Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The actions of ionophores with different ion specificities and of thrombin on the release of 14C-labeled 5-hydroxytryptamine, [3H]noradrenaline, and endogenous ATP were measured in human platelets suspended in media with various K+ and Na+ concentrations. Besides thrombin, those ionophores [monensin, nigericin, and the combination of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) with nonactin and/or valinomycin] which cause a rapid collapse of H+ gradients induced a fast and virtually total release of 14C-labeled 5-hydroxytryptamine and [3H]noradrenaline into the various media. FCCP alone, which causes an inversion of the membrane potential to inside negative values, induced a considerably slower amine release. Changes in the K+ and Na+ gradients did not lead to amine release, nor did interference with energy transduction by antimycin A with or without glycolysis inhibitors. Monensin and FCCP did not release ATP, whereas thrombin, added before or after incubation of platelets with FCCP and monensin, caused a marked liberation of the nucleotide. It is concluded that in intact human platelets (a) the intragranular storage of 5-hydroxytryptamine and noradrenaline mainly depends on the proton gradient across the granular membrane, and (b) ionophores causing a collapse of H+ gradients induce non-exocytotic release of 5-hydroxytryptamine and noradrenaline from intracellular storage granules.
Mol Pharmacol 1982 Jul
PMID:Storage of biogenic amines in intact blood platelets of man. Dependence on a proton gradient. 628 76

Human pro-coagulant alpha-thrombin may be proteolyzed under controlled conditions to the non-coagulant beta- and gamma-thrombin forms. These derivative forms nonetheless retain esterase and amidase activities with small substrates as well as several other thrombin functions. Structurally, human gamma-thrombin consists of three non-covalently associated fragments which retain structural integrity as measured by several spectroscopic criteria as well as enzymatic function. The protein folding characteristics of three-chain gamma-thrombin indicate that each fragment (domain) contains sufficient information to result in a correct renaturation of protein conformation. Those subtle structural differences which distinguish gamma- from alpha-thrombin are most likely the obstructions to fibrinogen binding which account for the loss of clotting activity.
Mol Cell Biochem 1984
PMID:Structure-function relationships in human alpha- and gamma-thrombins. 632 67

Two closely related crystal structures of alpha 1-proteinase inhibitor modified at the reactive site peptide bond Met358--Ser359 have been analysed. The crystal structure has been obtained from diffraction data at 3 A resolution, with phases originally from isomorphous replacement. The electron density map was substantially improved by cyclic averaging of the electron densities of the two crystal forms and allowed the chain to be traced in terms of the known chemical amino acid sequence. Energy restrained crystallographic refinement was initiated and resulted in conventional R-values of 0.251 for the tetragonal crystal form (6 to 3 A resolution) and 0.247 for the hexagonal crystal form (6 to 3.2 A resolution). The polypeptide chain is almost completely arranged in well-defined secondary structural elements: three beta-sheets and eight alpha-helices. The helices are preferentially formed by the first 150 residues. They are in proximity underneath sheet A. The chain ends Met358 and Ser359 of the nicked species are arranged in strands on opposite ends of the molecule indicating a major structural rearrangement upon modification of the intact inhibitor. It is suggested that the Met358 strand is in a different conformation removed from sheet A and approaches Ser359 in the intact inhibitor species. Glu342, which is exchanged by a lysine in the Z-variant is in a strategic position for such a rearrangement. The three carbohydrate chains of alpha 1-proteinase inhibitor have partly defined electron density close to their attachment sites at asparagine residues. The anti-thrombin and ovalbumin amino acid sequences can be accommodated in the alpha 1 inhibitor molecular structure. The intron-exon junctions of the ovalbumin and the alpha 1-proteinase inhibitor gene are all in surface loops of the mature protein.
J Mol Biol 1984 Aug 15
PMID:Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. 633 97

Fibrin-specific antibodies have been produced in rabbits which were immunized with synthetic peptides. Specificity against human fibrin monomer was achieved because the synthetic peptide haptens were derived from sites unique to fibrin as compared with fibrinogen. Two undecapeptides were chemically synthesized according to the amino acid sequence of the amino termini of human fibrin alpha- and beta-chains which are revealed by thrombin cleavage. Rabbits immunized with either an alpha- or beta-chain peptide conjugate produced anti-peptide sera which reacted with fibrin monomer. Following immunoadsorption of the rabbit sera with human fibrinogen-Sepharose, fibrin-specific antibodies were detectable by solid-phase radioimmunoassay that did not react with fibrinogen. Antisera elicited by clotted human fibrin contained antibodies that reacted with fibrin and fibrinogen when treated in a similar manner.
Mol Immunol 1983 May
PMID:Induction of fibrin-specific antibodies by immunization with synthetic peptides that correspond to amino termini of thrombin cleavage sites. 634 13

Among extracellular biological processes the spatial control of blood clotting is a unique phenomenon. Localization in space has very important consequences in both normal and pathological conditions. Under physiological circumstances a clot is formed only in the vicinity of injury, albeit the prerequisites of coagulation are almost completely given in the whole circulation. The local character of blood clotting is secured by the following major conditions: The regulatory signal initiating coagulation-the damaged vascular wall-is itself a surface on which the majority of clotting reactions take place. The first enzyme, factor XII, of the intrinsic coagulation pathway is activated on the collagen fibers exposed in the damaged vascular wall, although the significance of this reaction in respect of the clotting process is ambiguous. On the membrane of platelets adhered to the damaged blood vessel is activated factor XI, too, which is a well-established participant of the intrinsic clotting process. The further consecutive reactions of coagulation are confined to the surface produced by injury, because the enzymes involved contain gamma-carboxyl-glutamyl side chains which are anchored through calcium bridges to the phospholipids of the platelet membrane. The last enzyme of the sequence is thrombin, which is released from the surface. The reactions taking place on the surface form an enzyme cascade, which amplifies the relatively weak triggering signal by several orders of magnitudes. Amplification is ensured not only by the enzyme-substrate relationship of the consecutive reaction partners, but also by spatial confinement, which endows the process with higher efficacy than could be expected on a statistical basis from reactions in solution. It contributes to the efficiency of enzyme cascade that the non-enzymatic regulatory proteins increase the activity of factors IXa and Xa, and thereby the overall process. While the partner of factor IXa, factor VIII, is captured from plasma, factor V, the partner of factor Xa, is derived from the platelets adhered to the damaged surface and orients the binding of factor Xa. The surface localization ensures the protection of the members of clotting system: In the activator complexes found on the surface, the spatial arrangement of clotting factors prevents the inactivation of factors by physiological inhibitors or by proteolytic enzymes and specific antibodies that appear in the circulation in pathological conditions. Platelet factor 4, derived from platelets, binds heparin and thereby markedly decreases the reactivity of antithrombin III, the physiological inhibitor of clotting factors. The above two circumstances are
Mol Aspects Med 1983
PMID:Surface-governed molecular regulation of blood coagulation. 636 61

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.
Mol Cell Biol 1984 Nov
PMID:Xenopus fibrinogen: characterization of the mRNAs for the three subunits. 651 31

A stable hybridoma secreting homogeneous antibody (immunoglobulin class IgG2a) has been prepared by fusion using cells of immunoglobulin non-secreter myeloma (P3X63Ag8.653) and spleen cells of mice which had previously been immunized with the NH2-terminal CNBr fragment of human fibrinogen, the so-called N-DSK [(A alpha 1-51, B beta 1-118, gamma 1-78)2]. In competitive ELISA or radioimmunoassay this antibody (MAb/1-8C6) cross-reacted with intact fibrinogen, N-DSK, a des fibrinopeptide A (des FPA) variant of N-DSK, the so-called (B)N-DSK, as well as the intact B beta chain (B beta 1-118) obtained from N-DSK. Also, and mot importantly, cross-reactivity was observed with fibrinogen-free ethanol extracts of plasma obtained from patients known to contain high levels of fibrinogen or fibrin degradation products. In vitro thrombin digestion of any of these competitors resulted in complete loss of cross-reactivity. MAb/1-8C6 did not react with the A alpha or gamma-chains of N-DSK, free fibrinopeptide B(FPB), free B beta 15-42, as well as equimolar mixtures of the latter two peptides. These results suggest that MAb/1-8C6 may be to an epitope in or around the thrombin-susceptible B beta 14 Arg-25 Gly bond. Furthermore, due to its reactivity with patient plasma extracts, this antibody may be useful in clinical investigations dealing with fibrino(geno)lysis.
Mol Immunol 1983 Nov
PMID:A monoclonal antibody with ability to distinguish between NH2-terminal fragments derived from fibrinogen and fibrin. 665 69

Fine fibrin clots and coarse and fine fibrin films (both ligated and unligated), formed by shrinkage of clots in one dimension, were examined by electron microscopy. Specimens of clots were prepared by critical point drying and by embedding and sectioning; specimens of films were prepared by embedding and sectioning only. In the fine clots, network junctions appeared to be formed by fiber segments in which two or more protofibrils were gently twisted around each other for distances of the order of 200 nm and then diverged to give trifunctional branch points. This topology appeared to be preserved in the fine films. It is proposed that the strength of the junctions is primarily provided by the twisting topology, though reinforced by non-covalent bonding involving the B sites uncovered by thrombin. In coarse films, bundles of protofibrils, lying primarily in the film plane, had diameters of 40 to 200 nm and were gently twisted around each other to form thicker cables. Uniaxial stretching, up to 100%, of either fine or coarse film before fixing caused suprisingly extensive orientation of the protofibrils or bundles. However, random orientation was recovered if a stretched ligated film was allowed to retract to its original dimensions before fixing. In a stretched coarse film sectioned perpendicular to the stretch direction, fiber bundles could be seen in cross-section; these were roughly circular with scalloped edges. The changes with stretching and recovery are discussed in relation to possible mechanisms of deformation and elastic energy storage.
J Mol Biol 1984 Apr 05
PMID:Electron microscopy of fine fibrin clots and fine and coarse fibrin films. Observations of fibers in cross-section and in deformed states. 671 83

Differential scanning microcalorimetry was used to study the domain organization of calmodulin and its fragments obtained by trypsin and thrombin treatment of the protein. It has been shown that (1) at physiological concentrations of Ca2+ ions (10(-6) divided by 10(-5) M) the protein structure represents three cooperative blocks, one of which contains two Ca2+-binding domains and the two others contain one Ca2+-binding domain; (2) stability of the cooperative blocks strongly depends on the Ca2+ concentration and in the presence of 2 mM EDTA the cooperative block containing Ca2+-binding domain III melts already at room temperature; (3) in the absence of Ca2+ ions the addition of Mg2+ or Na+ ions to the buffer system (to the concentration of 2 mM and 150 mM, respectively) does not lead to stabilization of the cooperative structure of the block containing Ca2+-binding domain III; (4) judging by thermodynamic parameters of melting, the structure of cooperative blocks within the peptides coincides with their structure in the intact molecule.
Mol Biol (Mosk)
PMID:[The domain organization of calmodulin]. 685 59

In summary, in this review on the function of vitamin K in post-translational modification of precursor proteins by carboxylation of certain glutamyl residues, I have tried to cover in particular the recent work on the reaction, the enzymes involved and the mechanisms being considered. In doing this I have also considered vitamin K, its discovery, its functional form and the possible relation of its metabolism to the carboxylation reaction. Equally the various vitamin K-dependent gla-containing proteins currently known have been described. The carboxylation of synthetic small molecule exogenous substrates and the synthesis and metabolism of the products of carboxylation are of great help in studying the reaction. Structural specificity of vitamin K analogs in vivo and in vitro has been compared and the use of various antagonists in vivo and in vitro considered in attempts to gain an understanding of the overall reaction. The reactions subsequent to carboxylation, e.g., the activation of prothrombin to thrombin via serine proteases and the related activation of the other vitamin K-dependent proteins have not been considered in this review. The review has not covered prothrombin or other vitamin K-dependent protein isolation, nor the determination of these proteins. As the vitamin K-dependent protein carboxylation story has developed over the past six years, a number of reviews have been written which help in keeping up with the various aspects of the field as it has expanded. These reviews refer to many of the papers I have had to eliminate due to space limitations. They are referenced as 469-489. The review is in no sense comprehensive and many papers have been missed or only mentioned. I have tried to concentrate on the more recent work and, thus, much of the very fine work of the 1940's on vitamin K chemistry is hardly mentioned. Some redundancy has been built into the organization of the review so that a reader can obtain a reasonable view of any one section without having to search the whole review for all possible relevant information on any particular part of the field.
Mol Cell Biochem 1981 Aug 11
PMID:Post-translational carboxylation of preprothrombin. 702 26


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