Biomedical subjects

J Chao

Publications and source records attributed to J Chao.

At least 145 records · Page 8Linked to original sources

PubMed1995

Biochemistry, regulation and potential function of kallistatin.

Components of the tissue kallikrein-kinin system include tissue kallikrein, kallistatin (kallikrein-binding protein), kininogen, kinin, bradykinin B1 and B2 receptors, and kininases. Tissue kallikrein is a serine proteinase which is capable of cleaving kininogen substrate to release the vasoactive kinin peptide. The binding of kinin to its specific receptor at target organs can produce a wide spectrum of biological effects. Kinin generation is primarily determined by the activity and availability of kallikrein since the level of kininogen is not a rate-limiting factor. Kallikrein levels are controlled by its rate of synthesis, activation, inactivation and clearance. The synthesis of tissue kallikrein is regulated transcriptionally, and its activity is regulated through post-translational processing and inactivation by inhibitors. Kallistatin is a newly discovered serine proteinase inhibitor (serpin) which forms a specific and covalently-linked complex with tissue kallikrein. Kallistatin may regulate tissue kallikrein's activity, bioavailability and clearance rate at the post-translational level. The major site of kallistatin synthesis is the liver with lower expression levels in the pancreas and kidney. Unlike many other serpins which are only present in the plasma, kallistatin is found in various tissues, cells and bodily fluids. The fact that both tissue kallikrein and kallistatin are widely distributed in tissues suggests kallistatin's role as a potential regulator of kallikrein outside the circulation. Protein purification and molecular cloning techniques have been used to study the structure, regulation and function of the components of the kallikrein-kinin system and for exploring their roles in ion transport, inflammation and blood pressure regulation. Considerable progress has been made in recent years to achieve these goals. This article provides an overview of the biochemical properties and potential physiological and pathophysiological roles of kallistatin.

Animals↗

PubMed1994

Cloning, sequence analysis and expression of the gene encoding the mouse bradykinin B2 receptor.

The bradykinin B2 receptor (B2R) mediates most of the biological effects of kinins. In the present study, we have cloned and sequenced the gene (B2R) from a mouse embryonic stem (ES) cell genomic library. Mouse B2R is 7 kb in length containing three exons of 0.3, 0.23 and 3.7 kb separated by two introns of 2.5 and 0.41 kb. The first and second exons are noncoding while the third exon contains the full-length coding region and a long 3' noncoding region. An ATG translation start codon, TGA stop codon and a polyadenylation signal (AATAAA) were identified in the third exon. B2R encodes a protein of 41,470 Da and 366 amino acids (aa) forming seven transmembrane (TM) domains. At the aa level, B2R shares 91 and 82% sequence identity with the rat and human B2R, respectively. In the 5' flanking region, a consensus TATA box, a putative cAMP-response element, a putative phorbol ester-response element, a putative AP-1-binding site and a putative IL-6-response element were identified. Southern blot analysis following reverse transcription and PCR showed that B2R is expressed in most mouse tissues, except the liver and spleen, which is consistent with the wide distribution of B2R activity as deduced from pharmacological studies.

Amino Acid Sequence↗

PubMed1994

Structure and chromosomal localization of the gene (BDKRB2) encoding human bradykinin B2 receptor.

The bradykinin B2 receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth muscle contraction and relaxation. In the present study, we have cloned and sequenced the gene encoding human bradykinin B2 receptor from a human genomic library. The B2 receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B2 gene shares high sequence identity with rat and mouse B2 receptor genes and significant similarity with the gene encoding the angiotensin II type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5' flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B2 receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B2 receptor was found to be expressed in most human tissues.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, sequence analysis, and chromosomal localization of the human protease inhibitor 4 (kallistatin) gene (PI4).

The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, and alpha 1-antitrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5'-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-q32.1, close to the serpin genes encoding alpha 1-antichymotrypsin, protein C inhibitor, alpha 1-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and sequence analysis of rat bradykinin B2 receptor gene.

The bradykinin B2 receptor mediates the effect of kinin. In order to understand the structure and regulation of its expression, we have cloned and sequenced the gene encoding the rat bradykinin B2 receptor and its 5' flanking region from a rat genomic library. The B2 receptor gene spans 7.3 kb in length and contains three exons which are separated by two introns. It encodes a peptide of 366 amino acids. The transcription initiation site was mapped by the primer extension assay. A variant TATA box sequence, an IL-6 response element and a cAMP response element were identified in the 5' flanking region of the rat bradykinin B2 receptor gene.

Amino Acid Sequence↗

PubMed1994

Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.

Carbocyanines↗

PubMed1994

Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.

Base Sequence↗

PubMed1994

Prostasin is a novel human serine proteinase from seminal fluid. Purification, tissue distribution, and localization in prostate gland.

A novel serine proteinase, designated as prostasin, has been purified from human seminal fluid to apparent homogeneity by DEAE-Sepharose CL-6B and aprotinin-affinity chromatography. The purified protein migrates as two close bands with an apparent molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions. It can be labeled with [14C]diisopropyl fluorophosphate and has a pI ranging from 4.5 to 4.8. Sequence analysis reveals that the two protein bands have an identical NH2-terminal amino acid sequence which is different from any known protein sequence in the SwissPro or GenBank data base. The NH2-terminal 20-amino acid sequence shares 50-55% identity with human alpha-tryptase, elastase 2A and 2B, chymotrypsin, acrosin, and the catalytic chains of hepsin, plasma kallikrein, and coagulation factor XI. Prostasin has trypsin-like activity with a pH optimum of 9.0, hydrolyzing peptidyl fluorogenic substrates: D-Pro-Phe-Arg-MCA, D-Phe-Phe-Arg-MCA, D-Val-Leu-Arg-MCA, and Z-Gly-Pro-Arg-AFC. It is inhibited by aprotinin, antipain, leupeptin, and benzamidine. The tissue distribution of prostasin was determined by a newly developed radioimmunoassay. Linear displacement curves for immunoreactive prostasin in body fluids and tissues were parallel with the standard curve of purified prostasin, indicating their immunological identity. Immunoreactive prostatin levels were 8.61 +/- 0.42 microgram/ml in the seminal fluid and 0.201 +/- 0.029 microgram/ml in urine. Prostasin is present at high levels in the prostate gland (143.7 +/- 15.9 ng/mg protein), moderate levels (2-6 ng/mg protein) in colon, lung, kidney, pancreas, salivary gland, liver, and bronchi, but it is not detected in the brain, muscle, testis, ventricle, atrium, and aorta. Immunohistochemical localization reveals that prostasin is present in epithelial cells and ducts of the prostate gland. These studies indicate that prostasin purified from seminal fluid is a novel serine proteinase and originates from the prostate gland.

Amino Acid Sequence↗

PubMed1994

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.

Amyloid beta-Protein Precursor↗

PubMed1994

Subcellular distribution of tissue kallikrein and Na,K-ATPase alpha-subunit in rat parotid striated duct cells.

Intracellular protein distribution and sorting were examined in rat parotid striated duct cells, in which tissue kallikrein is apical, and Na,K-ATPase is basolateral. Electron-microscopic immunogold cytochemistry, with both polyclonal and monoclonal antibodies, demonstrated these enzymes at opposite poles of the cells and in distinct intracellular sites. Kallikrein was found within apical secretory granules, whereas Na,K-ATPase was present on basolateral cell membranes. In addition, kallikrein was localized throughout cisternae of all Golgi profiles, whereas Na,K-ATPase (alpha-subunit) was found only in small peripheral vesicles and/or lateral cisternal extensions of a basal subset of Golgi profiles. These differences in the subcellular distribution of the two marker antigens were most clearly seen with double immunogold labelling. Our results suggest that kallikrein, an apical, regulated secretory protein, and Na,K-ATPase, a basolateral, constitutively transported membrane protein, are segregated at (or prior to) the level of the Golgi apparatus rather than in the trans-Golgi network (TGN), as was expected.

Animals↗

PubMed1994

Human tissue kallikrein induces hypotension in transgenic mice.

We investigated the role of the kallikrein-kinin system in blood pressure control by developing transgenic mice overexpressing human tissue kallikrein. Two lines of transgenic mice carrying the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter were established. Human tissue kallikrein was identified in pancreas, salivary gland, kidney, liver, and spleen of the transgenic mice by a specific radioimmunoassay for human tissue kallikrein. The immunoreactive human tissue kallikrein reached high levels in the circulation. The linear displacement curves for the transgenic product were parallel with the human tissue kallikrein standard curve, indicating their immunologic identity. The expression of human tissue kallikrein transcript in the transgenic mice was further confirmed by Northern blot analysis and by reverse transcription-polymerase chain reaction followed by Southern blot. Both lines of transgenic mice had significantly lowered blood pressure (86.4 +/- 13.5 mm Hg [mean +/- SD], n = 8 and 78.9 +/- 12.4 mm Hg, n = 8) compared with control mice (100.9 +/- 5.0 mm Hg, n = 8). Induction with zinc did not lower the blood pressure further despite elevated expression of the transgene. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored the blood pressure of the transgenic mice but had no significant effect on control littermates. Our findings raise the possibility of tissue kallikrein being a powerful modulator of blood pressure and provide a new animal model for the study of blood pressure regulation.

Animals↗

PubMed1994

Detection of a kallikrein in the mouse lactating mammary gland: a possible processing enzyme for the epidermal growth factor precursor.

Kallikreins are a multigene subfamily of serine proteases that may have a role in processing precursors of polypeptide hormones and growth factors. The epidermal growth factor (EGF) immunoreactivity in mouse milk is derived from the membrane-bound EGF precursor located on the lumenal border of the alveolar cells in the mammary gland. Release of EGF into the milk requires the hydrolysis of the EGF precursor at Arg-X cleavage sites. We report the presence of a candidate EGF precursor-processing enzyme in the lactating mouse mammary gland. Kallikrein transcripts in the mouse lactating mammary gland were detected by primer-directed enzyme amplification of complementary DNA (cDNA). Primers to selected conserved regions of the kallikrein cDNA resulted in an amplified product of the predicted size (573 basepairs). Sequence analysis of the product over three nonconserved regions identified mGK-6 (mouse renal kallikrein) as the primary kallikrein in BALB/c mouse lactating mammary gland. Transcription products for the EGF-binding protein (mGK-9), mGK-1, MGK-3, and mGK-4 were not detected by enzyme amplification with specific primers corresponding to these kallikrein cDNAs. Positive immunohistochemical staining of the apical membrane of mammary alveolar cells was detected with a polyclonal antiserum to mouse kallikrein. Incubation of cell membranes isolated from lactating mammary glands released soluble EGF-immunoreactive material. Aprotinin partially inhibited the release of this material, whereas other protease inhibitors, such as leupeptin, benzamidine, and limabean trypsin inhibitor, had no detectable effect. These results support the hypothesis that the release of EGF-immunoreactive material into the milk is in part dependent upon a kallikrein enzyme (mGK-6) in the BALB/c mouse lactating mammary gland.

Animals↗

PubMed1994

Molecular cloning and characterization of a novel kallikrein transcript in colon and its distribution in human tissues.

1. We have cloned and characterized a new species of kallikrein cDNA from a human colon cDNA library. The new kallikrein cDNA clone contains a part of intron 2 of the tissue kallikrein gene which is spliced to the remaining exon sequences. It does not contain exons 1 and 2. 2. An in-frame open reading frame is present in the new kallikrein cDNA allowing translation of a 216-amino acid product. The intron-containing kallikrein transcript was detected in salivary glands, pancreas, kidney, colon, prostate gland, testis, spleen, and lung by reverse-transcription/polymerase chain reaction followed by Southern blot analysis using an intron-containing kallikrein-specific oligonucleotide probe. 3. The results indicate that the new species of kallikrein may be processed by alternative splicing or arises from a different transcription initiation site.

Amino Acid Sequence↗

PubMed1994

Histopathology of lymphatic tissues in transgenic mice expressing human tissue kallikrein gene.

BACKGROUND: Tissue kallikrein is a member of a family of closely related serine proteinases whose genetics are currently under intense investigation, but whose functions are still poorly understood. Functions of human tissue kallikrein, other than production of inflammatory kinins, are not readily amenable to investigation. A current mechanism for examining the function of a gene product is to introduce the gene with tissue-specific and/or inducible promoters into a suitable host that can then be experimentally manipulated. EXPERIMENTAL DESIGN: Five transgenic mouse lines were established by backcross matings of transgenic founder mice containing the human tissue kallikrein gene. The inserted genomic material included the full-length kallikrein coding sequence as well as 800 bp in the upstream promoter region and 300 bp in the 3' noncoding region (PHK). Two of the five strains contained, in addition to the complete coding region and 3' flanking sequence, an upstream, zinc-inducible metallothionein promoter (MRE-PHK). Tissues from animals containing human kallikrein gene constructs (as determined by Southern blot of tail DNA) were examined histologically, and compared with control tissues from siblings negative for the gene. RESULTS: Transgenic mice exhibited tissue pathology in several lymphatic organs. Cytoarchitecture was disrupted in both thymus and spleen. The distinction between cortex and medulla in the thymus was usually blurred, and cytolysis was common. Spleens exhibited decreased T cell-dependent zones (periarteriolar sheath), with active hematopoietic foci throughout the red pulp. In lymph nodes, cortical nodules were rare. The deep cortex (paracortical area) was usually normal in heterozygotes, but often depleted in homozygous animals. Comparable results were obtained in all five transgenic strains. CONCLUSIONS: Expression of human tissue kallikrein appears to exert a profound effect on the cytoarchitecture of lymphatic tissues and a general decrease in lymphocytes, particularly in T cell-dependent areas. These findings presumably reflect altered function of lymphatic tissues in transgenic mouse strains carrying the human kallikrein gene.

Animals↗

PubMed1994

Family physicians' disagreements with the US Preventive Services Task Force recommendations.

BACKGROUND: The 1989 recommendations of the US Preventive Services Task Force (USPSTF) represent an emerging consensus about which clinical preventive services should be delivered. However, practicing physicians disagree with a number of the recommendations in the Task Force prevention guidelines, and the reasons for disagreement have not been widely explored. METHODS: A survey questionnaire assessing physician agreement or disagreement with the USPSTF recommendations was sent to all 1784 active members of the Ohio Academy of Family Physicians in October 1990. A factor analysis was performed on the items with which at least 5% of physicians disagreed. Associations of physician demographics and attitudes with the factor scores were then examined. RESULTS: At least 5% of the 898 responding physicians disagreed with 67 of 150 USPSTF recommendations. Physicians disagreed with the USPSTF recommendations in three ways: (1) they believed that screening for some cancers is appropriate, even though not recommended by the USPSTF; (2) they believed that screening for other diseases in some populations is appropriate, even though not recommended by the USPSTF; and (3) they disagreed with some USPSTF recommendations for screening that is considered time-consuming or intrusive. Further analyses showed that practice setting and experience with the USPSTF guidelines were predictive of all three disagreement factors. Physician age, race, residency training, and reasons for disagreement were associated with two of the three factors. CONCLUSIONS: Physician disagreement with the USPSTF recommendations was not random but clustered into three distinct factors. An opportunity exists to design educational interventions for targeted subgroups of physicians. The views of practicing physicians should be incorporated into future guidelines.

Adult↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and sequence analysis of the monkey and human tissue kallikrein genes.

Cynomolgus monkey renal kallikrein cDNA and genomic human tissue kallikrein gene were cloned. The monkey gene encodes a 257 amino acid (aa) preprokallikrein and exhibits 95% and 92% homology to the human at nucleotide (nt) and aa level, respectively. The monkey gene encodes a 233-aa mature kallikrein versus a 238-aa in human. The human kallikrein gene and urinary kallikrein both contain a Lys-162 instead of the reported Glu-162. Human, monkey and rat renal/pancreatic kallikrein genes evolve with a N-glycosylation containing domain (aa 81-87) which is absent in porcine and is non-glycosylable in mice. Only human kallikrein evolves with an additional Thr-108 and with a N-glycosylation site at aa-141.

Amino Acid Sequence↗

PubMed1993

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.

Animals↗