Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.
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PMID:BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility. 1743 97

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin-Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.
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PMID:A thrombin inhibitor from the gut of Boophilus microplus ticks. 1771 May 57

Curry leaves (Murraya koenigii L. Spreng, Rutaceae) is an herbal species used in traditional medicine in eastern Asia. In this study, the antioxidant protein was purified by homogenization of curry leaves powder in Tris buffer, 65% ammonium sulphate precipitation and gel filtration on Sephadex G-75 column which resulted in three peaks (PI, PII and PIII). PII protein inhibited lipid peroxidation in human RBC ghost at 100 microg by 80%, whereas PI and PIII at 600 microg showed moderate antioxidant activity (40-50%). Homogeneity of PII was confirmed by rechromatographing on Sephadex G-75 and reverse phase HPLC. The antioxidant protein (PII) from curry leaves (APC) showed apparent molecular weight of 35 kDa by SDS-PAGE and MALDI/MS analysis. The protein nature of APC was established by the presence of amino acids and loss of antioxidant activity on heat and protease treatment. The APC at 0.8 microM inhibited lipoxygenase activity by 71%, effectively prevented diene, triene and tetraene lipids formation at 3 microM, and scavenged about 85% hydroxyl and DPPH radicals at 150-fold lesser concentration compared to BHA and alpha-tocopherol (400 microM). In addition, APC reduced cytochrome c and ferric ion, chelated ferrous ion, and inhibited ferrous sulfate: ascorbate-induced fragmentation and sugar oxidation to 80-90%. Thus, the present study demonstrates the in vitro characterization of antioxidant protein from the curry leaves (M. koenigii L.).
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PMID:Purification and characterization of approximately 35 kDa antioxidant protein from curry leaves (Murraya koenigii L.). 1808 61

In this study, recombinant allophycocyanin (rAPC) with a purity of 98% was transferred from a gel to a nitrocellulose (NC) membrane to develop a simple and efficient immuno-affinity membrane. Atomic force microscopy (AFM) was used to investigate the surface topography of the affinity membrane and its characterization indicated that rAPC easily forms trimers or hexamers on the membrane surface on use of the given transfer method. The hydrodynamic radius (R(h)) of the rAPC aggregation was equal to 103 nm or 365 nm according to dynamic light scattering (DLS), which was in agreement with the result obtained by AFM. Based on the specific immunological reaction of antigen and antibody, anti-APC antibodies were purified from rabbit polyclonal serum in a single step. The amount of absorbed antibody was 5.79 mg/g membrane according to analysis by ELISA methods. The purity of antibodies was up to 98% according to SDS-PAGE. The adsorption-desorption cycle of rAPC was repeated six times using the same immuno-affinity membrane, and there was no significant loss in adsorption capacity. The method provides a novel and efficient immunological affinity membrane for the purification of antibodies.
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PMID:Preparation of nitrocellulose (NC) immuno-affinity membrane for purification of rAPC antibody. 1838 93

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40+/-2% of donor, P<0.0001; failing 45+/-3% of donor, P<0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level. In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0-C2 (1-453) with PKA using (gamma32)P-ATP up to 3.5 mol Pi/mol C0-C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6+/-0.6 mol Pi/mol and 2.0+/-0.3 mol Pi/mol in myectomy samples. We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy.
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PMID:Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle. 1857 60

The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HV(T); 20 ml/kg) or low (LV(T); 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HV(T) induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HV(T) also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from HV(T)-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-beta(6)) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection.
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PMID:Activated protein C protects against ventilator-induced pulmonary capillary leak. 1936 21

Prosurfactant protein C (proSP-C) is a 197-residue integral membrane protein, in which the C-terminal domain (CTC, positions 59-197) is localized in the endoplasmic reticulum (ER) lumen and contains a Brichos domain (positions 94-197). Mature SP-C corresponds largely to the transmembrane (TM) region of proSP-C. CTC binds to SP-C, provided that it is in nonhelical conformation, and can prevent formation of intracellular amyloid-like inclusions of proSP-C that harbor mutations linked to interstitial lung disease (ILD). Herein it is shown that expression of proSP-C (1-58), that is, the N-terminal propeptide and the TM region, in HEK293 cells results in virtually no detectable protein, while coexpression of CTC in trans yields SDS-soluble monomeric proSP-C (1-58). Recombinant human (rh) CTC binds to cellulose-bound peptides derived from the nonpolar TM region, but not the polar cytosolic part, of proSP-C, and requires >/=5-residues for maximal binding. Binding of rhCTC to a nonhelical peptide derived from SP-C results in alpha-helix formation provided that it contains a long TM segment. Finally, rhCTC and rhCTC Brichos domain shows very similar substrate specificities, but rhCTC(L188Q), a mutation linked to ILD is unable to bind all peptides analyzed. These data indicate that the Brichos domain of proSP-C is a chaperone that induces alpha-helix formation of an aggregation-prone TM region.
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PMID:The Brichos domain of prosurfactant protein C can hold and fold a transmembrane segment. 1947 27

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5'-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.
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PMID:Comparative analysis of newborn and adult Bothrops jararaca snake venoms. 2081 86

Mechanically recovered meat is cheaper than raw meat and thus has been incorporated into many meat-derived products. EU regulations exclude mechanically recovered meat from the definition of meat; as a consequence analytical procedures are needed to differentiate it from hand-deboned meat. The present pilot study has utilized a proteomic approach to find potential markers for the detection of chicken mechanically recovered meat. Intact proteins were extracted from raw meat and then analyzed with OFF-GEL electrophoresis followed by SDS-PAGE and identification of potential markers by nano-LC-MS/MS. It was shown that it is possible to extract, separate and identify key proteins from processed meat material. Potential chicken mechanically recovered meat markers--hemoglobin subunits and those similar to myosin-binding protein C were also identified.
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PMID:Proteomic approach for the detection of chicken mechanically recovered meat. 2155 91

Protein C inhibitor was purified from human plasma by use of a dermatan sulfate or heparin column, followed by hydroxyapatite, gel filtration and ion exchange columns. A dimer of protein C inhibitor was detected by SDS-PAGE under reducing conditions, in addition to two forms of monomer species. One of the monomers, 52-kDa PCI, formed a stable complex with activated protein C, urokinase, plasma and tissue kallikrein, but the dimer species and 48-kDa PCI were inactive. When the monomer and dimer forms of protein C inhibitor were applied to 2D-PAGE, more than 20 spots were observed by Western blot analysis and were confirmed to be protein C inhibitor by MALDI-TOF mass spectrometry. The heterogeneity of the protein C inhibitor species was not due to glycosylation or phosphorylation.
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PMID:Diversity of human plasma protein C inhibitor. 2220 8


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