Kallikrein-binding Protein Research Articles

Intramuscular delivery of rat kallikrein-binding protein gene reverses hypotension in transgenic mice expressing human tissue kallikrein.

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure regulation. The activity of tissue kallikrein is controlled by a number of factors in vivo. Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor which binds to and inhibits tissue kallikrein's activity in vitro. We have recently developed several hypotensive transgenic mouse lines which express human tissue kallikrein. In order to investigate the role of RKBP in blood pressure regulation, we delivered the RKBP to these transgenic mice by intramuscular injection. Expression of the RKBP was detected in skeletal muscle by reverse transcription-polymerase chain reaction and Southern blot analysis at 10, 20, 30, and 40 days post-injection. Immunoreactive RKBP levels in the muscle and serum of these mice were quantified by a RKBP-specific enzyme-linked immunosorbent assay and Western blot analysis. The levels of RKBP mRNA and immunoreactive protein were detectable at 10 days post-injection and increased significantly at 20 and 30 days. During this period, RKBP delivery significantly increased systemic blood pressure in the kallikrein transgenic mice to a level comparable to that of normotensive control mice. The RKBP and vector DNA delivery had no effect on the blood pressure of normotensive control mice. No serum antibodies to RKBP or its DNA were detected in the mice 40 days post injection. These results suggest that the increase of systemic blood pressure by RKBP delivery in these hypotensive transgenic mice may be mediated by inhibiting tissue kallikrein activity.

Activation of serpins and their cognate proteases in muscle after crush injury

Direct muscle injury was induced in rats in order to evaluate alterations in the balance of serine proteases and inhibitors (serpins) as a response to tissue damage. It was previously found that certain proteases, specifically urokinase-like plasminogen activator (uPA) and others, required activation in order to effect regeneration. We hypothesized that the magnitude and temporal sequence of serpin activation would follow, pari passu, activation of their cognate proteases. In addition to uPA, tissue PA (tPA) and tissue kallikrein were the proteases studied. The serpins we analyzed were protease nexin I (PNI), PA inhibitor 1 (PAI-1, and the kallikrein-binding protein (KBP). uPA nearly doubled 48 h after injury, while there was no change in amidolytic activity after addition of fibrin monomer as an estimation of tPA activity. Tissue kallikrein activity, barely detectable in normal muscle, slowly increased, nearly tripling at 7 days after injury. Greater magnitude and more rapid changes in muscle serpins occurred over the same post-injury time course. By 24 h PNI increased threefold, while PAI-1 increased more slowly, reaching double the control values by 5 days after injury. Surprisingly, KBP, the serpin-class inhibitor of tissue kallikrein, had the most robust response, increasing tenfold over control 48 h after crush injury of muscle. These results further implicate the serpin:protease balance in tissue injury. Participation of complex receptors, such as the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP), various growth factors, cytokines, and other molecules, in regulating this balance is implicated by these data.

Kallistatin: a novel human serine proteinase inhibitor. Molecular cloning, tissue distribution, and expression in Escherichia coli.

We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities. In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction. The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide. The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein. Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser. Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin. In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84. A genomic Southern blot using the full-length kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied. The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli. The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa. The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E. coli provides means for studying its structure-function relationship through protein engineering.

Expression and characterization of rat kallikrein-binding protein in Escherichia coli

Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein. We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein [Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J. Biol. Chem. 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J. Biol. Chem. 266, 16029-16036]. In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system. A high level of expression was detected by an e.l.i.s.a. with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture. The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE. It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis. The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography. The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a. To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein. The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding. The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein. This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes.

Regulation of Kininogen Gene Expression and Localization in the Lung after Monocrotaline-Induced Pulmonary Hypertension in Rats

Pyrrolizidine monocrotaline (MCT) from plant seed produces pulmonary endothelial cell injury, pulmonary hypertension, and inflammation in rats, providing a useful animal model for studying progressive pulmonary vascular disease. Kininogen is the precursor of proinflammatory kinins and may also exert anti-inflammatory actions by inhibiting cysteine proteinases. Given the potential roles of kininogen in vascular injury and inflammation, we have investigated the regulation of kininogen gene expression in the MCT-induced pulmonary hypertensive rat model. Sprague-Dawley rats, in groups of six, were given a single subcutaneous injection of monocrotatine (60 mg/kg body wt) and sacrificed 10 and 20 days later. Northern blot hybridization using a kininogen cDNA probe showed kininogen gene expression in the liver, lung, and kidney. MCT treatment induced a time-dependent increase in kininogen mRNA levels, whereas it reduced rat alpha 1-antitrypsin and kallikrein-binding protein mRNA levels in the liver. Similarly, kininogen mRNA levels were low in the normal lung and were increased 7.5- and 13.7-fold, respectively, after MCT injection for 10 and 20 days. Immunoreactive kininogen levels in perfused liver and lung extracts of rats receiving MCT injection increased up to 20-fold, as measured by a T-kininogen radioimmunoassay. Western blot analyses showed that a 68-kilodalton immunoreactive kininogen increased in the serum and lung extracts of MCT-treated rats compared to those in the control rats. In control rats, immunostaining for kininogen in the lung was most marked in venous endothelial cells and alveolar macrophages. After MCT treatment, staining for kininogen increased dramatically throughout the lung tissues, often covering the epithelial surfaces of alveoli and bronchi. The present studies have shown that the toxin MCT altered the synthesis and distribution of pulmonary kininogen and suggest that the kininogen/kinin system may be associated with the pulmonary vascular injury, remodeling, and inflammation seen in this animal model.

23] Screening of complementary DNA library using radiolabeled antigen

The identification of bacterial colonies expressing fusion proteins encoded by cloned cDNA inserts in expression libraries can be achieved by several different approaches. Screening of cDNA clones with immunological methods is popular because only specific antibodies are required so that protein sequencing and oligonucleotide construction are bypassed. The immunoscreening of expression cDNA libraries with radiolabeled antigens is a sensitive and specific method for identifying cDNA clones encoding the protein of interest. This method is successfully in use to isolate cDNA clones coding for rat a α 1 -antitrypsin, rat kallikrein-binding protein, and human kininogen. However, screening expression cDNA libraries with immunological methods can detect only the fraction of cDNA inserts that produce proper protein products recognized by their specific antibodies. It is often necessary to use the isolated eDNA or oligonucleotides derived from the clones as probes for rescreening the library to obtain full-length cDNA clones. Nevertheless, the high degree of specificity of the immunoscreening method using antigen overlay makes it a valuable alternative to other screening methods, especially when nucleic acid probes are not available for the initial screening efforts.

Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and α1-proteinase inhibitor

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and α1-proteinase inhibitor (α1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 1 with association rate constants in the 10 3–10 4 M −1·s −1 range, whereas only ‘true’ tissue kallikrein rK1 was not suceptible to α1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the ony member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.

Inhibition of tissue kallikrein by protein C inhibitor. Evidence for identity of protein C inhibitor with the kallikrein binding protein.

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparin-dependent serine protease inhibitor present in plasma and urine. PCI inhibited the amidolytic activity (cleavage of H-D-valyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 x 10(4) M-1 s-1 and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PCI protein band (Mr = 57,000) and an increase in the intensity of the lower Mr (54,000) PCI form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PCI and with the formation of tissue kallikrein-PCI complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 micrograms/ml. This is in contrast to findings on the interaction of PCI with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of 125I-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PCI and this kallikrein binding protein we incubated 125I-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PCI and 125I-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PCI may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PCI may be identical to the previously described kallikrein binding protein.

Possible identity of kallikrein binding protein with protein C inhibitor.

Protein C inhibitor (PCI) inhibits tissue kallikrein by forming stable 1:1 complexes (k1 = 2.3 x 10(4)M-1s-1). Heparin inhibits the tissue kallikrein/PCI-interaction and complex formation of 125I-tissue kallikrein in serum. 125I-tissue kallikrein complexes formed in plasma can be immunoprecipitated with monoclonal anti-PCI IgG suggesting that PCI might be identical to the kallikrein binding protein described previously (J. Chao et al. 1986, Biochem. J. 239, 325-331).

Expression of kallikrein-binding protein and alpha 1-antitrypsin genes in response to sex hormones, growth, inflammation and hypertension.

We have recently purified rat kallikrein-binding protein (RKBP) and alpha 1-antitrypsin (alpha 1-AT) to homogeneity and isolated, sequenced cDNAs encoding these potential regulators of tissue kallikreins. Characterization of the cDNA and the gene has established the identity of the kallikrein-binding protein as a new member of the serpin (serine proteinase inhibitor) superfamily. Using the cDNA probes in Northern blot hybridization, we found a differential regulation of RKBP and alpha 1-AT gene expression in the liver. Ovariectomy results in a 67% reduction of RKBP mRNA levels but a 30% increase of alpha 1-AT mRNA levels. Estradiol or progesterone treatment of the ovariectomized rats increases RKBP transcripts by 2.5- and 6.5-fold, respectively, but reduces alpha 1-AT mRNA level by 30% and 45%, respectively. In contrast to kininogen expression, both RKBP and alpha 1-AT mRNA levels in the liver are at the lowest at birth and rapidly increase during growth and development. Rats injected with endotoxin from 4 to 24 h show a time-dependent decrease of RKBP mRNA levels while the same treatment induces alpha 1-AT gene expression. RKBP mRNA levels in the normotensive Wistar Kyoto (WKY) rats are higher than those in the spontaneously hypertensive rats (SHR) while there are no differences of alpha 1-AT mRNA levels between SHR and WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of kallikrein gene family proteases in rat tissues.

Monoclonal antibodies specific for three kallikrein gene family enzymes (tissue kallikrein, esterase A and tonin) have been used to determine the tissue and cellular distributions of these proteases as well as their association with other relevant molecules (kininogen, kallikrein-binding protein, and Na,K-ATPase alpha-subunit). Secretion of these enzymes from salivary glands was also analyzed. The results of these localization studies provide important clues to the functions of different members of this closely related family of serine proteases.

Molecular cloning and sequence analysis of the mouse kallikrein-binding protein gene

A genomic clone (MKBP-10) encoding the mouse kallikrein-binding protein (MKBP) was isolated from a mouse genomic DNA library by screening with a rat kallikrein-binding protein (RKBP) cDNA probe. The total sequenced region of the MKBP gene spans 8615 base pairs. The exon and intron locations of the RKBP gene were identified by similarity with the RKBP gene. The MKBP gene encodes a prepeptide of 417 amino acid residues which exhibits 71% homology with RKBP. A TATA box sequence was located in the 5' flanking region of the MKBP gene by similarity with the consensus sequence TATAAAA.

Molecular cloning and analysis of the rat kallikrein-binding protein gene

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.

Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats.

Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.

Differential interactions of human kallikrein-binding protein and α1-antitrypsin with human tissue kallikrein

The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.

A major difference of kallikrein-binding protein in spontaneously hypertensive versus normotensive rats

A unique tissue kallikrein-binding protein was identified and partially characterized in the brain and serum of Sprague-Dawley rats and in the serum-free conditioned media of mouse anterior pituitary cells (AtT 20) and rodent neuroblastoma x glioma hybrids (NG108-15). Kallikrein and kallikrein-binding protein(s) form SDS- and heat-stable complexes with a molecular weight (Mr) of approximately 92,000. The complex formation of 125I-labelled kallikrein and the binding protein in the serum and brain is inhibited by excess unlabelled rat urinary kallikrein, rat arginine esterase A (a kallikrein-like kininogenase), and human urinary kallikrein. When the active site of kallikrein was blocked by phenylmethylsulfonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl, no complex formation was detected. Kallikrein-binding protein only forms complexes with active kallikrein or trypsin-activated prokallikrein but not with prokallikrein. 125I-labelled kallikrein forms a 92-kilodalton protein with binding protein in various brain regions of perfused normotensive rats of the Wistar-Kyoto strain (WKY), including the cerebral cortex, cerebellum and brain stem; but complex formation was not found in corresponding brain regions of the spontaneously hypertensive rat (SHR). Similarly, the kallikrein-binding protein was identified in various tissues including thymus, lung, liver, prostate, Cowper's gland, adrenal gland, kidney, and pancreas of WKY rats but not in tissues of SHR. The results suggest a major difference in the kallikrein-binding protein in hypertensive versus normotensive rats. The role of this specific kallikrein-binding protein in cellular hemodynamic processes and blood pressure regulation remains to be investigated.

Identification of a new tissue-kallikrein-binding protein.

We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15). Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex. The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms. Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min. Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens. The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation. However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH. When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed. An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column. These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses. The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III. The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated.

Proteins binding to kallikrein and esterase A2 in the urine of salt-sensitive and salt-resistant rats.

Iodine-labeled ([125I]) rat urinary kallikrein and rat urinary TAME esterase A2 were used as probes to look for urinary and plasma proteins that bind to these enzymes. Such proteins are presumptive enzyme inhibitors. Complexes formed with labeled enzymes were identified by polyacrylamide gel electrophoresis followed by autoradiography. Urine from young (6 weeks old) Dahl salt-sensitive (S) rats showed no, or only traces, of protein binding to kallikrein. Concomitant with the slow development of hypertension and proteinuria in S rats fed normal rat chow, one of the six kallikrein-binding proteins demonstrable in plasma was readily found in S-rat urine. This kallikrein-binding protein was called "KBP-1." R rats showed either no or much less KBP-1 in the urine, compared to S rats up to 5 months of age. A partly purified preparation of KBP-1 was shown to inhibit the TAME esterase activity of rat urinary kallikrein in the radiometric TAME assay. Urine of proteinuric S rats also contained two TAME esterase-binding proteins, TEBP-1 and TEBP-2, detected with the [125I]-esterase A2 probe. As S rats aged from 3 to 8 months, free KBP-1 disappeared from the urine in spite of increased and marked proteinuria and the continued presence of KBP-1 in plasma. Concomitant with this age-related loss of urinary KBP-1 there was a marked shift in S urinary proteins binding to [125I]-esterase A2 from TEBP-1 to TEBP-2. It was speculated that KBP-1 and TEBP-1 were the same protein detectable with either labeled kallikrein or labeled esterase A2. The concomitant disappearance of free KBP-1 (TEBP-1) and the appearance of free TEBP-2 in the urine of old, hypertensive, proteinuric S rats suggests that: 1) most of the KBP-1 (TEBP-1) is bound to enzyme(s) in old rats; or 2) KBP-1 (TEBP-1) is largely converted to TEBP-2 in old rats; or 3) both are true and that binding of KBP-1 (TEBP-1) to enzymes is associated with the generation of TEBP-2.