Identification, purification, and localization of tissue kallikrein in rat heart.
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ABSTRACT:
SUBMITTER: Xiong W
PROVIDER: S-EPMC1131345 | biostudies-other | 1990 May
REPOSITORIES: biostudies-other
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Project description:A tissue kallikrein was purified from rat skeletal muscle. Characterization of the enzyme showed that it has alpha-N-tosyl-L-arginine methylesterase activity and releases kinin from purified bovine low-Mr kininogen substrate. The pH optimum (9.0) of its esterase activity and the profile of inhibition by serine-proteinase inhibitors are identical with those of purified RUK (rat urinary kallikrein). Skeletal-muscle kallikrein also behaved identically with urinary kallikrein in a radioimmunoassay using a polyclonal anti-RUK antiserum. On Western-blot analysis, rat muscle kallikrein was recognized by affinity-purified monoclonal anti-kallikrein antibody at a position similar to that of RUK (Mr 38,000). Immunoreactive-kallikrein levels were measured in skeletal muscles which have different fibre types. The soleus, a slow-contracting muscle with high mitochondrial oxidative-enzyme activity, had higher kallikrein content than did the extensor digitorum longus or gastrocnemius, both fast-contracting muscles with low oxidative-enzyme activity. Streptozotocin-induced diabetes reduced muscle weights, but did not alter the level of kallikrein (pg/mg of protein) in skeletal muscle, suggesting that insulin is not a regulator of kallikrein in this tissue. Although the role of kallikrein in skeletal muscle is unknown, its localization and activity in relation to muscle functions and disease can now be studied.
| S-EPMC1147924 | biostudies-other
Project description: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.
| S-EPMC1147284 | biostudies-other
Project description:We have cloned and characterized a full-length cDNA encoding tissue kallikrein from a human colon carcinoma cell line (T84). The nucleic acid sequence of the colon kallikrein cDNA is identical to that of renal/pancreatic or tissue kallikrein cDNA. Reverse-transcription PCR followed by Southern-blot analysis using specific oligonucleotide probes showed expression of tissue kallikrein in human colon, pancreas and kidney. Tissue kallikrein mRNA was localized in glandular epithelial cells (goblet cells) in colon by in situ hybridization histochemistry. Human colon kallikrein was purified to apparent homogeneity by DEAE-Sepharose Cl-6B, aprotinin-affinity, and HQ/M perfusion chromatography. The purified colon kallikrein migrated as a broad, 40-45 kDa band in SDS/PAGE and was recognized by antibodies to human tissue kallikrein. The linear displacement curves for the colon kallikrein in an RIA were parallel with the human tissue kallikrein standard curve, indicating their immunological identity. The N-terminal sequence of the purified colon kallikrein matches completely with that of purified urinary or tissue kallikrein. These results indicate that human colon kallikrein is transcribed from the tissue kallikrein gene.
| S-EPMC1136673 | biostudies-other
Project description:A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3' end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.
| S-EPMC1151144 | biostudies-other
Project description:Mitochondria are considered the main organelles in the cell. They play an important role in both normal and abnormal heart function. There is a supramolecular organization between the complexes of the respiratory chain (supercomplexes (SCs)), which are involved in mitochondrial respiration. Prohibitins (PHBs) participate in the regulation of oxidative phosphorylation (OXPHOS) activity and interact with some subunits of the OXPHOS complexes. In this study, we identified a protein whose level was decreased in the mitochondria of the heart in rats with heart failure. This protein was PHB. Isoproterenol (ISO) has been used as a compound to induce heart failure in rats. We observed that astaxanthin (AX) increased the content of PHB in rat heart mitochondria isolated from ISO-injected rats. Since it is known that PHB forms complexes with some mitochondrial proteins and proteins that are part of the complexes of the respiratory chain, the change in the levels of these proteins was investigated under our experimental conditions. We hypothesized that PHB may be a target for the protective action of AX.
| S-EPMC8699106 | biostudies-literature
Project description:PurposeIn this study, we aimed to investigate the relationship between hypothyroidism and sterile inflammation in rat heart tissue.MethodsGroups; control group (fed with standard rat chow diet and tab water) and the hypothyroid group (fed with a standard rat chow diet and tap water containing 0.05% 6-n-propyl-2-thiouracil for 6-weeks). At the end of the experiment, histopathologic examination was performed. The T3, T4, TSH and myocardial malondialdehyde (MDA) measurements were performed with an ELISA kit. TUNEL assay was performed to demonstrate apoptosis. Sterile inflammation markers, caspase-1 and NLRP3, were investigated by immunohistochemistry and western blot.ResultsIn histopathological examination, we observed leukocyte infiltration, myocardial atrophy, pyknotic nucleated cells and cytoplasmic vacuolization in hypothyroid group whereas the control group showed normal structure. MDA levels in myocardial tissue were significantly high in hypothyroid group when compared to the control group (P<0.05). Myocardial apoptosis increased in hypothyroid group when compared to the control group. NLRP3 and caspase-1 immunoreactivity was higher in the hypothyroid group. In ELISA results, we found significantly higher level of TSH and lower levels of T3 and T4 in hypothyroid group when compared to the control group.ConclusionHypothyroidism increased oxidative stress, and caused inflammatory alterations in cardiac tissue. In addition, our study also suggested that thyroid hormone deficiency would increase the amounts of cardiac NLRP3 and caspase-1 protein, which indicates that hypothyroidism exerts its destructive effects through sterile inflammation. Elucidation of sterile inflammation-associated pathways may produce promising results in the treatment of hypothyroidism-induced cardiac damage.
| S-EPMC6535312 | biostudies-literature
Project description:Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.
| S-EPMC2922146 | biostudies-literature
Project description:Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S(0) (20,w) 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1x10(6). Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.
| S-EPMC1158467 | biostudies-other
Project description:The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.
| S-EPMC1137214 | biostudies-other
Project description:Acute ischemic stroke (AIS) remains a major cause of death and disability throughout the world. The most severe form of stroke results from large vessel occlusion of the major branches of the Circle of Willis. The treatment strategies currently available in western countries for large vessel occlusion involve rapid restoration of blood flow through removal of the offending blood clot using mechanical or pharmacological means (e.g. tissue plasma activator; tPA). This review assesses prospects for a novel pharmacological approach to enhance the availability of the natural enzyme tissue kallikrein (KLK1), an important regulator of local blood flow. KLK1 is responsible for the generation of kinins (bradykinin and kallidin), which promote local vasodilation and long-term vascularization. Moreover, KLK1 has been used clinically as a direct treatment for multiple diseases associated with impaired local blood flow including AIS. A form of human KLK1 isolated from human urine is approved in the People's Republic of China for subacute treatment of AIS. Here we review the rationale for using KLK1 as an additional pharmacological treatment for AIS by providing the biochemical mechanism as well as the human clinical data that support this approach.
| S-EPMC6348491 | biostudies-literature