographic steps at a yield of 9.7%. This amounts to a 420,000-fold purification from a crude kidney extract. This pure human renin preparation has a specific activity of 830 Goldblatt unit/mg and is stable at pH 6.2 and 4°C at least for 3 weeks.     

Kinetic comparisons of amniotic fluid inactive renin and renal renin using synthetic and human renin substrates

Inactive renin has been isolated from pooled amniotic fluid and purified approximately 642-fold. Prior to activation the isolates had approximately 4% of the activity found after activation. The observation is similar to that reported for inactive renin from chorionic cell culture and suggests a placental origin of amniotic fluid inactive renin. Using plasma from an estrogen-treated woman, renin substrate was recovered free of renin and inactive renin and a portion was separated into NMW and HMW components. The NMW form constituted approximately 93% and the HMW form approximately 7% of the renin substrate. Amniotic fluid inactive renin was used for determinations of enzyme-substrate kinetics with the pooled, NMW, and HMW plasma substrate and tetradecapeptide synthetic substrate, and the results were compared to similar determinations using standard renal renin. Using synthetic substrate, the kinetics of renal renin and amniotic fluid inactive renin before and after activation were similar. The kinetics of renal renin with pooled, NMW, and HMW plasma substrate were also similar. Amniotic fluid inactive renin had a lower Km with pooled than with NMW substrate, however, which resulted from a significantly smaller Km with HMW component. Although the affinity constants with pooled substrate were not different for renin and inactive renin, the Km of inactive renin was significantly less with the HMW component of plasma renin substrate. The observations are compatible with a role for placental inactive renin in normal pregnancy and suggest the possibility of a further role in hypertensive pregnancy.     

Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, that is human renin specific and is resistant to cathepsin D

A synthetic tetradecapeptide, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, which corresponds to the 13 amino terminal residues of human angiotensinogen plus a carboxy terminal serine to replace a suggested site of carbohydrate attachment, has been shown to be a good substrate for human kidney renin. At pH 7.2 and 37 degrees C the KM or Michaelis constant was 8.4 +/- 2.9 microM, and the VM or velocity at infinite tetradecapeptide concentration was 11.3 +/- 2.4 mumol angiotensin I made per hour per milligram renin. The tetradecapeptide was highly resistant to cleavage by mouse submaxillary renin. The tetradecapeptide was also slowly cleaved by human liver cathepsin D, by rabbit lung angiotensin-converting enzyme, and by reconstituted human serum, but did not yield angiotensin I. Thus, this synthetic renin substrate should permit more specific measurement of human kidney renin activity.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

Characterization of recombinant human renin: Kinetics,pH-stability,and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P_3′ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (～1.8 µM) atpH ～7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Highly potent and specific inhibitors of human renin

We have designed and synthesized a series of small peptides containing a perfluoroalkyl ketone group at the C-terminal position of the angiotensin I sequence as inhibitors of human renin. From this series of compounds, 8 and 10 showed strong inhibition of human renin (IC₅₀ = 3 × 10⁻⁹, 7 × 10⁻⁹ M, respectively). Compound 10 did not inhibit pepsin and cathepsin D at 10⁻⁴ M. Comparison of the IC₅₀ of compound 8 and compound 11 (8.7 × 10⁻⁷ M) demonstrated the marked effect of the perfluoropropyl group on the potency of inhibition on renin, presumably due to the strong electron-withdrawing effect causing the ketone in 8 to exist predominantly as the hydrate — thus mimicking the tetrahedral transition state during hydrolysis of the scissile Leu¹⁰—Val¹¹ amide bond.     

Purification of human renin substrate.

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Substrate analog competitive inhibitors of human renin

Replacement with D-amino acids at the site of cleavage in the native sequence of tetradecapeptide renin substrate yields effective competitive inhibitors of renin. The present communication reports the solid phase synthesis and the kinetic parameters of analogs of des-Asp-tetradecapeptide renin substrate, in which L-Leu at position 10 and/or 11 has been replaced with D-Leu. The dissociation constant (K_i) for renin of the three inhibitors described here is about 10^?6 M. These synthetic substrate analogs are not significantly hydrolyzed by renin.     

Circadian variations of total renin, active renin, plasma renin activity and plasma aldosterone in clinically healthy young subjects

The direct assay of total renin (TRC) and active renin concentration (ARC) is a reality due to the availability of monoclonal antibodies against human renin. Because of this, a study has been performed in order to assess the circadian rhythmicity of TRC and ARC. The study was extended to plasma renin activity (PRA) and plasma aldosterone concentration (PAC) for a more complete assessment of the renin-angiotensin-aldosterone system (RAAS). Twelve clinically healthy subjects (6 males and 6 females, age from 20 to 25 years) volunteered for this study. Time-qualified data series were analysed by means of chronobiological procedures in order to validate the circadian rhythm and to correlate the sinusoidal profiles. The circadian rhythm was validated at a high significance for TRC, ARC, PRA and at a borderline significance for PAC. The periodic oscillations were significantly correlated, demonstrating that TRC, ARC, PRA and PAC cycles oscillate in synchronism during the 24-hour span.     

A novel purification method of human renin

Human renal renin was isolated from a partially purified preparation, Haas' preparation step 5 material, with a remarkably high yield of 28% by a newly developed method. This method consisted of only four steps including affinity chromatography on pepstatin-aminohexyl-agarose, chromatofocusing, gel filtration and DEAE-chromatography after the initial extraction and concentration. This method enabled us to obtain pure human renin without another affinity column used by other investigators. Pure human renin had a molecular weight of 40,000 daltons as estimated by gel filtration and sodiumdodecylsulfate-polyacrylamide gel electrophoresis. The pI of purified renin was 5.6.