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

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.
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PMID:Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. 788 15

Heparin has been widely investigated in medicine and biomedical applications as an anticoagulant. This paper described a covalent modification of poly (lactic acid) (PLA) films with heparin-carrying microcapsules. The heparin-carrying microcapsules were achieved by layer-by-layer (LbL) self-assembly technology by which poly (allylamine hydrochloride) (PAH) and heparin were alternately coated on the Ca(2+)-cross-linked alginate hydrogel. The microcapsules were characterized by scanning electron microscopy (SEM), zeta potential analysis and Fourier transform infrared spectroscopy (FTIR), and then grafted to PLA films by -CONH- linkage between surface PLA molecules with residual primary amino groups on the outer PAH layer of microcapsules, in the presence of catalysts of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). SEM and FTIR revealed the formation of -CONH- linkage. In vitro antithrombogenicity by the method of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests showed that surface-modified PLA films had superior coagulation properties to original PLA films. It is suggested that the present method would provide a potential effective tool for biomaterial modification.
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PMID:Construction of anticoagulant poly (lactic acid) films via surface covalent graft of heparin-carrying microcapsules. 1915 61

Electrospun polyacrylonitrile fiber membranes (EPFMs) were coated with multilayer films, assembled using the layer-by-layer (LbL) technique through the alternate deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA), to develop an antithrombogenic drug release membrane for hemodialysis. Methylene blue (MB) and heparin (HEP) were attached to the PAH and PAA multilayers, respectively, as model drug and antithrombogenic agent to investigate the dual functionality of the membranes. The positively (PAH, MB) and negatively (PAA, HEP) charged groups generated a supermolecular polyelectrolyte multilayer film (SPF) capable of loading high amounts of MB and HEP on the EPFMs at appropriate composition. The pH was fixed at 5.5 during assembly to stabilize the SPF. Heavy assembly of the PAH/PAA multilayer occurred at 10 wt% of both MB and HEP with 25 cycles of LbL deposition, and it exhibited long-term release of MB and low release of HEP at pH 7.4 in a circulatory system. The SPF-coated EPFMs also achieved low platelet attachment after 4 h of platelet rich plasma circulation and showed prolonged clotting times including thromboplastin, thrombin, and prothrombin times. Collectively, these observations suggest that SPF-coated EPFMs have great potential for use as hemodialysis membranes with positively charged drug loading.
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PMID:Preparation of biofiltration membranes by coating electrospun polyacrylonitrile fiber membranes with layer-by-layer supermolecular polyelectrolyte films. 3219 74