Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transglutaminase from guinea pig liver catalyzed the formation of cross-links between fibrinogen (or fibrin) and ribonuclease. Using transglutaminase, immoblized ribonuclease was prepared by two separate methods: (1) fibrinogen-ribonuclease conjugates formed by transglutaminase were treated with thrombin to make fibrin membrane bound covalently to the enzyme; (2) fibrin polymer formed from fibrinogen with thrombin was covalently bound to ribonuclease by transglutaminase to make fibrin-ribonuclease conjugates.
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PMID:Fibrin membrane endowed with biological function. IV. Formation of cross-links between fibrinogen (or fibrin) and ribonuclease by transglutaminase. 3 50

The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin-thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.
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PMID:Protein disulfide isomerase activity is released by activated platelets. 157 38

The venom from Crotalus molossus nigrescens contains many activities including: hyde powder azure proteinase; N-benzoyl-arginine-ethyl-ester hydrolase; phospholipase; phosphodiesterase; desoxyribonuclease; fibrinogen coagulase; collagenase, fibrinolytic activity, and hemorrhagic factors. The venom, assayed with amounts of venom up to 50 micrograms protein per assay, does not contain acetylcholinesterase, phosphatase, amylase, ribonuclease, tyrosyl-ester hydrolase or hyaluronidase activities. The venom is lethal to mice with an i.p. LD50 of 2.35 mg/kg mouse. Fractionation of soluble venom by Sephadex G-75 separates at least five families of components. Fractions I-III contains all the enzymes, and fraction V have six small peptides. Further separation of fractions II-III on diethyl-amino-ethyl-cellulose columns at pH 8.0 and 8.3 gave pure proteinase E with a mol. wt of 21,390 and the following N-terminal amino acid sequence; Phe-Ala-Lys-Arg-Tyr-Val-Glx-Leu-Val-Ile-Val-Ala. A thrombin-like enzyme with a mol. wt of 75,000 was also purified from this venom by means of affinity and ion exchange chromatographies.
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PMID:Characterization of the venom from Crotalus molossus nigrescens Gloyd (black tail rattlesnake): isolation of two proteases. 218 98

A new approach has been developed to circumvent the problems of false positive reactions in the Limulus Amoebocyte Lysate (LAL) assay for lipopolysaccharide (LPS) in root surface materials. These LAL-reactive materials include thrombin, thromboplastin, ribonuclease, ribonucleic acid, lipoteichoic acid and peptidoglycan fragments. In the present study, hot phenol/water extraction of these substances followed by ultracentrifugation of the resulting aqueous phases reduced their concentrations to very low levels. Furthermore, the application of Polymyxin B/Sepharose 4B affinity chromatography to these extracts enabled their intrinsic LAL-activity to be determined. Use of these techniques to assay root surface materials has identified LPS as being the major LAL-reactive material present. The mean LPS yield for the periodontally involved teeth was 4.13 micrograms/tooth, representing 2.82 micrograms/root. In contrast, the mean yield of LPS for the periodontally uninvolved teeth was 3.12 ng/tooth.
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PMID:Identity of limulus amoebocyte lysate-active root surface materials from periodontally involved teeth. 346 18

Gabonase, an enzyme which acts on fibrinogen and factor XIII in uniquely thrombin-like ways, was purified to electrophoretic homogeneity from the venom of Bitis gabonica. On sodium dodecyl sulfate-polyacrylamide electrophoresis, the reduced protein behaved as a single chain with Mr = 30,600. The enzyme contains 20.6% carbohydrate, no free sulfhydryl groups and hence, from amino acid analysis, five disulfide bonds. Its extinction coefficient (E1%1cm) at 280 nm is 9.6. Its pI is 5.3. Gabonase has an active serine residue, is inactivated by phenylmethanesulfonyl fluoride, and has an active histidine which reacts with the chloromethyl ketone of tosyl-L-lysine. Its NH2-terminal amino acid sequence (Val-Val-Gly-Gly-Ala-Glu-Cys-Lys-Ile-Asp-Gly-His-Arg-Cys-Leu-Ala-Leu-Leu -Tyr-) is homologous to the B chain of thrombin. The activity of the enzyme is stabilized by calcium ion. It exhibits strong N alpha-p-tosyl-L-arginine methyl esterase activity, hydrolyzes tripeptide nitroanilide derivatives weakly or not at all, and cleaves no peptide bonds in insulin, glucagon, or the S peptide of ribonuclease. Gabonase clots fibrinogen with a specific activity of 45 NIH thrombin-equivalent units/mg, releasing both fibrinopeptides A and B and showing substrate inhibition at fibrinogen concentrations of 3 mg/ml or greater. The enzyme also activates factor XIII. It is not inactivated by either heparin or hirudin.
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PMID:Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions. 352 80

Thrombin, via activation of vascular endothelial and smooth muscle cell thrombin receptors, modulates vascular wall healing. To understand the mechanisms that regulate human thrombin receptor (HTR) expression, we cloned and characterized the HTR gene. The HTR gene consists of Exon I, which contains the 5'-regulatory region and 85 nucleotides of coding sequence; a approximately 15-kb intron; and Exon II, which contains the remainder of the coding sequence and the entire 3'-untranslated region. Multiple transcription initiation sites were identified by S1 mapping and ribonuclease protection assay. DNA sequence analysis indicated the presence of two SP-1-AP-2 consensus binding sequences, near or within the transcription initiation sites, and consensus binding sequences for numerous regulatory proteins that potentially modulate HTR expression. Functional analysis of the HTR promoter was performed by transfecting human microvascular endothelial cells with HTR promoter region-luciferase constructs. The highest level of expression was obtained with a 0.7-kb promoter sequence and was progressively less with fragments of 0.54, 1.16, 1.6, and approximately3.2 kb. The data presented in this report provide a foundation for further characterization of the HTR gene and the mechanisms that regulate its expression within the blood vessel wall.
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PMID:Cloning and identification of regulatory sequences of the human thrombin receptor gene. 882 85

Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor-alpha, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up-regulated astrocyte beta-actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino-terminus of the thrombin receptor's tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase beta-actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombin's suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H-7 did prevent thrombin-induced reversal of astrocyte stellation and induction of beta-actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.
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PMID:Exposure of astrocytes to thrombin reduces levels of the metabotropic glutamate receptor mGluR5. 885 25

Rapid regeneration of the endothelium is a critical component of vascular wall repair because limitations of this process enhance early thrombotic and vasospastic complications, as well as late sequelae of recurrent lesion formation. We have postulated that direct activation of the thrombin receptor initiates both mitogenic and chemokinetic endothelial behavior which facilitates intimal repair. To characterize the role of the thrombin receptor in human endothelial cell (EC) proliferation and migration, we investigated the effects of both alpha-thrombin (0.5-10 U/ml) and its receptor-activating peptide (TRAP; 1-100 microM). Responses of human aortic (HAEC) and umbilical vein (HUVEC) were characterized using [3H]thymidine and 61Cr microcarrier bead assays of proliferation and migration, respectively. Expression of motility-related genes was evaluated using a ribonuclease protection assay. Thrombin exerts both of its chemokinetic and mitogenic effects differentially in human endothelial cells. Following 2 or 4 days in culture, HUVEC proliferation increased two- to threefold after exposure to thrombin, primarily in the low concentration range (P < 0.05). However, HACE proliferation was inhibited up to 50% after a 4-day incubation period (P < 0.005). These mitogenic effects, including the inhibition of aortic endothelial cell proliferation, were reproduced, in part, by thrombin receptor activation with TRAP. In contrast, thrombin stimulates migratory responses in HAEC, but not HUVEC. However, this behavior was not reproduced by TRAP. It is noteworthy that urokinase-plasminogen activator (u-PA) expression was much more strongly expressed in migrating HAEC than in the HUVEC population. Moreover, when stimulated with thrombin, u-PA gene expression was significantly augmented in HAEC. It has been speculated that an effective human thrombin receptor (HTR) antagonist may reduce the proliferation of vascular smooth muscle cells and the development of a restenotic lesion following arterial wall injury. Our data suggest that such an inhibitor will likely also accelerate intimal regeneration through a dominant effect on limiting the HTR inhibitory effect on endothelial proliferation.
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PMID:Endothelial cells exhibit differential chemokinetic and mitogenic responsiveness to alpha-thrombin. 918 72

We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, "active" dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247-267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.
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PMID:Thrombin induces the release of the Y-box protein dbpB from mRNA: a mechanism of transcriptional activation. 1139 Sep 77

Thrombin receptors, i.e. proteinase-activated receptors (PARs), are expressed in endothelial cells (ECs) and neutrophils and directly affect platelet function and thrombosis. Although endothelial dysfunction and neutrophil activation have been demonstrated in women with preeclampsia (PE), the expression and regulation of PARs have not been defined in PE. In this study, we measured the expression of PARs in ECs and in neutrophils derived from normal and preeclamptic pregnancies. We also examined the effects of placental factors on PAR expression in these cells in vitro. ECs were isolated from umbilical cords (human umbilical vein ECs) from normal and preeclamptic pregnancies. Neutrophils were isolated from blood obtained from nonpregnant, uncomplicated pregnant, and preeclamptic women. Total RNA was extracted from the first-passage (P1) ECs (normal and PE) and from normal P1 ECs incubated with conditioned media derived from normal and preeclamptic placental cultures. The mRNA expression of thrombin receptor (PAR1), PAR2, and PAR3 was measured by ribonuclease protective assay. The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for each sample. We found that: 1) PAR1 expression was enhanced in ECs from PE, compared with ECs from normal pregnancies; 2) PAR2 expression was expressed in PE ECs but not in normal ECs; 3) neutrophils from nonpregnant women, normal, and preeclamptic pregnancies expressed PAR2, whereas only neutrophils from normal and preeclamptic pregnancies expressed PAR1; and 4) factors released from preeclamptic placenta up-regulated PAR1 and PAR2 expression in ECs but not in neutrophils. We conclude that mRNA expression of PAR1 and PAR2 is increased in ECs derived from preeclamptic pregnancies. Up-regulation of thrombin receptor expression in neutrophils may be a unique phenomenon during pregnancy but not apparently unique to PE. Factors released from the placenta are likely candidates in regulating PAR expression in ECs and may contribute to the platelet activation and vascular endothelial dysfunction in PE.
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PMID:Expression of thrombin receptors in endothelial cells and neutrophils from normal and preeclamptic pregnancies. 1216 2


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