Purification and properties of NADP+-dependent isocitrate dehydrogenase from the corpus luteum

Jennings, Gary T.; Sadleir, John W.; Stevenson, Patricia M.

doi:10.1016/0304-4165(90)90080-g

Cited by 23 publications

(11 citation statements)

References 20 publications

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“…Immunoblot analyses were conducted as described previously using an antiserum prepared against yeast Idp1p 7, which cross-reacts with yeast Idp2p and mamIdp1p, and an antiserum prepared against mamIdp2p (29), which exhibits no cross-reactivity with yeast isozymes. Immunoreactivity was detected using radiolabeled protein A (7) or the enhanced chemiluminescent method (ECL, Amersham Pharmacia Biotech) and autoradiography.…”

Section: Methodsmentioning

confidence: 99%

Sources of NADPH and Expression of Mammalian NADP+-specific Isocitrate Dehydrogenases in Saccharomyces cerevisiae

Minard

Jennings

Loftus

et al. 1998

Journal of Biological Chemistry

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To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.

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Section: Methodsmentioning

confidence: 99%

Sources of NADPH and Expression of Mammalian NADP+-specific Isocitrate Dehydrogenases in Saccharomyces cerevisiae

Minard

Jennings

Loftus

et al. 1998

Journal of Biological Chemistry

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“…Consequently, if the shapes of the unknown and standard proteins are dissimilar, the determination of the apparent mo1 wt from the elution volume can be in error. An anomalous native mo1 wt calculated from gel-filtration data has been described previously for NADP-ICDH from other sources (Kelly and Plaut, 1981;Jennings et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

et al. 1994

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Creen, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. lheir elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves. lCDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anionexchange and affinity chromatography. l h e isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833-and 1328-fold for ICDH1 and ICDH2, respectively. Polyclonal antibodies were raised against each isoform. The ICDHZspecific antibody was used to localize tobacco leaf lCDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDHl and lCDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits. Similar apparent K,,, values for NADP, D(+)isocitrate, and Mgz+ were found for the two enzymes when assayed with Mgz+ as the metal cofactor.

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“…In an attempt to solve these questions, Pandolfi et a/. (39) have disrupted the GGPD gene in male mouse embryonic stem cells by homologous recombination and have isolated located in mitochondria (40), and the malic enzyme has been suggested to be most important in the functioning of pancreatic islets(41~42).…”

Section: Ggpd and Oxidative Stressmentioning

confidence: 99%

A new lease of life for an old enzyme

Martini

Ursini

1996

BioEssays

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We review here some recent data about glucose-6-phosphate dehydrogenase (G6PD), the first and key regulatory enzyme of the pentose phosphate pathway. New evidence has been presented to suggest that malaria is a selective agent for G6PD deficiency, which is the most common enzymopathy in man, and that G6PD deficiency, generally considered to be a mild and benign condition, is significantly disadvantageous in certain environmental conditions. At the molecular level, the enzyme structure has recently been elucidated and mechanisms regulating G6PD gene expression have been determined. A G6PD knock-out mutation introduced in mouse cells makes them exquisitely sensitive to oxidative stress, indicating that this ubiquitous metabolic enzyme has a major role in the defence against oxidative stress, even in eukaryotic nucleated cells, which have several alternative routes for providing the same protection. Because of the high prevalence of G6PD deficiency in many populations, it is expected that these findings will prompt further studies to ascertain the putative role of G6PD deficiency in conditions such as carcinogenesis and ageing.

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Purification and properties of NADP+-dependent isocitrate dehydrogenase from the corpus luteum

Cited by 23 publications

References 20 publications

Sources of NADPH and Expression of Mammalian NADP+-specific Isocitrate Dehydrogenases in Saccharomyces cerevisiae

Sources of NADPH and Expression of Mammalian NADP+-specific Isocitrate Dehydrogenases in Saccharomyces cerevisiae

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

A new lease of life for an old enzyme

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