Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68-113) resulting from the V8 digestion. A synthetic peptide corresponding to the region having the amino acid sequence 72-86 of follistatin was able to bind both heparin and sulfate cellufine, as well as compete with recombinant follistatin for binding to heparin. These findings further define the location of the heparin and heparan sulfate-binding site of follistatin at the basic amino acid-rich region comprising the amino acid sequence Lys75-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys-Pro-Arg86.
Mol Cell Endocrinol 1992 Dec
PMID:Localization of the heparin binding site of follistatin. 130 90

The high prevalence of glucose 6-phosphate dehydrogenase (G6PD) deficiency in African populations is due almost entirely to the enzyme variant A-, which differs from the wild-type G6PD B by two amino acid replacements, 68 Val-->Met and 126 Asn-->Asp. The non-deficient polymorphic variant G6PD A contains only the mutation 126 Asn-->Asp. The frequencies of the G6PD A and of the G6PD A- genes in parts of Africa are both about 0.2. The 68 Val-->Met mutation has not been found in a B background. This could be because the 68 Val-->Met mutation happened to arise in an A gene in the first instance, or because the 68 Val-->Met mutation alone is not sufficient to cause G6PD deficiency. We have approached this question by producing G6PD B, A, A-, and G6PD 68 Val-->Met in a bacterial expression system and analysing their biochemical properties. With each single mutation we found a slight decrease in both the specific activity and the yield of enzyme when compared to G6PD B. When both mutations were introduced together, there was a roughly additive effect on specific activity, but a much more drastic effect on enzyme yield (4% of normal). This synergistic effect was also demonstrated on thermal stability, especially at low NADP concentrations. Comparable results were produced when the replacement 119 Gln-->Glu was studied instead of 126 Asn-->Asp. We infer that the coexistence of the two mutations is responsible for enzyme deficiency in G6PD A- because they act synergistically in causing instability of the enzyme.
Hum Mol Genet 1992 Jun
PMID:Both mutations in G6PD A- are necessary to produce the G6PD deficient phenotype. 130 73

Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R. E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a "control" analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket.
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PMID:The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis. 132 82

Peptides that are derived from the processing of proopiomelanocortin were isolated in pure form from the brain of the frog Rana ridibunda. The primary structure of the most abundant of those peptides was established as: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. This amino acid sequence is identical to that of mammalian and frog pituitary alpha-melanocyte-stimulating hormone (MSH) and the peptide co-eluted with synthetic desacetyl alpha-MSH, indicating that it is COOH-terminally alpha-amidated. A second component, which exhibited a shorter retention time, co-eluted with the glycine-extended form of desacetyl alpha-MSH [ACTH(1-14)]. The primary structure of the third peptide isolated in pure form from the brain extract was established as: Lys-Tyr-Val-Met-Ser-His-Phe-Arg-Trp-Asn-Lys-Phe-NH2. This sequence corresponds to Lys-gamma 1-MSH as predicted from the nucleotide sequence of frog proopiomelanocortin. The presence of substantial amounts of desacetyl alpha-MSH and Lys-gamma 1-MSH in the frog brain supports the concept that, in amphibia, melanotropins may act as neurotransmitters and/or neuromodulators as well as hormonal peptides.
Brain Res Mol Brain Res 1992 Sep
PMID:Isolation and structural characterization of peptides related to alpha- and gamma-melanocyte-stimulating hormone (MSH) from the frog brain. 133 55

In homogenates of guinea pig lung, binding of 125I-Bolton-Hunter-labeled substance P (BHSP), Bolton-Hunter-labeled eledoisin (BHELE), and [125I]iodohistidyl neurokinin A (INKA) was investigated. Equilibrium dissociation constants (derived from "cold" saturation experiments) for BHSP, INKA, and BHELE were 0.96 +/- 0.15, 1.61 +/- 0.26, and 1.98 +/- 0.12 nM, respectively. Specific binding of all three radioligands was increased 2-3-fold by 10 microM phosphoramidon. The rank order of potency of unlabeled tachykinins in competing against BHSP was substance P (SP) greater than [Sar9,Met(O2)11]-SP greater than SP methyl ester greater than neuropeptide gamma greater than neurokinin A greater than or equal to neurokinin B = kassinin greater than or equal to eledoisin greater than or equal to scyliorhinin II much greater than neuropeptide K, indicating binding to sites with the general characteristics of NK1 receptors. Similar rank potency orders were observed for INKA and BHELE, showing binding to NK1 sites, rather than to NK2 or NK3 sites, which are labeled with high affinity by these radioligands in other tissues. For all radioligands, competition curves for SP and the NK1-selective agonist [Sar9,Met(O2)11]-SP could be resolved into two components, representing high and low affinity binding sites. These were present in the approximate ratios 2:3 (for BHSP), 1:1 (for INKA), and 8:1 (for BHELE). Other agonist competition curves also yielded high and low affinity components. The data suggest that BHSP and INKA bind partly and BHELE predominantly to high affinity NK1 receptors. The nature of the low affinity site(s) could be another tachykinin receptor or a low affinity state of the NK1 receptor. Binding to a "classical" NK2 receptor is unlikely, because selective NK2 receptor antagonists and analogs were very weak competitors. Our data suggest that, in addition to the NK1 receptor, another type of tachykinin receptor may exist in this tissue. The inability to detect NK2 binding sites is strikingly at variance with functional studies.
Mol Pharmacol 1992 Jan
PMID:Radioiodinated substance P, neurokinin A, and eledoisin bind predominantly in NK1 receptors in guinea pig lung. 137 Jul 5

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA(Met) containing the acceptor stem of tRNA(fMet) and the remaining of the structure of tRNA(mMet). In addition, more than 30 variants of the genes encoding tRNA(mMet) or tRNA(fMet) have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA(mMet) became formylatable by the only change of the G1.C72 base-pair into C1-A72. It was possible to render tRNA(mMet) as good a substrate as tRNA(fMet) for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2.C71, C3.G70 and G4.C69 are positive determinants for the specific processing of methionyl-tRNA(fMet) by the formylase while the occurrence of a G.C or C.G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA(fMet).
J Mol Biol 1992 Mar 20
PMID:Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase. 137 94

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini. 139 Jun 48

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.
Mol Biol (Mosk)
PMID:[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3]. 143 86

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH2-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.
Plant Mol Biol 1992 Dec
PMID:Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase. 146 36

Spatial structures of proteolytic segment A (sA) of bacterioopsin of Halobacterium halobium (residues 1-36) solubilized in the mixture of methanol-chloroform (1:1), 0.1 M LiClO4 or in perdeuteriated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. Most of the resonances in 1H-NMR spectra of fragment A were assigned using DQF-COSY, TOCSY and NOESY spectra. Deuterium exchange rates for amide protons were measured in series of NOESY spectra. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in mixture of organic solvents and SDS micelles, respectively. The sA structure was determined by local structure analysis, distance geometry calculation with program DIANA and systematic search for energetically allowed side chain rotamers consistent with NOESY cross-peak volumes. The structures of sA are similar in both milieus. These structures have the right-handed alpha-helical region from Pro-8 to Met-32 with root mean square deviation (RMSD) of 0.25 A between back bone heavy atoms and fit well with Pro-8 to Met-32 alpha-helical region in electron cryo-microscopy (ECM) model of bacteriorhodopsin [4]. The C-terminal region Gly-33-Asp-36 is disordered in both milieus, while N-terminal region Ala-2-Gly-6 in organic solvents has a fixed structure (RMSD of 0.25 A) stabilized by the Thr-5 NH...O=C Gln-3 and Ile-4 NH...O = C Ala-2 hydrogen bonds. This region of sA in SDS micelles has disordered structure with RMSD of 1.44 A for back bone heavy atoms. Torsion angles chi 1 of sA were unequivocally determined for 72% of side chains in the alpha-helical region and are identical in both milieus.
Mol Biol (Mosk)
PMID:[Spatial structure of (1-36)bacterioopsin solubilized in a methanol-chloroform mixture with sodium dodecylsulfate micelles]. 149 81


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