High Molecular Weight Renin Research Articles

Effects of Nucleotides on the Interaction of Renin with GlcNAc 2-Epimerase (Renin Binding Protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.

Assignment of the gene encoding renin binding protein (Renbp) to rat chromosome Xq37 by in situ hybridization and radiation hybrid mapping

The renin-angiotensin system (RAS) plays a crucial role in the regulation of blood pressure (Laragh, 1995). Within the RAS, the physiological relevance of renin binding protein (RnBP) has been controversial for several years (Schmitz et al., 2000). Originally, RnBP was identified as a protein in the kidney that was capable of binding renin in renal homogenates giving rise to a complex designated high molecular weight renin (Takahashi et al., 1983). It was subsequently proposed that RnBP might act as an endogenous cellular renin inhibitor. Recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase (Takahashi et al., 1999). Moreover, gene-targeting experiments in the mouse excluded a specific interaction between RnBP and renin in the kidney or in the circulating RAS of the mouse (Schmitz et al., 2000). So far, the gene encoding RnBP has been mapped on chromosome X in different mammals: Xq28 in human (van den Ouweland et al., 1994), X 29.53 cM in mouse (http:// www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=19703) and X in canine (Stoy et al., 1998). As mapping data for the rat homolog Renbp is still lacking, it is unclear whether this gene is located in blood pressure quantitative trait loci (QTL) termed BP/SP-2 and SS-X on rat chromosome X. BP/SP2 has been identified in an intercross between the stroke prone spontaneously hypertensive rat (SHRSP) and the normotensive Wistar-Kyoto (WKY) rat (Hilbert et al., 1991) and SS-X has been characterized in the Sabra hypertension-prone rat (Yagil et al., 1999). We therefore set out to test the chromosome location of Renbp by performing chromosomal mapping analysis in the rat using fluorescence in situ hybridization (FISH) and radiation hybrid (RH) mapping.

Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein).

Renin-binding protein (RnBP) is a highly specific renin inhibitor first isolated from porcine kidney. Our recent studies demonstrated that the human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have developed a new assay method for GlcNAc 2-epimerase activity using a system of N-acyl-D-hexosamine oxidase coupled with peroxidase and employed this method to study the effects of renin on GlcNAc 2-epimerase activity. The recombinant human (rh) RnBP existed as a dimer and its GlcNAc 2-epimerase activity was strongly inhibited by the purified renin concomitant with the formation of RnBP-renin heterodimer, so-called high molecular weight (HMW) renin. The renin activity was also inhibited by rhRnBP in a dose-dependent manner. These results indicate that renin is an inhibitor of GlcNAc 2-epimerase, and the renin-RnBP heterodimer HMW renin is an inactive form of both renin and GlcNAc 2-epimerase activities.

Normal Blood Pressure and Plasma Renin Activity in Mice Lacking the Renin-binding Protein, a Cellular Renin Inhibitor

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.

Structure and function of renin binding protein

Renin binding protein (RnBP) is a proteinous renin inhibitor. This protein binds to renin forming a protein complex, so-called high molecular weight (HMW) renin. The protein complex has been confirmed by purification and characterization of HMW renin [1] and RnBP from porcine kidney [2, 3]. Recently, cDNA clones encoding RnBPs were isolated from porcine, human and rat kidney cDNA libraries [4, 5]. The amino acid sequences, which were deduced from the nucleotide sequences, were highly homologous and characterized by the presence of a conserved leucine-zipper motif. This motif plays an essential role in the formation of an RnBP homodimer and an RnBP-renin heterodimer [6]. The present study describes the biochemical and molecular biological properties of RnBPs.

Biosynthesis of a renin binding protein

The biosynthesis of a porcine renin binding protein (RnBP), which specifically binds to renin and forms an inactive high molecular weight renin, was investigated. mRNAs from various porcine tissues were used to investigate in vitro protein synthesis. The kidney mRNA directed the synthesis of a high level of RnBP, whereas the liver, adrenal and pituitary gland mRNAs gave a low but significant level of it. The in vitro synthesized RnBP as well as the immunologically detected RnBP synthesized in vivo had the same molecular weight, 42,000, as that of the purified protein. Moreover, both the human and rat kidney mRNAs directed the synthesis of this protein identified with an anti-porcine RnBP antibody. These results strongly indicate that RnBP, present in various mammalian species, is synthesized in renin-producing tissues as the mature size and undergoes binding with renin without proteolytic processing.

Maintenance of SHR Substrains and Development of a New Rat Strain

Comparative studies were performed on four SHR substrains; SHR C, SHR B2, SHRSP and M-SHRSP. When the blood pressure increased to 210 mmHg and above in SHRSP and MSHRSP, the plasma renin concentration, cerebrospinal fluid pressure and brain water content began to increase alongside of these elevations in blood pressure and showed significantly positive correlations. High molecular weight renin was also recognized after the blood pressure elevation above 210 mmHg. Blood pressures for SHR C and SHR B2 were below 210 mmHg.Continued successive selective brother-sister breeding of SHC and M-SHRSP hybrids produced a colony with severe hypertension (blood pressure above 210 mmHg) and marked hyper-cholesterolemia (plasma cholesterol level above 400 mg/dl) .

Changing renin substrates in human pregnancy.

Renin substrate of high molecular weight was identified in plasma from pregnant women, non-pregnant subjects and in samples of umbilical arterial and venous plasma, using gel filtration chromatography. The proportion existing in the high molecular weight form in pregnant women was significantly increased above that of non-pregnant subjects by 11 weeks after the last menstrual period and continued to rise to term. In contrast, the proportion in fetal plasma was significantly lower than that of the non-pregnant group. High molecular weight renin substrate was itself heterogeneous and appeared to consist of four sub-groups. In non-pregnant adults the first peak, eluting near the void volume of the column, was the largest. In the fetus this profile was reversed. The elution profile in maternal plasma had changed markedly by as early as 8 weeks of gestation. Thereafter, it was possible to see increasing dominance of the minor 'intermediate' peaks visible in the non-pregnant adult until, in the third trimester, the high molecular weight form was eluted as a large broad band with little evidence of the individual peaks. Oestrogens are the most likely stimulus for the increased production of the high molecular weight renin substrate in pregnant women. In the fetus, although the same form of renin substrate is stimulated, the actual amount in the high molecular weight range is very small. This may be because oestrogens are sulphated in the fetus or because the fetal liver is not very responsive to the stimulation. The similarity between the profiles of mother and fetus may indicate a common source of production of renin substrate in the feto-placental unit.

Renal high-molecular-weight renin: unusual formation in the aged stroke-prone spontaneously hypertensive rats.

A high-molecular-weight renin (HMWR) was detected in the plasma (molecular weight 50,600) and renal cortical homogenate (molecular weight 57,000) of 25-week-old male stroke-prone spontaneously hypertensive rats (SHRSP), in contrast to the renin with a molecular weight of 38,000 in normotensive Wistar Kyoto rats (WKY) and 5-week-old SHRSP. However, renin granules contained only the renin with a molecular weight of 38,000, indicating that the renal HMWR is not a native form but is probably a renin/renin-binding protein complex. Such HMWR was not produced when the renal cortex was homogenized with an equal amount of renal cortex of WKY. Further, the HMWR of the aged SHRSP was converted into the 38,000-dalton renin, when incubated with the extract of renal cortex of WKY. Thus, the existence of a renal cortical substance that converts the renal HMWR into the 38,000-dalton renin was evidenced. This substance was fractionated with DEAE-cellulose and was characterized as a putative SH enzyme. We conclude that a deficiency in the HMWR-converting substance may be attributed to the unusual formation of HMWR in the aged SHRSP.

High molecular weight renin in the plasma and kidney of SHRSP

High molecular weight renin in the plasma and kidney of SHRSP

Conversion of low molecular weight renin into the high molecular weight form by Cu2+ in the rat kidney.

The low molecular weight form of renin (M.W. 40000) in the extract of rat kidney cortex was converted into the high molecular weight form (M.W. 60000) in the presence of Cu2+ and this conversion was abolished following pretreatment of the extract with disodium ethylenediaminetetraacetic acid. Fe3+, Fe(CN)6(3-), Cd2+ and Zn2+ had no effects on the reaction of the molecular weight conversion of this enzyme. The reactivity in the formation of high molecular weight renin catalyzed by Cu2+ increased when the extract was previously dialyzed, indicating the presence of substance(s) that interfere with the binding reaction of renin. The high molecular weight renin produced by Cu2+ was stable against dithiothreitol, suggesting that the formation of disulphide bridge(s) is not involved in the reaction of renin with the renin binding substance. We also confirmed that for formation of the high molecular weight renin, Cu2+ must react with both the renin enzyme and the renin binding substance. Our findings provide new evidence related to the mechanism of molecular weight conversion of renin in the rat kidney.

An increase in high-molecular weight renin substrate associated with estrogenic hypertension

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form ( K m = 1800 ± 290 versus 3520 ± 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.

Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP).

The high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80 degrees C for 4 months. The specific activity was 3.97 mg of angiotensin I formed/mg of protein per h at 37 degrees C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39,000 and 32,000, respectively, by gel permeation liquid chromatography in 6 M guanidine-HCl. A modified rapid method for purification of renin is also presented.

Purification and characterization of renin binding protein (RnBP) from porcine kidney.

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.

Differences in the kinetic rate constants of normal and high molecular weight renin substrate from term pregnancy human plasma.

We have previously reported on the differences in physical and chemical characteristics between the high-molecular weight renin substrate (HMS greater than 150,000 daltons) and the normal substrate (NMS = 60,000). In this study, the kinetic constants were determined in both HMS and NMS which were prepared by gel exclusion chromatography from the plasma of pregnant women at term. Renin substrate (angiotensinogen) levels were expressed by radioimmunoassay of angiotensin I after incubation of samples with added semi-purified human kidney renin in the presence of angiotensinase inhibitors. The kinetic constants (Km and Vmax) were determined by the method of Line-weaver-Burk plots and also the method of Wilkinson. The Km for the HMS was 1.79 micrograms angiotensin 1 equivalents ( AIeq )/ml and the Vmax = 41.2 ngAIeq /ml/h, and the Km for the NMS was 3.52 micrograms AIeq /ml and the Vmax = 138 ng/ml/h. When adding small amounts of the HMS to the NMS, the production of angiotensin I was found to increase more than that in the NMS alone. It was also observed that the renin substrate reactivities of the plasma of pregnant women, which contained small amounts of the HMS, were higher than that found in the plasma of normotensive women not taking oral contraceptives. It is suggested that the existence of small amounts of the HMS may therefore contribute to the elevation in blood pressure under the influence of estrogens.

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A 0, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A 0, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.

High molecular weight renin in the mouse kidney.

This study was conducted to determine whether or not high molecular weight renin is present in the mouse kidney. The molecular weight of renin in the cytosol fraction of mouse kidney cortex was approximately 40000 as determined by gel filtration. Renin in this fraction was partially converted into high molecular form (54000) in the presence of sodium tetrathionate or N-ethylmaleimide, which are sulfhydryl group blockers. This high molecular weight renin was converted into regular size renin by acidification and trypsin treatment. In contrast, the molecular weight of renin in the granules was 40000, regardless of the absence or presence of sulfhydryl group blockers. Furthermore, neither change in molecular weight nor change in activity was observed after acidification. These results indicate that regular size renin is stored in the granules and may be converted into high molecular weight renin when it is released from the granules and reacts with some substance in the cytosol fraction of renal cortical tissue.

In vitro biosynthesis of human renin: evidence for a high molecular weight form.

Primary cultures of human chorionic cells have been developed. They secrete an inactive form of renin which can be activated by trypsin treatment. In biosynthesis experiments forming labelled renin the inactive renin exhibited a molecular weight (54 KD) higher than active kidney renin (44 KD). The same high molecular weight renin was obtained during biosynthesis experiments performed on tissue slices of human infarcted kidney. Therefore it is postulated that 54 KD biosynthetized renin might be the precursor of active renin.

Biochemical properties of the renin binding substance of rat kidney.

Renin extracted from isolated renin granules of the rat is a low molecular weight form (40,000 daltons). The renin binding substance which is capable of binding with the low molecular weight renin to form a high molecular weight renin (60,000 daltons) under sulfhydryl oxidation was found to be contained in the crude extract of rat renal cortex. This substance is presumably a protein with molecular weight of over 47,000 daltons by gel filtration. The most striking event was that rat renin binding substance was bound with dog renin and vice versa.