Charles E. Odya

Kinin and angiotensin metabolism by purified renal post-proline cleaving enzyme

Post-proline cleaving enzyme (PPCE; EC 3.4.21.26) is a proline specific endopeptidase capable of hydrolyzing biologically active peptides. The present studies examined the hydrolysis of kinin- and angiotensin-related peptides by cytosolic PPCE purified from porcine kidney. PPCE hydrolysis of the synthetic substrate Z-Gly-Pro-MCA (30.7 +/- 0.3 mumol . min-1 . mg-1) was competitively inhibited by saralasin, bradykinin, des(Arg9)bradykinin, [Leu8], des(Arg9)bradykinin and angiotensin II (IC50 = 0.5 to 7.0 microM). Qualitative TLC studies demonstrated that each peptide was degraded by hydrolysis on the carboxyl side of proline residues (positions 7 or 8). Quantitative HPLC studies established that peptide degradation was optimal at pH 8.2 to 8.7 and was inhibited by the specific PPCE inhibitor Z-Pro-prolinal (IC50 = 0.8 +/- 0.1 nM). Conversely, degradation was unaffected by inhibitors of aminopeptidases (amastatin), neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin I converting enzyme (captopril). Apparent Km values, obtained from Lineweaver-Burk analysis, were comparable for all kinin and angiotensin peptides (Km = 5.5 to 12.8 microM), whereas Vmax values ranged from 1.7 mumol . min-1 . mg-1 for angiotensin II to 0.44 mumol . min-1 . mg-1 for saralasin. These data are consistent with a role for PPCE in the degradation of kinins and angiotensin in vivo.

Further studies of myometrial bradykinin receptor-like binding

[125I-Tyr1]Kallidin (T1K), a bradykinin (BK) analog with biological potency comparable to BK, was used as a probe for BK receptor-like binding from bovine uterine myometrium. BK binding exhibited a high affinity, Kdissoc = 1.65 X 10(-10) M. The specificity of T1K binding was examined with forty-four BK analogs. Comparison of the binding inhibitory potencies with the relative biological potencies of these analogs on isolated rat uterus resulted in a good correlation, r = 0.87. BK binding activity was solubilized with CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent. The solubilized binding activity exhibited a BK binding affinity, Kdissoc = 2.25 X 10(-10) M, and a specificity for three 125I-labeled kinins similar to those of the particulate BK receptor-like binding activity.

Development of a radioimmunoassay for [DES-ARG9]-bradykinin.

Antisera to [des-Arg9]-bradykinin were elicited in rabbits immunized with the peptide conjugated to thyroglobulin and/or ovalbumin. Sera were screened for the presence of antibody with three radioactive antigens, mono-125I-labeled derivatives of [Tyr1, des-Arg]-kallidin, [Tyr, des-Arg]-bradykinin, and [Tyr, des-Arg]-bradykinin that were prepared by treating mono-125I-labeled [Tyr]-kallidin, [Tyr]-bradykinin, and [Tyr]-bradykinin with carboxypeptidase B. Of the six animals immunized, five produced antibodies to [des-Arg]-bradykinin as evidenced by the ability of their sera to bind at least 33% of the added radioactivity at a final dilution of 1:500. Sensitivity and specificity studies were performed with each labeled antigen and a dilution of antiserum that bound 30-50% of the radioactivity. The best labeled antigen-antibody combination, with respect to titer, sensitivity, and specificity was obtained with [mono-125I-Tyr, des Arg]-bradykinin and serum from a rabbit immunized with [des-Arg]-bradykinin conjugated to ovalbumin with toluene diisocyanate. The lowest concentration of [des-Arg]-bradykinin inhibiting 50% of this radioactive antigen binding was 0.23 ng/ml and the lowest concentration which could be distinguished from no [des-Arg]-bradykinin added was 67 pg/ml. This antiserum cross-reacts with bradykinin and lysyl-bradykinin about 9% but not with methionyl-lysyl-bradykinin.

Interactions of kinins with angiotensin I converting enzyme (kininase II)

Angiotensin I converting enzyme (ACE) was purified to homogeneity from porcine kidney in order to determine whether iodobradykinins bind to the enzyme and, if so, whether pGlu-Trp-Pro-Arg-Pro-Gin-Ile-Pro-Pro, SQ20881, a competitive ACE inhibitor, changes the conformation of the enzyme in such a way that it binds kinins with an affinity and specificity expected of a bradykinin (BK) receptor, i.e. where the BK potentiating action of SQ20881 involves an increase in the number of BK receptors due to a conformational change in ACE. 125I-Labeled derivatives of [Tyr1]-kallidin and [Tyr-8]-bradykinin bound to the EDTA-inhibited enzyme, and binding was inhibited by nonradioactive BK. [125I-Tyr5]-BK was not bound by the enzyme. Specificity of [125I-Tyr5]-kallidin (T1K) binding was tested with forty-eight BK analogs, and the concentrations of analogs that inhibited 50% of T1K binding were determined. BK at 1.6 +/- 0.3 X 10(-8) M inhibited 505 of T1K binding. In addition, the concentrations of analogs that decreased by 50% the rate of [3H]-Hip-Gly-Gly ([3H]-HGG) hydrolysis by ACE were assessed. BK at 1.2 +/- 0.2 X 10(-6) M decreased the rate of [3H]-HGG hydrolysis by 50%. A comparison between these concentrations of analogs for inhibition of T1K binding and [3H]-HGG hydrolysis yielded a high correlation coefficient (r = 0.85). The specificity of ACE binding was clearly different from that expected of a BK receptor. Compounds structurally unrelated to BK, such as 5Q20881, pGlu-Lys-Trp-Ala-Pro-OH (BPP5a) and angiotensin I, inhibited T1K binding and [3H]-HGG hydrolysis by ACE.

Characterization of soluble bradykinin receptor-like binding sites

Bradykinin (BK) receptor-like binding sites were solubilized from a particulate fraction of bovine uterine myometrium (BUM) using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). Scatchard analysis of [125I-Tyr1]kallidin (T1K) binding revealed a single class of soluble binding sites with KD = 0.35 nM and Bmax = 0.13 pmol/mg protein. The soluble binding sites exhibited a kinin-binding specificity comparable to that of the particulate BUM receptor-like sites. Soluble T1K binding association kinetics were first-order at the four T1K concentrations examined. A plot of the pseudo-first order rate constant (Kobs) versus T1K concentration was linear, and values for the association (k1) and dissociation (k-1) rate constants were obtained. These rate constants yielded a kinetically derived equilibrium dissociation constant (KD = 0.64 nM) which was comparable to that obtained by Scatchard analysis. Biphasic dissociation of bound T1K was resolved into rapid and slow dissociation phases. The rate constant (k-1) of the rapid dissociation phase was comparable to the dissociation rate constant determined in association experiments. A biphasic loss of soluble T1K binding activity was observed with storage at 10 degrees C.

Specific, high-affinity bradykinin binding by purified porcine kidney post-proline cleaving enzyme

Post-proline cleaving enzyme (PPCE) was purified from porcine kidney cytosol. The purified enzyme bound [125I-Tyr5]-bradykinin but neither [125I-Tyr1]-kallidin nor [125I-Tyr8]-bradykinin. Scatchard analysis of the data was consistent with a single class of binding sites with a Kassoc = 1.3 +/- 0.1 X 10(8) M-1. The optimal pH for [125I-Tyr5]-bradykinin binding was 6.8. The specificity of binding was evaluated with sixty-seven bradykinin analogs. The catalytic activity of the enzyme was measured with N-benzyloxycarbonyl-Gly-Pro-methylcoumarinyl-7-amide (Z-Gly-Pro-MCA). The optimal pH for hydrolysis of this substrate was broad and centered at 8.3. The apparent Km and Vmax were obtained from Lineweaver and Burk plots and were 4.8 +/- 0.4 X 10(-5) M and 42 +/- 5 mumoles X mg-1 X min-1 respectively. The IC50 values for bradykinin, diisopropylfluorophosphate (DFP), and N-benzyloxycarbonyl-Pro-Prolinal (Z-Pro-Prolinal) to inhibit Z-Gly-Pro-MCA hydrolysis by PPCE were 5.9 +/- 1.4 X 10(-7) M, 8.8 +/- 3.1 X 10(-7) and 7.9 +/- 0.3 X 10(-9) M respectively. Corresponding values for inhibition of [125I-Tyr5]-bradykinin binding by PPCE were 5.1 +/- 2.3 X 10(-9) M, 1.2 +/- 0.3 X 10(-6) M and 1.4 +/- 0.6 X 10(-8) M.

Immunoassays for des-Arg9-bradykinin

Splenocytes from a female, BALB/c mouse immunized with bradykinin conjugated to ovalbumin with toluene diisocyanate were fused with mouse myeloma cells, X63/Ag8.653, using polyethylene glycol. Seventy-nine hybridomas were identified by ELISA to be making kinin reactive antibodies. In preliminary specificity studies it was determined that all of these hybridomas were producing antibodies more reactive with des-Arg9-bradykinin than with bradykinin. ELISAs were developed with the five clones that displayed the highest affinities for des-Arg9-bradykinin. Radioimmunoassays were developed for 3 of these 5 clones as well as with 5 monoclonal antibodies previously described (Odya and Lee 1990). The most sensitive des-Arg9-bradykinin assay developed was a radioimmunoassay in which carboxypeptidase B-treated [Tyr5]-bradykinin was the labeled antigen, clone OLNBK-5 was the antibody, and dextran-coated charcoal was used to separate bound from free radioactivity. The concentration of des-Arg9-bradykinin that inhibited 50% of the radioactive peptide binding was 0.08 +/- 0.03 nM. The relative specificity of this assay (des-Arg9-bradykinin = 100%) was: 29% bradykinin and about 1% with each of the following: lysyl-bradykinin, methionyl-lysyl-bradykinin, des-Arg1-bradykinin and des-Phe8-Arg9-bradykinin.

Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes

There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a kappa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2-3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously.