Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.
J Mol Biol 1992 May 05
PMID:Binding of hirudin to human alpha, beta and gamma-thrombin. A comparative kinetic and thermodynamic study. 137 1

We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function.
Mol Immunol 1992 May
PMID:Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides. 137 44

C5b-8 binding sites in C9 were examined using mAbs raised against C9. Among 16 mAbs, two, designated P40 and X197, blocked C9-mediated EAC1-8 lysis. C9 pretreated with the mAbs failed to bind to EAC1-8 at 4 degrees C. In addition, the mAbs became inaccessible to the C9 that had been incorporated into EAC1-8 at 4 degrees C. These findings suggest that C9 binding to EAC1-8, but not its membrane spanning or polymerization, is blocked by mAbs. By immunoblotting analysis using alpha-thrombin proteolytic fragments derived from C9 [a N-terminal fragment of mol. wt 25,000 (C9a) and a C-terminal one of mol. wt 37,000 (C9b)] and tryptic fragments of C9 (mol. wts 53,000 (C9a') and 20,000 (C9b')), the epitopes of P40 and X197 were mapped to the N-terminal and C-terminal regions of C9b, respectively. Both P40 and X197 bound to the C9 polymerized with Zn2+ in the fluid phase, whereas X197 but not P40 reacted with the membrane attack complex (MAC) formed on membranes. The results suggest that two distinct epitopes are involved in C9 binding to EAC1-8, and behave in a different manner for globular C9 bound to EAC1-8 at 4 degrees C, C9 assembled in MAC, or poly-C9 induced by Zn2+. These mAbs may be useful in clarifying the conformational states of C9 and in analyzing the molecular interaction between C9 and its inhibitors.
Mol Immunol
PMID:Analysis of C5b-8 binding sites in the C9 molecule using monoclonal antibodies: participation of two separate epitopes of C9 in C5b-8 binding. 137 34

Platelets from diabetic humans and animals have been found previously to be hypersensitive to agonists, including thrombin, in vitro but it is unclear if this hypersensitivity also occurs in vivo and leads to a greater thrombotic tendency. In the present study, the effect of diabetes was examined on thrombus formation and vessel wall responses which result from continuous intimal injury induced by indwelling aortic catheters in rabbits. Platelet and fibrin(ogen) associated with the thrombus and damaged aortae were examined. Control or alloxan-induced diabetic rabbits (9-12 months after initial treatment) were injected with 51Cr-labeled autologous platelets and 125I-labeled fibrinogen (prepared from control rabbits) before insertion of indwelling aortic catheters. The anesthetized rabbits were perfused-fixed after 20 hr or 4 days. The dry weight of thrombus that formed was determined and platelet and fibrin(ogen) accumulation in thrombi and on injured aortae were calculated from the associated 51Cr and 125I, respectively. In diabetic rabbits, more platelets accumulated in the thrombi which formed after either 20 hr or 4 days, although the weight of thrombus and net fibrin(ogen) incorporation into the thrombus were not different from corresponding control rabbits. Net platelet and fibrin(ogen) association with the injured aortae were not different between control and diabetic rabbits. It is likely that the increased platelet accumulation in arterial thrombi in diabetic rabbits which results from continuous injury to aortae is a consequence of hypersensitivity of these platelets to thrombin generated in the thrombus and at the sites of vessel injury.
Exp Mol Pathol 1992 Oct
PMID:Increased platelet, but unaltered fibrinogen, accumulation in experimental thrombi in alloxan-induced diabetic rabbits. 142 57

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.
J Mol Biol 1992 Nov 05
PMID:The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli. 144 91

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.
Cell Mol Biol 1992 Aug
PMID:High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli. 146 11

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.
J Mol Biol 1992 Dec 20
PMID:Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51). 147 86

The fungal toxin associated with Dutch elm disease, cerato-ulmin, has been produced in the bacterium Escherichia coli by the assembly of oligonucleotides according to the unpublished amino acid sequence of the toxin. This toxin was produced at approximately 80 micrograms/L of cell culture as a fusion to glutathione S-transferase. We synthesized the toxin as a fusion protein to improve purification and stability. Recombinant cerato-ulmin was analyzed by immunoblot analysis and then separated from its fusion partner by thrombin. We incorporated this molecule into an appropriate medium to test the activity of the toxin on the growth of American elm callus cultures.
Mol Plant Microbe Interact
PMID:Expression of a modified Dutch elm disease toxin in Escherichia coli. 147 6

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.
J Mol Biol 1992 Aug 20
PMID:Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics. 151 46

The action of many agonists results in rapid production of sn-1,2-diacylglycerol (DAG). Using platelets as a model system, we previously identified a delayed phase of DAG accumulation that is temporally associated with secondary aggregation and secretion. In the present study, we examined the quantitative relationship between this delayed DAG accumulation and platelet aggregation and secretion. To quantitate the low levels of DAG in platelets, we used the sensitive DAG kinase assay and simultaneously compared DAG levels with aggregation and ATP secretion. In platelets stimulated by gamma-thrombin or collagen, there was a dose response between concentration of agonist and DAG accumulation. Significantly, a dose response was observed between DAG accumulation and extent of aggregation and secretion in platelets stimulated by either agonist. A concentration of either gamma-thrombin or collagen that caused secondary aggregation and secretion was associated with DAG accumulation above 0.2 pmol of DAG/nmol of phospholipid. Subthreshold concentrations of gamma-thrombin or collagen resulted in DAG levels less than 0.2 pmol/nmol of phospholipid. Thus, these data suggest that a to occur. Moreover, secretion was blocked when DAG production was blocked with aspirin or when protein kinase C was inhibited with sphingosine. We conclude that endogenously formed DAG plays a critical role in regulating secondary aggregation and secretion and, therefore, represents an important target for future antiplatelet agents.
Mol Pharmacol 1992 Feb
PMID:Quantitative analysis of diacylglycerol second messengers in human platelets: correlation with aggregation and secretion. 153 14


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