Molecular cloning and deduced amino acid sequences of the <i>γ</i>-subunits of rat and monkey NAD+-isocitrate dehydrogenases

Nichols, Benjamin J.; Hall, Len; Perry, Anthony C.F.; Denton, Richard M.

doi:10.1042/bj2950347

Cited by 48 publications

(48 citation statements)

References 24 publications

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“…IPMDH contains two cysteine residues, Cys167 and Cys185. Because one of them, Cys185, is conserved in several mitochondrial NAD-dependent ICDHs (all subunits of the monkey enzyme [26], 3/4 subunit of the bovine enzyme [41], and ␥ subunits of the pig [11] and the rat [27] enzymes), it is possible that these Cys residues form an intersubunit disulfide bond, contributing to the formation of their quaternary structure. To investigate this possibility, a quantitative analysis of free SH groups was performed.…”

Section: Resultsmentioning

confidence: 99%

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

et al. 1997

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The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.3-Isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85) is the third enzyme in the leucine biosynthetic pathway and catalyzes the oxidative decarboxylation of threo-D S -3-isopropylmalate to 2-oxoisocaproate (32). Genes coding for the enzyme have been cloned and sequenced from various bacteria (eubacteria) and eukarya (eukaryotes). IPMDHs so far investigated are NAD dependent and homodimeric (20,39).It has been suggested that the catalytic mechanism of IPMDH is similar to that of bacterial homodimeric NADPdependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) a key enzyme in the tricarboxylic acid cycle. The latter catalyzes the oxidative decarboxylation of threo-D S -isocitrate to oxoglutarate (9). The primary structures of IPMDHs are homologous to those of the homodimeric bacterial NADP-dependent ICDHs (42). In addition, IPMDHs show sequence similarities to mitochondrial NAD-dependent ICDHs. The eukaryotic NAD-dependent ICDHs are either heterotetrameric (e.g., ␣2␤␥ for the mammalian mitochondrial enzymes) (33, 41) or heterooctameric (e.g., ␣4␤4 for the yeast mitochondrial enzyme) (21), consisting of subunits homologous to each other.Recently, the X-ray crystal structure of Thermus thermophilus HB8 IPMDH has been determined (13,15,19). The structure resembles that of Escherichia coli 14). In particular, both enzymes are unique in lacking the Rossmann fold motif which is frequently found in NAD(P)-dependent enzymes. In addition, most of the functional key residues proposed by the crystallographic studies are conserved among IPMDHs, bacterial NADP-dependent ICDHs, and mitochondrial NAD-dependent ICDHs (42). The structural evidence strongly supports the idea (13, 42) that these enzymes originated from a gene duplication that preceded the divergence of the three domains of life.To clarify the phylogenetic relationship among IPMDHs and ICDHs, the structural and functional data of archaeal enzyme are absolutely required, since Archaea (archaebacteria) comprises the third independent domain of life, which is only distantly related to both the Bacteria and the Eukarya (38). Recently, we have isolated and preliminarily characterize...

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Section: Resultsmentioning

confidence: 99%

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

et al. 1997

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“…To test whether mitochondria are more abundant in the surface epithelium, we performed immunohistochemical staining for the g subunit of the NAD 1 -dependent isocitrate dehydrogenase complex (isocitrate dehydrogenase 3-g, IDH3G). This enzyme is a ubiquitously expressed mitochondrial matrix protein in mammals (22,23), and catalyzes the oxidative decarboxylation of isocitrate. This reaction is an irreversible and energy-yielding step in the Krebs cycle.…”

Section: Mitochondrial Distribution Is Highly Skewed Toward Surface Ementioning

confidence: 99%

Differential Gene Expression in Human Conducting Airway Surface Epithelia and Submucosal Glands

Fischer¹,

Goss²,

Scheetz³

et al. 2009

Am J Respir Cell Mol Biol

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Human conducting airways contain two anatomically distinct epithelial cell compartments: surface epithelium and submucosal glands (SMG). Surface epithelial cells interface directly with the environment and function in pathogen detection, fluid and electrolyte transport, and mucus elevation. SMG secrete antimicrobial molecules and most of the airway surface fluid. Despite the unique functional roles of surface epithelia and SMG, little is known about the differences in gene expression and cellular metabolism that orchestrate the specialized functions of these epithelial compartments. To approach this problem, we performed large-scale transcript profiling using epithelial cell samples obtained by laser capture microdissection (LCM) of human bronchus specimens. We found that SMG expressed high levels of many transcripts encoding known or putative innate immune factors, including lactoferrin, zinc a-2 glycoprotein, and proline-rich protein 4. By contrast, surface epithelial cells expressed high levels of genes involved in basic nutrient catabolism, xenobiotic clearance, and ciliated structure assembly. Selected confirmation of differentially expressed genes in surface and SMG epithelia demonstrated the predictive power of this approach in identifying genes with localized tissue expression. To characterize metabolic differences between surface epithelial cells and SMG, immunostaining for a mitochondrial marker (isocitrate dehydrogenase) was performed. Because greater staining was observed in the surface compartment, we predict that these cells use significantly more energy than SMG cells. This study illustrates the power of LCM in defining the roles of specific anatomic features in airway biology and may be useful in examining how disease states alter transcriptional programs in the conducting airways.Keywords: airway epithelia; microarray; submucosal glandThe airways maintain a dynamic interface with the environment. The continuity of the airway epithelium serves as a physical barrier to inhaled pathogens, while the secreted protein and peptide products of the tissue, in concert with ciliary function, support mucociliary clearance and host defense. The epithelium of the intrapulmonary airways contributes to pulmonary innate immunity as a first responder and as an important site of signal amplification. Through a variety of receptor systems, the surface epithelial cells sense and respond to inhaled chemical, microbial, thermal, and particulate stimuli to maintain health. Perturbation of the function of this tissue is involved in the pathogenesis of several diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF).The epithelial tissue of the conducting airways includes many specialized cell types and the spatial division of functions. Two anatomically distinct conducting airway compartments are the airway surface epithelium and submucosal glands (SMG). The surface epithelium of the cartilaginous conducting airways is composed of ciliated, nonciliated, goblet, and basal cell ty...

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“…The residue location of the pig heart sequence is assigned based on the position of the same peptide sequence in the ␥-subunit of the rat enzyme, for which the complete amino acid sequence is available (33). Underlined boldface residues in the NADP ϩ -specific isocitrate dehydrogenase sequence are involved in NADP ϩ binding (12).…”

Section: Coli Isocitrate Dehydrogenase (Icd) (B) Residue Identities mentioning

confidence: 99%

Affinity Purification and Kinetic Analysis of Mutant Forms of Yeast NAD+-specific Isocitrate Dehydrogenase

Zhao

McAlister-Henn

1997

Journal of Biological Chemistry

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Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD ؉ -specific isocitrate dehydrogenase (IDH). The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme. This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding. The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP. In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in V max due to a 70-fold increase in the S 0.5 value for NAD ؉ . These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e. that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands. In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects. A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.The oxidative decarboxylation reaction catalyzed by mitochondrial NAD ϩ -specific isocitrate dehydrogenase (IDH) 1 is believed to be a rate-limiting step in the tricarboxylic acid cycle. The enzyme isolated from Saccharomyces cerevisiae has been described as responsive to energy charge due to allosteric activation by AMP and inhibition by ATP and NADH (1). Mammalian enzymes exhibit similar regulatory properties, but utilize ADP as a positive regulator (2).The catalytically active form of IDH from yeast is an octamer composed of two subunits in a 1:1 ratio (3, 4). Independent nuclear genes encoding the IDH1 and IDH2 subunits have been cloned, and gene disruption studies show that both subunits are essential for catalytic activity (5, 6). Disruption of either or both of the IDH1 and IDH2 genes results in an inability to grow with acetate as a carbon source, a phenotype shared with several other tricarboxylic acid cycle mutants in yeast (7,8).The deduced amino acid sequences and amino-terminal sequence analyses indicate that the mature IDH1 and IDH2 polypeptides contain 349 and 354 amino acid residues, respectively, and share 42% residue identity. The yeast polypeptides share ϳ32% sequence identity with Escherichia coli NADP ϩ -specific isocitrate dehydrogenase (9), a homodimeric enzyme analyzed in several crystallographic studies (10 -12).Equilibrium binding and kinetic analyses (13, 14) conducted with purified yeast IDH led to the characterization of four binding sit...

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Molecular cloning and deduced amino acid sequences of the γ-subunits of rat and monkey NAD+-isocitrate dehydrogenases

Cited by 48 publications

References 24 publications

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

Differential Gene Expression in Human Conducting Airway Surface Epithelia and Submucosal Glands

Affinity Purification and Kinetic Analysis of Mutant Forms of Yeast NAD+-specific Isocitrate Dehydrogenase

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