Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A stretch of about 150 amino acids located between the heme and the calmodulin recognition sequence of nitric oxide synthase (NOS) has been strongly conserved within isoforms and was proposed to participate in pteridine binding because of sequence similarities to the folate binding site of dihydrofolate reductase (DHFR). In the present study we tested four synthetic peptides corresponding to sequences located within the putative DHFR domain of rat neuronal NOS for their effects on catalytic and binding activities of the recombinant enzyme purified from baculovirus-infected insect cells. Three of the selected peptides had no effects at concentrations of up to 0.1 mM, but one peptide, corresponding to amino acid residues 564-582 of neuronal NOS, led to a concentration-dependent inhibition of L-citrulline formation. The potency of the peptide decreased with increasing assay concentrations of NOS, pointing to a competitive interaction with a specific structure of the enzyme. The peptide was not competitive with L-arginine and H4biopterin, did not antagonize binding of radiolabeled NG-nitro-L-arginine or H4biopterin, and had no effect on Ca2+/calmodulin-dependent reduction of cytochrome c. However, the presence of the peptide led to a pronounced inhibition of NADPH oxidation in the absence of L-arginine and prevented stimulation of this reaction by the amino acid substrate. These results indicate that sequence 564-582 of neuronal NOS does not contribute to L-arginine or H4biopterin binding but is critically involved in the electron transfer from the reductase domain to the heme.
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PMID:A synthetic peptide corresponding to the putative dihydrofolate reductase domain of nitric oxide synthase inhibits uncoupled NADPH oxidation. 970 Oct 44

The authors evaluated whether supplementation of tetrahydrobiopterin (BH4) or the BH4 precursor in vascular smooth muscle cells (VSMC) that are deficient in de novo BH4 production affected the ability of these cells to synthesize nitric oxide (NO). GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for the de novo synthetic pathway of BH4. The selective GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) restricts the de novo synthesis of BH4 in VSMC. Thus, treatment with DAHP and cytokines should lead to the intracellular accumulation of inactive inducible nitric oxide synthase (iNOS) apoenzyme (relative to BH4), which can be reactivated upon the repletion of BH4. Using such VSMC deficient in de novo BH4, the authors evaluated their ability to synthesize NO by supplementing with BH4 or the BH4 precursor sepiapterin or dihydrobiopterin. Adding BH4 to VSMC increased NO production in a concentration-dependent manner. Sepiapterin (SEP) or 7,8-dihydrobiopterin (BH2) also dose-dependently induced NO generation. Nitric oxide was produced in the order SEP >BH2>> BH4 at half-maximal concentrations for stimulation of 0.05, 0.1, and 1 micromol/L, respectively. Addition of BH4 or SEP substantially induced iNOS enzyme activity, which was quantified as the formation of [3H]L-citrulline from [3H]L-arginine. SEP or BH2 in the presence of methotrexate, a selective inhibitor of dihydrofolate reductase, no longer induced NO production. In contrast, although the amount of NO induced by supplemented BH4 was substantially depressed, higher concentrations of BH4, but not SEP or BH2, significantly caused NO production even in the presence of methotrexate. Thus, BH4 produced from BH2 is largely responsible for NO production in VSMC that are deficient in de novo BH4. NOS would preferentially use BH4 that is regenerated from BH2 via dihydrofolate reductase, rather than BH4 or BH2 obtained from the cytosolic milieu.
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PMID:Exogenous biopterins requirement for iNOS function in vascular smooth muscle cells. 1288 22