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S N Murthy

Publications and source records attributed to S N Murthy.

At least 37 records · Page 2Linked to original sources
PubMed1998

A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of gamma chains by factor XIIIa.

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its alpha chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIIIa-catalyzed end-to-end ligation of the gamma chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIIIa reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of alpha chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.

Cross-Linking Reagents↗
PubMed1998

Properties of purified lens transglutaminase and regulation of its transamidase/crosslinking activity by GTP.

On account of its protein crosslinking activity, the Ca2+-dependent transglutaminase of the lens is likely to be involved in the formation of cataracts. We have now purified the rabbit lens enzyme to near homogeneity as judged by SDS-PAGE (Mr approximately 78 kDa), and a key feature of the procedure was the use of a highly selective affinity chromatographic step with a fibronectin fragment as ligand. The catalytic activity of the lens transglutaminase, measured by the incorporation of dansylcadaverine into dimethylcasein, was compared with those of two similar enzymes isolated from human red cells and from guinea pig liver, respectively. All three enzymes were inhibited by GTP, but the lens enzyme was most sensitive to inhibition by the nucleotide. Moreover, GTP was also shown to inhibit the formation of the approximately 55 kDa betacrystallin dimers in the Ca2+-treated rabbit lens homogenate, proving that the nucleotide is a negative regulator for the crosslinking activity of transglutaminase in this tissue.

Animals↗
PubMed1998

Cross-linking sites of the human tau protein, probed by reactions with human transglutaminase.

A portion of the neurofibrillary tangles of Alzheimer's disease has the characteristics of cross-linked protein. Because the principal component of these lesions is the microtubule-associated protein tau, and because a major source of cross-linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (tau23 and tau40) capable of participating in human TGase-mediated isopeptide bond formation. Using dansyl-labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule-binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross-linked during the TGase-catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.

Amino Acid Sequence↗
PubMed1997

Hierarchy of lens proteins requiring protection against heat-induced precipitation by the alpha crystallin chaperone.

Gel filtration of the water-soluble extract from bovine lens yields a group of proteins, emerging between the peaks of beta H and beta L crystallins, which show a considerably greater sensitivity to heat-induced aggregation/precipitation than the far more abundant beta and gamma crystallins. However, the small heat shock protein: alpha crystallin was effective in protecting these trace constituents of the lens from precipitating out of solution at 55 degrees C (measured under the standard conditions in a pH 7.5 buffer containing 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA and 0.05% NaN3). Prominent components of the precipitate, formed in the absence of a recombinant alpha B crystallin chaperone could be resolved by one- and two-dimensional electrophoresis. Identification by amino acid sequencing revealed that the heat-sensitive group of lens proteins comprised glyceraldehyde-3-phosphate dehydrogenase (M(r) approximately 39 kDa), enolase (approximately 48 kDa), leucine aminopeptidase (approximately 52 kDa) and aldehyde dehydrogenase (approximately 53 kDa). These findings indicate for the first time that the aggregation of such minor lens constituents could possibly contribute to initiating the process of opacification in the development of cataracts.

Amino Acid Sequence↗
PubMed1995

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Animals↗
PubMed1995

The fibronectin-binding domain of transglutaminase.

Guinea pig liver transglutaminase (EC 2.3.2.13) displays a Ca(2+)-independent binding (Ka = 10(7) M-1) to the same gelatin-binding domain of human plasma fibronectin that is known to form a very tight complex with the human red cell enzyme. The fibronectin-combining site of the liver transglutaminase was investigated by testing fragments obtained from the parent protein by controlled digestion with endoproteinase Lys-C. Overlay assays, probed with anti-fibronectin antibody, revealed that the fibronectin binding ability of the transglutaminase was encoded in a linear sequence in its 28-kDa N-terminal domain. Removal of the first 7 residues by further digestion of the purified 28-kDa material with endoproteinase Glu-C generated a 27-kDa fragment that, however, showed no binding activity. Thus, residues 1-7 in the liver enzyme seem to be of particular importance for influencing its ability to bind to fibronectin.

Amino Acid Sequence↗
PubMed1994

Residue Gln-30 of human erythrocyte anion transporter is a prime site for reaction with intrinsic transglutaminase.

Elevating the intracellular concentration of Ca2+ ions in human erythrocytes leads to the formation of membrane-associated polymers, composed of skeletal and cytoplasmic proteins. The anion transporter band 3 serves as a membrane anchor for this N epsilon-(gamma-glutamyl)lysine cross-linked polymer. The reaction is catalyzed by an intrinsic transglutaminase, and it can be observed in broken cells merely by the addition of Ca2+. Certain primary amines, such as dansylcadaverine, inhibit the process by virtue of competition against the epsilon-lysyl or donor functionalities of the protein substrates. Dansylcadaverine itself becomes incorporated into the enzyme-specific gamma-glutaminyl or acceptor residues, blocking these from participating in the formation of protein-to-protein cross-links. This labeling procedure, coupled with an anti-dansyl antibody affinity procedure to isolate dansyl-labeled compounds, was employed to identify Gln-30 as the preferred transglutaminase-reactive acceptor site in the band 3 protein.

Amino Acids↗
PubMed1993

Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.

Antibodies, Monoclonal↗
PubMed1993

Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin.

The use of oral and intravenous cyclosporin represents a significant advance in the therapy of refractory inflammatory bowel diseases (IBD). However, oral administration of cyclosporin is fraught with improper delivery of cyclosporin to the colon for its topical action. Because of unpredictable metabolism by cytochrome P-450 IIIA, the targeted blood level for systemic effect is not reached at low doses. Furthermore, the doses that have been used for therapy of IBD have been shown to induce several adverse side effects. Thus, an alternate method of delivering cyclosporin to the colon is desirable. In this study, the effect of intracolonically administered cyclosporin was tested for its efficacy to heal mucosal erosions in dextran sulfate sodium (DSS)-induced colitis in mice. Both acute and chronic colitis was induced by feeding female Swiss-Webster mice with 5% DSS (30,000-40,000 mol wt) for five or seven days, respectively. Therapy was advocated prophylactically, prophylaxis plus therapy and therapeutically during the acute and chronic phase of the disease and therapeutically during the chronic phase of the disease. Intracolonic cyclosporin given prophylactically showed adverse effects by increasing the damage to the colonic mucosa. However, intracolonic cyclosporin given therapeutically in 2.5, 5, and 10 mg/kg after the induction of colitis resulted in dramatic responses in terms of reducing the disease activity and histologic scores, corroborated by complete histological resolution compared to oral cyclosporin given at identical doses. Intracolonic cyclosporin (5 mg/kg) was also very effective in reducing the chronic inflammation. The results of this study highlight the application of this animal model for therapeutic research. Furthermore, cyclosporin administered as an enema provides a new stratagem for the therapy of IBD because of its rapid onset of action at very low doses without the risk inherent in oral or systemic administration.

Acute Disease↗
PubMed1993

Clinicopathologic study of dextran sulfate sodium experimental murine colitis.

BACKGROUND: We undertook this study in order to fully characterize the clinical and histopathology features of the dextran sulfate sodium (DSS) model of experimental murine colitis and to discover the earliest histopathologic changes that lead to colitis. EXPERIMENTAL DESIGN: Acute colitis was induced in Swiss-Webster mice by 7 days of oral DSS with animals sacrificed daily. Chronic colitis was induced by: (a) 7 days of oral DSS followed by 7 days of H2O (for 1, 2, and 3 cycles) and (b) 7 days of oral DSS followed by 14 and 21 days of H2O. In each experimental group, the entire colons were examined histologically and correlated with clinical symptoms. RESULTS: Acute clinical symptoms (diarrhea and/or grossly bloody stool) were associated with the presence of erosions and inflammation. More importantly, the earliest histologic changes which predated clinical colitis was loss of the basal one-third of the crypt (day 3), which progressed with time to loss of the entire crypt resulting in erosions on day 5. The earliest changes were very focal and not associated with inflammation. Inflammation was a secondary phenomena and only became significant after erosions appeared. Animals treated with only 7 days of DSS followed by 14 and 21 days of H2O developed a chronic colitis with the following histologic features: areas of activity (erosions and inflammation), inactivity, crypt distortion, florid epithelial proliferation and possible dysplasia. These changes were similar to animals given 3 cycles of DSS. The clinical disease activity index correlated significantly with pathologic changes in both the acute and chronic phases of the disease. CONCLUSIONS: The mechanism of DSS colitis is presently unknown. However, the finding of crypt loss without proceeding or accompanying inflammation suggests that the initial insult is at the level of the epithelial cell with inflammation being a secondary phenomena. This may be a good model to study how early mucosal changes lead to inflammation and the biology of the colonic enterocyte. Chronic colitis induced after only 7 days of DSS may serve as a useful model to study the effects of pharmacologic agents in human inflammatory disease and mechanisms of perpetuation of inflammation. Finally, we believe that this model has the potential to study the dysplasia cancer sequence in inflammatory disease.

Animals↗
PubMed1992

Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of chicken erythrocyte transglutaminase.

We report the sequences of cDNAs encoding chicken erythrocyte transglutaminase (EC 2.3.2.13). The complete mRNA consists of 3345/3349 nucleotides and predicts a single open reading frame. Nine peptide sequences derived from partial digests of the isolated protein agreed with the corresponding translation of the open reading frame. Approximately 60% identities between the avian protein and three related mammalian enzymes were found. Chicken erythrocyte transglutaminase mRNA is most abundant in red blood cells and kidney, and it accumulates during erythroid cell differentiation.

Amino Acid Sequence↗
PubMed1992

Amide bond cleavage monitored continuously through detection of a dansylcadaverine leaving group.

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

Amides↗
PubMed1992

Visualization of purified fibronectin-transglutaminase complexes.

It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830.

Animals↗
PubMed1992

Increased phospholipase A2 activity in peritoneal leukocytes in rat experimental colitis.

Activated leukocytes in the peritoneal cavity have been shown to release phospholipase A2(PLA2) activity and increased production of eicosanoids. It is not known whether a similar phenomenon occurs in inflammatory bowel diseases. We measured PLA2 activity in the peritoneal leukocytes and eicosanoid content in the peritoneal cavity of rats with experimental colitis induced by dinitrochlorobenzene, immune complex-mediated colitis, and acetic acid-induced colitis. In all models of colitis, proteins in the peritoneal cavity were increased by 60-260% above control, leukocyte number was elevated by 3- to 10-fold, the PLA2 activity by 4- to 32-fold, and anti-PLA2 activity was absent. Eicosanoid concentrations were 3- to 4-fold higher than controls. In vitro studies confirmed that the peritoneal leukocytes were the source of PLA2. Treatment of rats with dexamethasone and leukocytes in vitro reduced PLA2 significantly, but they were well above control rats without colitis. However, dexamethasone did not improve the histology. These studies suggest that peritoneal leukocytes may serve as an additional source of inflammatory mediators, which may further enhance the inflammatory response in the rat colon.

Acetates↗
PubMed1991

Intramolecular crosslinking of monomeric fibrinogen by tissue transglutaminase.

In addition to generating polymeric products from human fibrinogen, human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) was shown to catalyze the intramolecular reaction of crosslinking two of the constituent chains within monomeric fibrinogen itself. This internally fused protein derivative contains appreciable amounts of the N epsilon-(gamma-glutamyl)lysine bridge peptide and displays the A alpha.gamma hybrid chain pattern of crosslinking, characteristic for the actions of tissue transglutaminases on fibrinogen. Diagnostic analysis in pathological situations, where such enzymes might have escaped from cells into the plasma environment, should include a search for the internally crosslinked soluble fibrinogen monomer.

Chromatography, Gel↗
PubMed1991

Azathioprine reduces extravasation and neutrophil trafficking in immune complex-mediated inflammation in the rat colon.

Azathioprine (AZ) has been used in the treatment of refractory inflammatory bowel disease. The mechanism by which AZ decrease colonic inflammation is not known. It is alluded that AZ may be effective in the maintenance of remission. We examined whether AZ in non-immunosuppressive doses reduces extravasation and neutrophil trafficking in a rat model of colonic inflammation. Rats were treated with I.P. injection of AZ (1 mg/kg) for 6 weeks. At the end of 2 and 6 weeks rats were injected I.V. immune complex and on the following day the proximal colon was perfused with 2.5% formaldehyde (local irritant 3 ml/hour for 5 mins). Extravasation was measured by Evans' blue technique and neutrophil concentration in the tissue was determined by measuring myeloperoxidase (MPO). AZ did not inhibit extravasation and MPO after 2 weeks of therapy. However, after 6 weeks, AZ reduced extravasation to 20 +/- 2 micrograms/gm compared to untreated animals (51 +/- 6 micrograms/gm tissue) and MPO levels to 0.3 +/- 13 compared to untreated rats (0.8 +/- 0.32 mU/gm). There was a good correlation between extravasation and MPO levels. These results suggest that long-term treatment with AZ may prevent extravasation and cause reduction in neutrophil trafficking. Such an effect may be beneficial for maintaining remission in IBD.

Aminosalicylic Acids↗