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.     

Analysis of inactive renin by renin profragment monoclonal antibodies

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.     

Conversion between renin and high-molecular-weight renin in the dog.

Two forms of renin, one of mol.wt. 43,000 and the other 60,000, were found in the dog kidney. Conversion between the two forms of renin was reversible at neutral pH. Though the molecular weight of renin in kidney-cortex homogenate was 43,000, it was completely converted into high-molecular-weight renin in the presence of substances that react with thiol groups. On the contrary, stored renin in the granules was the form of normal size (mol. wt. 43,000) regardless of the absence or presence of such substances. The present experiments indicated that renin is stored in the granules as the form of normal size and might be converted into high-molecular-weight renin when it is released from the granules and attached to some substance in the soluble fraction of renal-cortical tissue.     

Biochemistry of renin

Biochemical features of renin have been studied. Determination of the amino acid sequence and catalytically essential groups in the active sites of mouse submandibular gland revealed the similarity of renin with acid proteases. Yet stringent substrate specificity, neutral pH optimum of its enzyme activity and the unique structure of the activation peptide distinguish it from digestive enzymes. Inactive renin was identified as renin zymogen by complete purification and translation in vitro.     

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.     

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.     

Purification of pig renin

1. A new method of purification of renin is described. This method employs the following procedures: ethanol precipitation; saline extraction; precipitation of renin with 40% ammonium sulphate; precipitation of impurities with 3% ammonium sulphate at pH2.5; chromatography on DEAE-cellulose and CM-Sephadex; gel filtration on Sephadex G-100 (both normal and superfine grade); finally, starch-gel electrophoresis. 2. The final renin preparation had a specific activity 10(4) times that of the initial saline extract. 3. A single band of stained protein corresponding to the renin activity was present on starch-gel electrophoresis in the final step and a single precipitin line was obtained to this material with rabbit anti-(pig renin) serum. 4. Double diffusion in agar with rabbit anti-(pig renin) serum showed one major precipitin line, probably due to renin-anti-renin complex, and in addition two minor components.     

Fluorimetric assay of renin

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 reaction reacted with the free NH2 of the tetrapeptide generated to induce a fluorimetric reaction detected at 395--405 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.     

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.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).     

A radiochemical renin assay

1. A synthetic 3-([(14)C]valine)-labelled tetradecapeptide renin substrate was used to measure renin concentration. Renin liberated (14)C-labelled angiotensin I, which was separated from the labelled substrate by paper chromatography. The conversion of substrate into angiotensin I was quantitated by liquid-scintillation counting of radioactivity. 2. The rate of conversion of the substrate into angiotensin I was shown to be linearly related to renin concentration and time under suitable conditions. Angiotensin generation measured in this system agrees well with that measured by bioassay. 3. It is suggested that the use of a pure substrate offers advantages that include the standardization of current units of renin measurement.