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)

Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD+ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H2O2 production for every mole of NADH that was oxidized. Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5'-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of VIV species by reduction of VV of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.
Mol Cell Biochem 1984 Jun
PMID:A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide. 608 22

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.
Mol Biol (Mosk)
PMID:[Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups]. 609 23

The covalent binding of the ultimate carcinogen (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [alpha]pyrene (BPDE) to enriched ovalbumin messenger RNA (mRNAov) of known sequence was examined. Incubation of mRNAov with elevated concentrations of labeled BPDE in TE buffer (0.02 M Tris X HCl, 1 mM EDTA, pH 7.2) containing 0.1 M KCl and 10 mM MgCl2 resulted in approximately 30 BPDEs covalently bound per RNA molecule. Covalent binding in the absence of KCl and MgCl2 resulted in a significant increase in binding to 110 BPDEs bound per molecule or modification of 12% of the total guanosine and adenosine nucleotides present. The nucleoside adducts formed were nearly all guanosine and adenosine in a ratio of 1.6:1.0. It was also observed that digestion of mRNAov with T2 RNase prior to reaction with BPDE resulted in a 52% decrease in guanosine adduct formation and a 93% decrease in adenosine adducts compared with undigested controls. Comparison of the binding of labeled BPDE to 18 S and 28 S ribosomal RNAs and to mRNAov revealed that the guanosine adduct to adenosine adduct ratio and the number of BPDEs bound increased with increasing G-C content. The results reported here show that ionic composition of the medium, G-C content, and the presence of a polymeric state can significantly influence the quantitative and/or qualitative nucleoside BPDE adducts formed.
Mol Pharmacol 1984 Sep
PMID:Factors influencing the covalent binding of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to ribonucleic acids. 620 22

Myosin from chicken pectoralis muscle consists of isozymes that differ in their alkali light chains. It is possible to isolate alkali 1 (A1) and alkali 2 (A2) homodimers of native myosin by immunoadsorption methods, and to compare their steady-state kinetics as well as their assembly into synthetic filaments under a variety of ionic conditions. Bipolar filaments of the isozymes formed at low salt concentrations had a narrow length distribution and did not differ from controls made from unfractionated myosin. Chicken myosin also assembles into highly homogeneous minifilaments similar to those formed by rabbit myosin in a citrate/Tris buffer. Analytical ultracentrifugation and electron microscopy showed that A1-homodimer, A2-homodimer and unfractionated myosin assembled into 0.3 micron short, bipolar minifilaments, which were indistinguishable from one another in size and shape. The steady-state myosin ATPase activity of the two homodimeric isozymes was identical in K+(EDTA) and Ca2+ assay media. The actomyosin Mg2+ ATPase measured at 25 and 55 mM-KCl (pH 8.0) showed only minor differences in both Vmax and Kapp. Actomyosin activity was also determined for the more homogeneous minifilament preparations of the isozymes and these, as well, produced essentially indistinguishable kinetic parameters. Thus we find no evidence to support the hypothesis that a particular alkali light chain of myosin can affect either the structure of the filaments or the steady-state rate of ATP hydrolysis.
J Mol Biol 1983 Oct 25
PMID:Assembly and kinetic properties of myosin light chain isozymes from fast skeletal muscle. 622 5

The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polymerase holoenzyme (RNAP) and the lambda PR promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 degrees C, 37 degrees C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at PR by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction. The association reaction between RNAP and lambda PR was investigated under ionic conditions where the process is essentially irreversible, and under pseudo first-order conditions of excess polymerase. The pseudo first-order rate constant is directly proportional to the concentration of active polymerase over the entire range investigated (2 to 10 nM) at both 25 degrees C and 37 degrees C, within experimental uncertainty. Second-order association rate constants (ka), calculated from these data at standard ionic conditions (0.12 M-KCl, 0.01 M-MgCl2, 0.04 M-Tris (pH 8)), were strongly temperature-dependent: ka = (2.6 +/- 0.4) X 10(6) M-1 S-1 at 37 degrees C and ka = (7.2 +/- 1.4) X 10(5) M-1 s-1 at 25 degrees C, corresponding to an activation energy of the association reaction of approximately 20 +/- 5 kcal. In addition, ka decreases strongly with increasing KCl concentration, corresponding to the net release of the thermodynamic equivalent of at least nine monovalent ions prior to or during the rate-limiting step of the association reaction. This strong dependence of ka on the ionic environment suggests that inorganic cations should be considered as possible regulators of in vivo transcription initiation. Dissociation rate constants (kd) were also measured under irreversible reaction conditions. At the standard ionic conditions, kd = (2.2 +/- 0.3) X 10(-5) s-1 at 37 degrees C and kd = (4.0 +/- 0.4) X 10(-5) s-1 at 25 degrees C. The increase in kd with decreasing temperature corresponds to a negative activation energy of dissociation (-9 +/- 4 kcal). In addition, kd increases with increasing KCl concentration, corresponding to the net uptake of the thermodynamic equivalent of at least six monovalent ions in or prior to the rate-limiting step of the dissociation reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1984 Jul 15
PMID:Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter. 623 75

The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.
Mol Immunol 1983 Jan
PMID:Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers. 630 2

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.
J Mol Biol 1984 Jan 25
PMID:Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. 631 16

The relationship between crossbridge release and alpha-helix-coil transition in myosin has been investigated by employing synthetic myosin and rod minifilaments prepared in 10 mM-citrate/Tris buffer at pH 7.0 and 8.0. Initial sedimentation velocity and turbidity measurements have established that the minifilament structures obtained at pH 7.0 and 8.0 are relatively similar in size and homogeneity, and can be used in comparative circular dichroism studies. Chemical crosslinkings and proteolytic digestions carried out at pH 7.0 and 8.0 verify that myosin and rod minifilaments undergo the same pH-induced changes as myosin filaments, i.e. a decrease in the rate of subfragment-2 crosslinking to the filament surface, and an increase in proteolytic susceptibility of the light meromyosin-heavy meromyosin hinge at alkaline pH. These results suggest charge-induced release of the S-2 element from the myosin and rod minifilament surface. Circular dichroism measurements reveal a reduced alpha-helical content of myosin (5%) and rod minifilaments (10%) at pH 8.0 compared to the respective pH 7.0 structures. These results establish a direct link between crossbridge release and alpha-helix-coil transition in myosin.
J Mol Biol 1983 Sep 15
PMID:Crossbridge release and alpha-helix-coil transition in myosin and rod minifilaments. 635 54

Crystals for Fab fragments from a monoclonal antibody to HPr of the phosphoenopyruvate:sugar phosphotransferase system of Escherichia coli have been obtained from 14% polyethylene glycol 6000, 5 mM-Tris X HCl, 50 mM-sodium phosphate and 0.2 M-sodium chloride at pH 8.0. The space group is P2(1) with a = 110.85 A, b = 66.18 A, c = 67.21 A, beta = 113.0 degrees and Z = 4. The crystals exhibit the forms [100], [011] and [011] and the solvent content is 47%.
J Mol Biol 1984 Aug 05
PMID:Preliminary crystallographic data for a monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. 637 99

Trypanosoma (Schizotrypanum) cruzi epimastigotes (EP stock) grown in complex LIT medium rapidly consume the glucose present but, under aerobic conditions, continue growth in its absence with the concomitant excretion of ammonia, suggesting the utilization of amino acids for energy production. A search for metabolic pathways responsible for amino acid oxidation led to the detection of a NAD+-dependent glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase, E.C.1.4.1.2) which is different from an NADP+-dependent enzyme previously reported. The enzyme has been partially purified and its kinetic and regulatory properties studied in both directions of the reaction. Km values were 3.6 mM for alpha-ketoglutarate, 0.170 mM for NADH and 16 mM for NH+4, Vmax = 0.67 mumol min-1/mg-1 protein for aminative reduction; Km values were 23.5 mM for L-glutamate and 2.9 mM for NAD+, Vmax = 0.02 mumol min-1 mg-1 protein for deaminative oxidation, Tris buffer, pH 7.6. The enzyme is strongly inhibited by ATP, GTP, ADP and GDP (50% inhibition at 0.75 mM ATP, 3 mM MgCl2). S-Acetyl-CoA is also a potent inhibitor of the enzyme. The results demonstrate the presence of a specific pathway for the oxidation of amino acids, which is tightly regulated by the energy charge and the Krebs cycle activity in T. cruzi epimastigotes.
Mol Biochem Parasitol 1984 Apr
PMID:Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. II. NAD+-dependent glutamate dehydrogenase. 637 48


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