The Journal of biological chemistry
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Activation by phenylalanine and reduction by the co-factor (6R)-tetrahydrobiopterin (BH4) are required for formation of active liver phenylalanine hydroxylase. This work describes effects of the activation and redox state on substrate and effector recognition of this enzyme, it establishes relationships among the pterin and phenylalanine binding sites on the different forms of the enzyme, and it provides a quantitative description of the enzyme's presumptive regulatory and catalytic sites. BH4, 7,8-dihydrobiopterin (BH2), 6-methyltetrahydropterin, and 5-deaza-6-methyltetrahydropterin were found to bind to unactivated phenylalanine hydroxylase with a stoichiometry of 1/enzyme subunit and with hyperbolic kinetics; all appear to compete for the same binding site on the enzyme, and all appear to bind in the proximity of, but not to, the enzyme's non-heme iron. ⋯ The pterin- and phenylalanine-binding sites on activated phenylalanine hydroxylase appear to be part of the enzyme's active site. Despite large effects on substrate binding, neither chelator binding ability nor solvent accessibility of the iron are affected by activation; activation appears to affect the nearby environment of the enzyme's iron but not the iron itself. Studies of oxidized and reduced phenylalanine hydroxylase indicate that the redox state is not a major determinant of pterin and phenylalanine association with enzyme.
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Tetrahydropterins react with phenylalanine hydroxylase at a redox site, a regulatory site, and the catalytic site, but neither the properties of nor relationships among these sites are well understood. We have studied the redox site using the fluorescent iron chelators 2,3-dihydroxynaphthalene and bathophenanthroline; these compounds act as site-specific reporter groups for reactions on oxidized and reduced enzyme, respectively. The chelators bind reversibly and specifically to the enzyme's iron with 1:1 stoichiometry, high affinity (Kd values approximately 1 nM), and complete quenching of their own fluorescence. ⋯ Studies of the reaction of tetrahydrobiopterin (BH4) at the enzyme's redox site showed that BH4 reduces the enzyme more slowly than 6-methyltetrahydropterin under catalytic and non-catalytic conditions. Reduction occurs at a distinct site whose binding determinants and reaction characteristics are different from those of the BH4 regulatory or catalytic sites, and phenylalanine-activated enzyme is reduced more rapidly than unactivated enzyme. In reducing phenylalanine activated enzyme, BH4 donates one electron/subunit (1/iron atom); the reduction kinetics suggest a trihydrobiopterin-free radical as a reaction intermediate.
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Iron regulatory factor (IRF) is a cytoplasmic mRNA-binding protein with specificity for iron-responsive element (IRE) RNA stem-loops. IRF post-transcriptionally regulates intracellular iron levels via binding to IREs in the untranslated regions of ferritin, transferrin receptor, and erythroid 5-aminolevulinic-acid synthase mRNAs. Specific IRE nucleotides are phylogenetically conserved: those of the 6-base loop (5'-CAGUGN-3') and an unpaired "bulge" cytosine. ⋯ This novel finding predicts base pairing within the IRE loop between positions 1 and 5, thus facilitating the formation of a specific loop structure in which nucleotides at positions 2-4 are made accessible for protein interaction. Nucleotide substitution at these loop positions, or at the position of the bulge cytosine, decreased binding by 36-99%. In addition, we demonstrate a preferred IRE bulge structure and report a striking difference in the RNA binding specificity of rat IRF compared with that of the related IRE-binding protein, IRFB.
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Comparative Study
Molecular characterization of a new metabotropic glutamate receptor mGluR7 coupled to inhibitory cyclic AMP signal transduction.
A cDNA clone for a new rat metabotropic glutamate receptor termed mGluR7 was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic receptor (mGluR) family and by the subsequent screening of a rat forebrain cDNA library. The cloned mGluR7 subtype consists of 915 amino acid residues and exhibits a structural architecture common to the mGluR family with a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR7 shows the highest sequence similarity to mGluR4 and mGluR6 among the members of the mGluR family. Similar to mGluR4 and mGluR6, mGluR7 inhibits forskolin-stimulated cyclic AMP accumulation in response to agonist interaction and potently reacts with L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate in Chinese hamster ovary cells transfected with the cloned cDNA. RNA blot and in situ hybridization analyses of mGluR7 mRNA indicated that it is widely expressed in many neuronal cells of the central nervous system and is thus different from the more limitedly expressed mGluR4 or mGluR6 mRNA. mGluR7 together with mGluR4 thus corresponds to the putative L-2-amino-4-phosphonobutyrate receptor which plays an important role in modulation of glutamate transmission in the central nervous system.
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As an approach to elucidate the role of collagen XIV, which is still unclear, molecules exhibiting affinity for this collagen have been sought in connective tissue. Extracts from fetal bovine tendon were resolved by gel electrophoresis and electrophoretically transferred to nitrocellulose. The blot was overlaid with native collagen XIV and the collagen XIV-binding molecules revealed by immunodecoration with a monoclonal antitype XIV collagen antibody. ⋯ By solid phase assays we have studied this newly described association between decorin and type XIV collagen and shown that it is a saturable process. In addition, preliminary determination of the domains of the two molecules involved in the association has been performed. The possible role of these interactions is discussed.