Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Characterization of the anti-DnaJ monoclonal antibodies and their use to compare immunological properties of DnaJ and its human homologue HDJ-1

Escherichia coli DnaJ (Hsp40) is suspected to participate in rheumatoid arthritis (RA) pathogenesis in humans by an autoimmune process. In this work a set of 6 anti-DnaJ monoclonal antibodies (mAbs) was raised and localization of the epitopes recognized by the mAbs was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) experiments showed that the mAbs efficiently bound only native antigen. Using DnaJ mutant proteins with deletions of specified domains and ELISA, we found that AC11 mAb reacted with the best conserved in evolution N-terminal J domain, whereas BB3, EE11, CC5, CC8, and DC7 bound to the C-terminal part after residue 200. Mapping performed with the use of a random peptide library displayed by filamentous phage indicated that (1) AC11 mAb bound to a region between residues 33-48, including D-34 which belongs to the HPD triad, present in all DnaJ homologues, (2) BB3 recognized residues localized in the 204-224 region, (3) EE11 recognized the 291-309 region, (4) CC5--the region 326-359, and (5) CC8--the 346-366 region. All these mAbs, as well as the polyclonal antibodies against the N- or C-terminal domain, bound efficiently to HDJ-1, human Hsp40. These results show the presence of a significant immunological similarity between bacterial DnaJ and human HDJ-1, which is not restricted to the evolutionarily conserved parts of the proteins, and suggest that HDJ-1 could be a possible target of immune response triggered by DnaJ.     

A synthetic peptide spontaneously self-assembles to reconstruct a group-specific, conformational determinant of hepatitis B surface antigen.

A cysteine-rich peptide of sequence 124 to 147 of the major protein of hepatitis B surface Ag (HBsAg) was synthesized. On cleavage and subsequent work-up it was found that all of the cysteine sulfhydryl groups had spontaneously formed disulfide bonds to yield a heterogenous mixture of multiple forms with molecular masses ranging from 8 to 35 kDa (peptide OS[124-147]). In a direct ELISA peptide OS[124-147] showed a high degree of cross-reactivity with polyclonal anti-HBsAg antiserum whereas the HBsAg-related antigenicity of its disulfide-reduced analogs was insignificant. Peptide OS[124-147] was also recognized by all 15 of the anti-HBsAg-positive human sera tested. Further studies revealed that peptide OS[124-147] represents the conformational, disulfide-dependent "a" determinant of HBsAg and elicits antibodies that cross-react with a variety of HBsAg subtypes. Anti-peptide antibodies bound to the corresponding native epitope with an apparent affinity higher than that of homologous antisera. Finally, polyclonal anti-OS[124-147] antibodies could also immunoprecipitate purified Dane particles in solution. Together these studies indicate that peptide OS[124-147] represents an excellent candidate component of a peptide-based vaccine for hepatitis B.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

A Continuous Fluorescence Assay of Renin Activity

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate K_m 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability.     

Effect of point mutations in the native simian virus 40 tumor antigen, and in synthetic peptides corresponding to the H-2Db-restricted epitopes, on antigen presentation and recognition by cytotoxic T lymphocyte clones.

The effects on CTL recognition of individual amino acid substitutions within epitopes I, II, and III of SV40 tumor Ag (T Ag) were examined. Epitope I spans amino acids 207 to 215, and epitope II/III is within residues 223 to 231 of SV40 T Ag. An amino acid substitution at position 207 (Ala----Val) or 214 (Lys----Glu) of SV40 T Ag expressed in transformed cells resulted in loss of epitope I, recognized by CTL clone Y-1. The amino acid substitution at residue 214 in the corresponding synthetic peptide, LT207-215(214-Lys----Glu), also led to loss of recognition by CTL clone Y-1. The recognition, by CTL clone Y-1, of peptides LT207-215 and LT207-217 with an Ala----Val substitution at position 207 was severely affected. Peptides LT205-215 and LT205-219 with the Ala----Val substitution at residue 207 were, however, recognized by CTL clone Y-1, suggesting that residues 205 and 206 may be involved in presentation of site I. Alteration of residue 224 (Lys----Glu) in the native T Ag resulted in loss of recognition by both CTL clones Y-2 and Y-3. However, a peptide corresponding to epitope II/III with an identical amino acid substitution at residue 224 provided a target for CTL clone Y-3 but not clone Y-2. A change of Lys----Gln at residue 224 in both the native protein and a synthetic peptide caused loss of recognition by CTL clone Y-2 but not CTL clone Y-3. Further, an amino acid substitution of Lys----Arg at position 224 of the native T Ag decreased recognition of epitope II/III by CTL clones Y-2 and Y-3 but had no effect on recognition of a synthetic peptide bearing the same substitution. These results indicate that the mutagenesis approach, resulting in identical amino acid substitutions in the native protein and in the synthetic peptides, may provide insight into the role of individual residues in the processing, presentation, and recognition of CTL recognition epitopes.     

The His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin

The amino acid sequence His-Pro-Phe as N-terminal residues 6-8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5-10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9-11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

Physicochemical characteristics of human high molecular weight angiotensinogen

A high molecular weight angiotensinogen (Mr. 332,000 daltons) was prepared from plasma of pregnant women by gel filtration on Sephacryl S-300. The molecular weight was reduced to 81,000 by treatment with dithiothreitol (DTT), but not by treatment with SDS. DTT-treated high molecular weight (HMW) angiotensinogen was very similar to low molecular weight (LMW) angiotensinogen with respect to molecular weight, pH profile for angiotensin formation by human kidney renin, thermostability, Km value and isoelectric point. The antibody against LMW-angiotensinogen completely cross-reacted with HMW-angiotensinogen. These results suggest that HMW-angiotensinogen is probably a complex of LMW-angiotensinogen and other protein(s) which might be bound by disulfide bond.     

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.     

Purification and characterization of rat urinary renin

Rat urinary renin was purified by a procedure involving ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B chromatography, ion exchange chromatography and gel filtration. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 39000 by SDS-gel electrophoresis and 40000 by gel filtration. The optimum pH determined with rat angiotensinogen was 7.0, and the Km was 3.6 microM. These properties agreed well with those of purified rat renal renin. The activity of urinary renin was specifically inhibited by anti-renin antibody. These results suggest that urinary renin may originate in the kidney.     

A primate bioassay for the determination of renin inhibitory peptides in serum

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.     

Primary structure requirements for the binding of human high molecular weight kininogen to plasma prekallikrein and factor XI

We recently identified residues 185-224 of the light chain of human high molecular weight kininogen (HMWK) as the binding site for plasma prekallikrein (Tait, J.F., and Fujikawa, K. (1986) J. Biol. Chem. 261, 15396-15401). In the present study, we have further defined the primary structure requirements for binding of HMWK to factor XI and prekallikrein. In a competitive fluorescence polarization binding assay, a 31-residue synthetic peptide (residues 194-224 of the HMWK light chain) bound to prekallikrein with a Kd of 20 +/- 6 nM, indistinguishable from the previously determined value of 18 +/- 5 nM for the light chain. We also prepared three shorter synthetic peptides corresponding to different portions of the 31-residue peptide (residues 205-224, 212-224, and 194-211), but these peptides bound to prekallikrein more than 100-fold more weakly. Factor XI also bound to the same region of the HMWK light chain, but at least 58 residues (185-242) were required for optimal binding (Kd = 69 +/- 4 nM for the light chain; Kd = 130 +/- 50 nM for residues 185-242). The four synthetic peptides inhibited kaolin-activated clotting of blood plasma with potencies paralleling their affinities for prekallikrein and factor XI. Peptide 194-224 can also be used for rapid affinity purification of prekallikrein and factor XI from plasma.     

Direct photoaffinity labeling of cysteine 211 or a nearby amino acid residue of beta-tubulin by guanosine 5'-diphosphate bound in the exchangeable site

Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.     

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.     

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.     

Purification of Canine Plasma Renin by Affinity Chromatography

An affinity column for the purification of canine plasma renin was prepared using goat anti-renin (dog kidney) γG globulins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide-activated Sepharose. Using the anti-renin globulin-coupled Sepha-rose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1, 000-fold purity.