UNIPROT:Q07644 - FACTA Search

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Query: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.
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PMID:Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. 164 26

Previously, the synthesis and validation of [32P]2N3NAD+ as an active site directed photoaffinity probe for glutamate dehydrogenase (GDH) was reported (8). This report shows that 2N3NAD+ is also an effective probe for the NAD+ binding site of lactate dehydrogenase (LDH). With the appropriate photolabeling procedures and immobilized boronate column chromatography the active site peptides of GDH and LDH involved in the adenine base binding domain have been isolated and sequenced. With both GDH and LDH a single photolabeled peptide, which contained the majority of the photoinserted radiolabel, was isolated. Additionally, these peptides had UV spectra that were markedly different from the nonphotolabeled peptides. The modified peptide from GDH corresponded to Cys270 through Lys289. Both sequencing and compositional analysis identified Glu275 as the site of photoinsertion. Sequencing of this peptide aborted at Glu275 after five rounds of analysis, indicating that insertion was blocking further progress. Compositional analysis showed that the entire sequence from residues 270 to 289 was present except that the single Glu residue was missing. This is interpreted as indicating that the photoinsertion is into the polypeptide backbone at the Glu site. The peptide isolated from LDH corresponded to Asp82 through Arg90. Sequencing of this peptide could be completed throughout with only the round at Tyr83 giving no identifiable residue. Compositional analysis of this peptide was in agreement with the peptide from Asp82 to Arg90 with the exception that the single Tyr residue was missing. This indicates that the photoinsertion is into the tyrosine side chain. This data was found to be in agreement with X-ray crystallographic results identifying the NAD(+)-binding domains.
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PMID:Identification of peptides in the adenine ring binding domain of glutamate and lactate dehydrogenase using 2-azido-NAD+. 168 10

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.
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PMID:Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution. 174 99

The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined. In the 5' noncoding region of the P. stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106. The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR). The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae. Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae. None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein. Transformants of S. cerevisiae containing XYL1 of P. stipitis synthesize an active XR. Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S. cerevisiae and Escherichia coli.
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PMID:Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis. 175 86

Identification and characterization of the Na+/Pi co-transporter in the renal brush-border membrane (BBM) has proved to be difficult in part because of the lack of a specific covalent label. NAD is a competitive inhibitor of Na+/Pi co-transport, and we have explored its potential use as a specific label. We describe the synthesis and use of a highly reactive azido derivative of NAD. This derivative (AB-NAD), like the parent NAD molecule, acts as a competitive inhibitor of Na+/Pi co-transport by isolated BBM vesicles. After photoirradiation, the inhibition changes to noncompetitive, as would be expected if the label was bound covalently. This was confirmed by use of [3H]AB-NAD. Photoirradiation produced a 4-fold increase in acid-stable incorporation of 3H into BBM vesicles compared to controls which were not exposed to light. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that photoirradiation with [32P]AB-NAD produced labeling of several different protein bands, but almost one-half of the 32P was recovered in two bands corresponding to molecular masses of 97 and 70 kDa. Labeling of these bands was markedly reduced in the presence of Na+ and phosphonoformic acid, a specific inhibitor of Na+/Pi co-transport. Chromatography of solubilized BBM proteins indicated that the protein fraction which is photolabeled by AB-NAD is co-eluted with the protein fraction which exhibits Na(+)-dependent binding of phosphonoformic acid. The 97- and 70-kDa polypeptide bands may contain components of the intact Na+/Pi co-transport system.
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PMID:Photoaffinity labeling of brush-border membrane proteins which bind phosphonoformic acid. 182 42

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3. 184 55

Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.
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PMID:Characterization and sequence of a novel nitrate reductase from barley. 189 7

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

The energy-transducing NADH--quinone oxidoreductase (NDH-1) isolated from Thermus thermophilus HB-8 is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN and at least three iron-sulfur clusters [Yagi, T., Hon-nami, K., and Ohnishi, T. (1988) Biochemistry 27, 2008-2013]. When NDH-1 of T. thermophilus HB-8 was irradiated by short UV light in the presence of [adenylate-32P]NADH or [adenylate-32P]NAD, radioactivity was incorporated into a single polypeptide of Mr 47,000. The labeling of the Mr 47,000 polypeptide was diminished when UV irradiation of the enzyme complex with [adenylate-32P]NAD was carried out in the presence of NADH or deamino-NADH which act as substrates for the NDH-1, but not in the presence of NADP(H) or AMP which act neither as substrates nor as competitive inhibitors. These results strongly suggest that the Mr 47,000 polypeptide is an NADH-binding subunit of the NDH-1 of T. thermophilus HB-8.
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PMID:Identification of the NADH-binding subunit of energy-transducing NADH-quinone oxidoreductase (NDH-1) of thermus thermophilus HB-8. 189 72

The chloroplast genomes of Marchantia polymorpha, Nicotiana tabacum, and Oryza sativa contain open reading frames (ORFs or potential genes) encoding homologues of some of the subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I). Seven of these subunits (ND1-ND4, ND4L, ND5, and ND6) are products of the mitochondrial genome, and two others (the 49- and 30-kDa components of the iron-sulfur protein fraction) are nuclear gene products. These findings have been taken to indicate the presence in chloroplasts of an enzyme related to complex I, possibly an NAD(P)H:plastoquinone oxidoreductase, participating in chlororespiration. This view is reinforced by the present work in which we have shown that chloroplast genomes encode a homologue of the 23-kDa subunit, another nuclear-encoded component of bovine complex I. The 23-kDa subunit is in the hydrophobic protein fraction of the enzyme, the residuum after removal of the flavoprotein and iron-sulfur protein fractions. The sequence motif CysXXCysXXCysXXXCysPro, which provides ligands for tetranuclear iron-sulfur centers in ferredoxins, occurs twice in its polypeptide chain and is evidence of two associated 4Fe-4S clusters. This is the only iron-sulfur protein identified so far in the hydrophobic protein fraction of complex I, and so it is possible that one of these centers is that known as N-2, the donor of electrons to ubiquinone. The sequence of the 23-kDa subunit is closely related to potential proteins, which also contain the cysteine-rich sequence motifs, encoded in the frxB ORFs in chloroplast genomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A homologue of a nuclear-coded iron-sulfur protein subunit of bovine mitochondrial complex I is encoded in chloroplast genomes. 190 Oct 22

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