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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).
J Mol Biol 1991 Jul 20
PMID:Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase. 185 54

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgCl2 and 0.1 microM S-adenosyl-L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in Km for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1 microM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.
Mol Cell Biochem 1991 Apr 24
PMID:Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma. 185 47

We constructed a 66 base-pair DNA template capable of supporting transcription by T7 RNA polymerase. This template had a psoralen cross-link downstream from a T7 promoter such that a 36 (+1) nucleotide transcript was synthesized at the time the T7 polymerase came to a stop at the cross-link. The stability of elongation complexes formed on this template, and the effect of different factors that are known to affect polymerase-DNA interactions was investigated by non-denaturing gel electrophoresis and gel filtration chromatography. We found that an elongation complex could lose its RNA component but the T7 polymerase still remained attached to the DNA template for extended periods of time (at least up to 18 h). This type of an elongation complex, bereft of its nascent RNA transcript, is called a quasi-elongation complex. DNase I footprinting within gel slices indicated that the polymerase molecules were arrested at the psoralen cross-link on the DNA template in the quasi-elongation complexes. The quasi-elongation complexes were found to be extremely stable in 0.5 M-NaCl and in 0.2 M-NaCl plus 60 mM-MgCl2, and could withstand temperatures up to 42 degrees C. The quasi-elongation complexes were destabilized by heparin and excess calf thymus DNA. Excess tRNA caused only a minimal degree of disruption. Non-promoter-containing plasmid DNAs did not have a destabilizing effect on the quasi-elongation complexes. Interestingly, it was observed that in a T7 ternary transcriptional complex arrested by a psoralen cross-link, the nascent RNA transcript could be stabilized from release by the presence in trans of a plasmid DNA bearing a T7 promoter and a T7 terminator. Such a stabilization against RNA release was not observed with plasmid DNAs containing either only a promoter or a terminator. The elongation complexes were stable during gel filtration through Sephacryl S-300 HR. However, it was found that 30% to 45% of the labeled RNA was retained during gel filtration as RNA that was apparently free from ternary complexes.
J Mol Biol 1991 Oct 20
PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. II. Stability and some properties of elongation complexes. 194 45

There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns. This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a "pseudoknot" and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof. In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU). Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule. While P11 is not essential for self-splicing activity in vitro, mutants with disrupted P11 require higher concentration of MgCl2 for self-splicing. By contrast, mutants with a reinforced P11 pairing (via introduction of several G.C base-pairs) self-splice more efficiently than the wild-type at 55 degrees C. Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.
J Mol Biol 1991 Oct 20
PMID:Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA. 194 46

Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pf1 with single-stranded DNA. The c.d. spectrum of the Pf1 gene 5 protein is consistent with the absence of any significant alpha-helical content. The negative c.d. peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT). Likewise, the c.d. peak at 265 nm arising from the poly(dT) decreases when the Pf1 gene 5 protein is bound, c.d. titrations of poly(dT) with Pf1 gene 5 protein indicate strong binding with a stoichiometry (n) of four nucleotides per protein subunit. In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions. The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides. The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess. The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice. Further experiments in which the order of addition of components is changed, and the concentration of MgCl2 is varied, show that both of these factors are important in determining the dominant binding mode. In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein. This transition is suppressed in the presence of greater than 3 mM-MgCl2.
J Mol Biol 1991 Feb 20
PMID:Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT). 200 18

The small oligomers formed from Mg-G-actin under favorable conditions were studied by sedimentation velocity ultracentrifugation. The critical concentration of actin at pH 7.8 in the presence of 100 microM MgCl2 and 200 microM ATP was 12.5 +/- 2.8 microM. Under these conditions, about 15% of 7.5 microM Mg-actin was converted to oligomers of subunit size four to eight in 5 h at 20 degrees C. In 100 microM MgCl2 and no free ATP, the critical concentration was about 6.5 microM, and about 22% of 7.5 microM Mg-actin was converted to dimers in 80 min. There were no detectable higher oligomers or F-actin present in either case. As determined by the analysis of ATP hydrolysis, most, if not all, of the oligomer subunits contained ATP. When 28.5 microM actin was polymerized to steady state in 100 microM MgCl2 and 200 microM ATP, about 50% of the actin was present as F-actin, consistent with the critical concentration (approximately 12.5 microM), about 50% as oligomers as large as seven subunits, and only about 5% as monomers. When solutions containing oligomers were diluted the oligomers dissociated. Alternatively, when the MgCl2 concentration was raised to 1 mM, the solutions containing oligomers polymerized more rapidly than monomeric Mg-G-actin and to the same final steady state. These data are entirely consistent with the condensation-elongation model for helical polymerization proposed by Oosawa and Kasai (Oosawa, F., and Kasai, M. (1962) J. Mol. Biol. 4, 10-21) according to which, under certain conditions, substantial amounts of short linear and helical oligomers should be formed below the critical concentration and linear oligomers should coexist with monomers and F-actin at steady state.
 ...
PMID:The formation of actin oligomers studied by analytical ultracentrifugation. 201 96

Because adenosine narrows asthmatic airways, is released during hypoxia and by mast cells, and is antagonized by theophylline, it may play a role in asthma. I characterized the first step in pulmonary responses to adenosine: its adenosine receptor. Plasma membranes, prepared from macroscopically normal human peripheral lung, were incubated with 10 nM 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) and various concentrations of competing ligand under experimentally determined optimal conditions: 4 degrees C, pH 7.4, 5 mM MgCl2, 1.8 mg protein/ml, 30-min incubation time. Bound and free ligand were separated by rapid vacuum filtration, and the radioactive counts were analyzed using a weighted, non-linear, least-squares curve-fitting program, LIGAND. Analyzed together, the data from the lungs of 6 patients revealed a single binding site with a dissociation constant (Kd) for NECA of 200 nM +/- 14% and a receptor concentration of 543 fmol/mg protein +/- 13%. Analyzed separately, the individual Kds ranged from 133 to 430 nM and the receptor concentrations from 338 to 811 fmol/mg protein. Adenosine receptor ligands competed with NECA in an A2 rank order of potency: NECA greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than theophylline greater than N6-L-phenylisopropyladenosine greater than N6-D-phenylisopropyladenosine greater than N6-cyclohexyladenosine. Theophylline bound to the receptor with an inhibition constant (Ki = 70.9 microM +/- 28%) near the therapeutic range (28 to 56 microM). Cromolyn also bound with high affinity (Ki = 5.42 microM +/- 47%). I conclude that human lung adenosine receptors: (1) are single-site receptors, probably of the A2 subtype and (2) bind to both theophylline and cromolyn.
Am J Respir Cell Mol Biol 1990 Feb
PMID:Characterization of the human peripheral lung adenosine receptor. 240 76

The following DNA fragments were studied: T5A5, U5G5, C5G5, RI (CCGAATTCGG), and RV (CCGATATCGG). The ligated decamers were run on 8% polyacrylamide gel. The PAGE anomaly was measured by the R-factor. Under standard conditions R is maximum for A5G5 and U5A5 (R = 1.9-2.0); R has intermediate values for RI and T5A5 (R200 = 1.05-1.10). T5A5 exhibits unusual non-monotonous temperature dependence of the R-factor, which reaches its maximum at 35 degrees C. Based on this fact we make two conclusions: (i) PAGE anomaly is highly sensitive to the DNA winding angle tau, (ii) tau = 36 degrees for T5A5 under these conditions. For A5G5, RI and RV the temperature dependence of the R-factor is conventional: R monotonously decreases. Conceivably tau less than 36 degrees for these sequences. In the presence of 10 mM of MgCl2 the anomaly increase for all fragments, except for T5A5; e.g. for C5G5R150 R200 = 1.2. This testifies to the moderate curvature of C5G5. The results of the measurements were interpreted on the basis of the wedge AA and junction An/B models. To get a better agreement with the experiment these models should be supplemented with the bends in the YR dimers (TA, CG, CA) directed into the major groove and with the bends in AT directed into the minor groove.
Mol Biol (Mosk)
PMID:[The effect of temperature and ionic strength on the electrophoretic motility of synthetic DNA fragments]. 216 94

Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.
J Mol Biol 1990 Feb 20
PMID:Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament. 231 98

Reassembly of the neurofilament (NF) in vitro was studied by low-angle rotary shadowing electron microscopy. Various intermediate stages of the reassembly were reconstructed from the smallest molecular mass subunit (NF-L) under controlled reassembly conditions. NF-L in 6 M-urea took the form of spherical particles with a diameter of about 12 nm. NF-L aggregated into rodlets of 70 to 80 nm long in a low-salt solution at alkaline pH. By reducing the pH of the dialyzing solution to 6.6, a pair of rods was formed by association side-by-side. Increasing the temperature of low-salt solutions from 4 degrees C to 35 degrees C did not produce intermediate-sized filaments. The addition of Mg2+ to the dialyzing solution resulted in the formation of short intermediate-sized filaments even at 4 degrees C. Further dialysis of the short intermediate-sized filaments against reassembly solution containing both NaCl and MgCl2 at 37 degrees C failed to elongate them into longer filaments, suggesting that annealing does not contribute to the elongation of neurofilaments. Different roles for Mg+ and NaCl in neurofilament reassembly were indicated. While Mg2+ strengthened the lateral association between 70 to 80 nm rods, NaCl appeared to promote the end-to-end association of filaments preferentially. Longer filaments were formed by increasing the NaCl concentration. By dialyzing NF-L against a buffer containing 50 mM-NaCl in the absence of Mg2+, unraveled filaments were formed. The many unraveled filaments were composed of four 8 nm wide filaments, which have been called the subfilament or the protofibril. Time-course experiments of the reassembly were performed in the absence of Mg2+, in which condition the rate of neurofilament reassembly appeared to be reduced. Star-like clusters, about four protofibrils joined together at one end, were suggested to be the initial stage of the intermediate-sized filament formation. The following two-step elongation mechanism of neurofilaments was deduced from these results. The pairs of rods were added to the ends of the protofibrils of neurofilaments, and after all four protofibrils were elongated they were then packed into neurofilaments. Distribution of larger molecular mass subunits, NF-M and NF-H, was studied. Addition of NF-M or NF-H to NF-L did not change the assembly properties of neurofilaments. Unraveled filaments reconstituted from NF-L plus either NF-M or NF-H indicated that NF-M and NF-H are incorporated evenly into each protofibril.
J Mol Biol 1990 Feb 20
PMID:Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro. 231 99


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