Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.
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PMID:Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol. 1047 Dec 77

Factor Xa is the serine protease component of prothrombinase, the enzymatic complex responsible for thrombin generation. Production of recombinant factor X/Xa has proven to be difficult because of inefficient gamma-carboxylation, a critical post-translational modification. The affinities of the vitamin K-dependent propeptides for the gamma-carboxylase vary over 2 logs, with the propeptide of factor X having the highest affinity followed by the propeptides of factor VII, protein S, factor IX, protein C, and prothrombin [Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944]. On the basis of this observation, it was hypothesized that exchanging the propeptide of factor X with one that binds the gamma-carboxylase with a reduced affinity would enhance gamma-carboxylation by allowing greater substrate turnover. A chimeric cDNA consisting of the human prothrombin signal sequence and propeptide followed by mature human factor X was generated and stably transfected into HEK 293 cells, and modified factor X was purified from conditioned medium. The results indicate that on average 85% of the total factor X produced with the prothrombin propeptide was fully gamma-carboxylated, representing a substantial improvement over a system that employs the native factor X propeptide, with which on average only 32% of the protein is fully gamma-carboxylated. These results indicate that the affinity of the gamma-carboxylase for the propeptide greatly influences the extent of gamma-carboxylation. It was also observed that regardless of which propeptide sequence is directing gamma-carboxylation (factor X or prothrombin), two pools of factor X are secreted; one is uncarboxylated and a second is fully gamma-carboxylated, supporting the notion that the gamma-carboxylase is a processive enzyme.
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PMID:Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. 1108 81

Factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we investigated the molecular basis of FX deficiency in a Chinese pedigree. The proposita showed a markedly prolonged activated partial thromboplastin time and a mild prolongation of prothrombin time. The levels of FX antigen and FX activity were 58.6% and 2.5%, respectively. Molecular analysis revealed that the proposita was compound heterozygous for two novel mutations: IVS1 + 1G > A and G1185A (Arg347His). The aberrant transcripts from the IVS1 + 1G > A mutant allele were not detected by analyzing the splicing pattern of ectopic transcripts in leukocytes of the patient with nested polymerase chain reaction after reverse transcription. We thus hypothesize that the mRNA molecules originating from the IVS1 + 1G > A mutation were rapidly destroyed in vivo. Site-directed mutagenesis of FX cDNA was used to introduce FXG1185A mutation, and wild-type as well as mutant FX proteins were expressed by transient transfection in HEK 293 cells. Normal FX antigen levels both in the conditioned media of cells expressing the mutant and in cell lysates were detected by an enzyme-linked immunoadsorbent assay. Evaluation of wild-type and mutant coagulant activity demonstrated that the FX molecules carrying the Arg347His mutation have dramatically decreased activity.
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PMID:Molecular characterization of two novel mutations causing factor X deficiency in a Chinese pedigree. 1566 Sep 86

Several mutations in factor XII have been reported in patients with factor XII deficiency. Here, we described three mutations in the F12 gene (c. 6635G more than A (p. G259E), c. 6658G more than C (p. R267G) and c. 8489G more than A (p. E521K)) of five patients with congenital FXII deficiency. Among these, two were heterozygous mutations. All five patients had prolonged activated partial thromboplastin time, as well as markedly decreased FXII activity and antigen levels. In vitro studies in transiently transfected HEK 293T cells demonstrated that these mutations significantly lowered the FXII levels in the culture media, but had no impact on transcription. Further protein degradation inhibition experiments with various inhibitors suggested that the three mutants were degraded intracellularly through the proteasome pathway in the pre-Golgi compartment. Moreover, G259E and R267G mutations exhibited dominant negative effects, consistent with the phenotypes observed in the heterozygous carriers. Such dominant negative effects were not due to the dimerization of FXII. Our findings suggest that the three mutations in the F12 gene are the causing reasons for the cross-reactive material-negative FXII deficiencies.
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PMID:Novel mutations in congenital factor XII deficiency. 2670 83