Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.
J Mol Biol 1992 Jul 20
PMID:Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum. 164 Apr 69

The cluster of streptomycin (SM) production genes in Streptomyces griseus was further analysed by determining the nucleotide sequence of genes strFGHIK. The products of the strF and/or strG genes may be involved in the formation of N-methyl-L-glucosamine, and that of the strH gene in the first glycosylation step condensing streptidine-6-phosphate and dihydrostreptose. The putative StrI protein showed strong similarity to the amino-terminal NAD(P)-binding sites of many dehydrogenases, especially of the glyceraldehyde-3-phosphate dehydrogenases. The product of the strK gene strongly resembles the alkaline phosphatase of Escherichia coli. It was shown that S. griseus excretes an enzyme that specifically cleaves both SM-6-phosphate and--more slowly--SM-3''-phosphate ate during the production phase for SM. The identity of this enzyme with the StrK protein was demonstrated by expression of the strK gene in Streptomyces lividans 66. Further evidence for an involvement of these genes in SM biosynthesis came from the fact that genes homologous to them were found in the equivalent gene cluster of the hydroxy-SM producer Streptomyces glaucescens; these, however, were in part differently organized. The ca. 5 kb DNA segment downstream of strI in S. griseus which contains the strK gene was found to be located in inverse orientation between the homologues of the aphD and strR genes in S. glaucescens.
Mol Gen Genet 1991 Sep
PMID:Genetics of streptomycin production in Streptomyces griseus: nucleotide sequence of five genes, strFGHIK, including a phosphatase gene. 165 2

NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent Kms for alpha-ketoglutarate, NADPH and NH4+ are 1.2 mM, 9.7 microM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 microM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn2+ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 microM) yet N-ethylmaleimide does not. In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.
Mol Cell Biochem 1991 Jun 26
PMID:Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum. 165 3

The mechanism of stimulation of 17 beta-estradiol (E2) formation from estrone (E1) by 5 alpha-dihydrotestosterone (5 alpha-DHT) in placental villi was investigated by examining; (1) if dehydroepiandrosterone (DHA) was stimulatory, (2) if NAD(P)H-generating, non-steroidal substrates stimulated E2 formation, (3) the subcellular localization of the effect, (4) if NAD(P) or NAD(P)H was required and (5) rates of 5 alpha-DHT oxidation by villi and microsomes. Although 5 alpha-DHT and DHA both inhibited the E2 to E1 reaction in villi and microsomes, only 5 alpha-DHT stimulated the conversion of E1 to E2. Glucose and lactate were slightly stimulatory when compared with 5 alpha-DHT. Stimulation of E2 formation was observed with microsomes but not with cytosol, and NAD or NADP was required. The results indicate that neither inhibition of the back reaction, E2 to E1, nor NADH or NADPH formation via the 3 beta-hydroxysteroid dehydrogenase/5-ene-3-ketosteroid isomerase reaction can account for the stimulation. It is proposed that the mechanism of stimulation involves one or more forms of membrane-bound 17 beta-hydroxysteroid oxidoreductase with NADH or NADPH formed as a product of 5 alpha-DHT oxidation being used as the cofactor for E1 reduction. This may involve a direct transfer of reduced pyridine nucleotide between enzyme molecules without equilibration with intracellular coenzyme pools.
J Steroid Biochem Mol Biol 1991 Nov
PMID:Regulation of human placental 17 beta-hydroxysteroid oxidoreductase: mechanism of stimulation of 17 beta-estradiol formation from estrone by 5 alpha-dihydrotestosterone in homogenates and villi in vitro. 165 69

Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am-, acu- double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am+ (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3- mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.
Mol Gen Genet 1991 Oct
PMID:Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme. 168 13

Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit.
Plant Mol Biol 1991 Nov
PMID:A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana. 171 78

DNA fragments encoding streptococcal NADH peroxidase (NPXase) have been amplified, cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The NPXase gene (npr) comprises 1341 base-pairs and is preceded by a typical ribosome binding site. Upstream from the structural gene, putative -10 and -35 promoter regions have been identified, as has a possible factor-independent terminator that occurs in 3'-flanking sequences. The deduced relative molecular mass (Mr = 49,551), amino acid composition and isoelectric point of NPXase are in good agreement with previous values obtained with the purified enzyme. In addition, three sequenced peptides totaling approximately 20% of the protein were located in the npr gene product. From the sequencing data the deduced NPXase sequence shares low but significant homology with the flavoprotein disulfide reductase class of enzymes ranging from 21% for glutathione reductase (GRase) to 28% for thioredoxin reductase. Alignment of NPXase to Escherichia coli GRase allowed the identification of three previously reported fingerprints for the FAD, NADP+ and central domains of GRase, in the peroxidase sequence. In addition, Cys42 of NPXase, which is present as an unusual stabilized cysteine-sulfenic acid in the oxidized enzyme, aligns favorably with the charge-transfer cysteine in E. coli GRase, and both residues closely follow FAD-binding folds found near their respective amino termini. Such sequence characteristics can also be seen in mercuric reductase, lipoamide dehydrogenase and trypanothione reductase, suggesting that all these enzymes may have originally diverged from a common ancestor. Sequences that are on average 50% identical with three previously reported peptides of the related streptococcal NADH oxidase were also identified in the NPXase primary structure, suggesting a strong similarity between these flavoenzymes. Using the E. coli phage T7 expression system the npr gene has now been overexpressed in an E. coli genetic background. The resultant overexpressing clone produced a recombinant NPXase that was catalytically active and immunoreactive to NPXase antisera.
J Mol Biol 1991 Oct 05
PMID:Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. 171 12

The present studies on initial velocity of testosterone reduction by hepatic 5 beta-reductase (4-en-3-oxosteroid 5 beta-reductase) of chicken and mode of inhibition of the 5 beta-reduction by 5 beta-dihydrotestosterone and NADP+ indicated that the reduction of testosterone occurred after the 5 beta-reductase bound firstly to NADPH and then to testosterone, forming a ternary complex. After 5 beta-reduction, 5 beta-dihydrotestosterone and then NADP+ were liberated from the complex, following a mechanism of "ordered Bi-Bi". Effect of (4R)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione (a steroidal 5 alpha-reductase-inhibitor or Secosteroid), diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (the other steroidal 5 alpha-reductase-inhibitor or 4-MA), and glycyrrhetinic acid (3 beta-hydroxy-11-oxoolean-12-en-30-oic acid, a 5 beta-reductase-inhibitor) was examined upon the 5 beta-reductase activity by double reciprocal plots. The mode of inhibition against testosterone by 4-MA and glycyrrhetinic acid was found to be competitive, while that by Secosteroid was non-competitive.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Kinetic mechanism of reduction of testosterone by hepatic 5 beta-reductase of chicken and inhibition of the reductase activity by a secosteroid, an azasteroid and glycyrrhetinic acid. 173 34

We have isolated and sequenced the gene for a putative NADP-dependent glutamate dehydrogenase from the extremely halophilic archaebacterium Halobacterium salinarium. This gene is transcribed as a unique RNA molecule of about 1700 nucleotides. The 5' end of the transcript contains characteristic consensus transcription initiation and promoter sequences observed in halophilic archaebacteria. The encoded polypeptide, with a predicted length of 435 amino acids, shows significant overall homology and conservation of functional domains when compared with different eubacterial and eukaryotic glutamate dehydrogenases. Surprisingly, the archaebacterial protein shares a larger number of identical amino acid residues with homologous polypeptides from higher eukaryotes than with those from unicellular eukaryotes and eubacteria.
Mol Gen Genet 1991 Dec
PMID:The gene for a halophilic glutamate dehydrogenase: sequence, transcription analysis and phylogenetic implications. 176 32

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.
Mol Cell Biochem 1991 Sep 18
PMID:Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds. 178 72


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