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1.
J Bacteriol ; 201(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570530

RESUMO

The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.


Assuntos
Proteínas de Bactérias/fisiologia , Proteína Receptora de AMP Cíclico/fisiologia , Regulação Bacteriana da Expressão Gênica , Glicerol Quinase/fisiologia , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Mycobacterium smegmatis/metabolismo , Óperon , Fator sigma/fisiologia , Fatores de Transcrição/fisiologia , Ácidos Glicéricos/farmacologia , Mycobacterium smegmatis/genética
2.
Mol Biol Cell ; 30(17): 2134-2140, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31141459

RESUMO

The yeast high-osmolarity glycerol (HOG) stress-activated protein kinase Hog1 is activated in response to hyperosmotic stress, inducing the production and retention of glycerol to restore osmotic balance. Hog1 promotes retention of glycerol through closure of the plasma-membrane glycerol channel Fps1. Treatment of yeast with the toxic trivalent metalloid arsenite (As(III)) also activates Hog1 as part of a protective response in which Hog1 closes Fps1, the main entry port for As(III). In this study, we investigated how cells treated with As(III) avoid creating a new stress caused by the accumulation of glycerol in the absence of hyperosmotic stress conditions. We found that As(III) treatment did not induce glycerol accumulation and, in fact, blocked the accumulation of glycerol induced by constitutive Hog1 activity. We demonstrated that As(III) blocks glycerol production indirectly after its metabolic activation to methylarsenite (MAs(III)), which is a potent inhibitor of glycerol-3-phosphate dehydrogenase. Finally, we used a biotinylated arsenic probe to establish that Cys306 of yeast Gpd1, a highly conserved residue within the active site, is the key target of MAs(III). Conservative mutations at this residue greatly diminished Gpd1 activity. This study offers insight into mechanisms by which SAPK outputs are tailored to specific stressors.


Assuntos
Arsenitos/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Arsenitos/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mutação , Pressão Osmótica/fisiologia , Fosforilação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
3.
Biochim Biophys Acta ; 1837(1): 73-84, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23933018

RESUMO

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.


Assuntos
Complexo I de Transporte de Elétrons/química , Transporte de Elétrons , Glicerolfosfato Desidrogenase/química , NADH Desidrogenase/química , Oxirredutases/química , Sequência de Aminoácidos , Respiração Celular/fisiologia , Debaryomyces/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/fisiologia , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
4.
Biochim Biophys Acta ; 1827(3): 401-10, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23220394

RESUMO

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.


Assuntos
Glicerolfosfato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Regulação Alostérica , Animais , Ácidos Graxos/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicólise , Humanos , Especificidade de Órgãos , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo
5.
Circ Res ; 108(7): 884-97, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21454796

RESUMO

Variations in the gene encoding for the major sodium channel (Na(v)1.5) in the heart, SCN5A, has been shown to cause a number of arrhythmia syndromes (with or without structural changes in the myocardium), including the long-QT syndrome (type 3), Brugada syndrome, (progressive) cardiac conduction disease, sinus node dysfunction, atrial fibrillation, atrial standstill, and dilated cardiomyopathy. Of equal importance are variations in genes encoding for various subunits and regulatory proteins interacting with the α-subunit Na(v)1.5 and modifying its function. Based on detailed studies of genotype-phenotype relationships in these disease entities, on detailed studies of the basic electrophysiological phenotypes (heterologous expressed wild-type and mutant sodium channels and their interacting proteins), and on attempts to integrate the obtained knowledge, the past 15 years has witnessed an explosion of knowledge about these disease entities.


Assuntos
Arritmias Cardíacas/genética , Proteínas Musculares/genética , Mutação/genética , Fenótipo , Canais de Sódio/genética , Arritmias Cardíacas/fisiopatologia , Proteínas de Ligação ao Cálcio/fisiologia , Caveolina 3/fisiologia , Glicerolfosfato Desidrogenase/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5
6.
Mol Reprod Dev ; 77(9): 773-83, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20602492

RESUMO

A tyrosine phosphoproteome study of hamster spermatozoa indicated that glycerol-3-phosphate dehydrogenase 2 (GPD2), is one of the proteins that enables tyrosine phosphorylation during sperm capacitation. Further, enzymatic activity of GPD2 correlated positively with sperm capacitation [Kota et al., 2009; Proteomics 9:1809-1826]. Therefore, understanding the function of GPD2 would help to unravel the molecular mechanism of sperm capacitation. In this study, involving the use of spermatozoa from Gpd2(+/+) and Gpd2(-/-) mice, it has been demonstrated that in the absence of Gpd2, hyperactivation and acrosome reaction were significantly altered, and a few changes in protein tyrosine phosphorylation were also observed during capacitation. Evidence is provided to demonstrate that GPD2 activity is required for ROS generation in mouse spermatozoa during capacitation, failing which, capacitation is impaired. These results imply that GPD2 is involved in sperm capacitation.


Assuntos
Glicerolfosfato Desidrogenase/fisiologia , Capacitação Espermática/fisiologia , Reação Acrossômica/fisiologia , Animais , Cricetinae , Glicerolfosfato Desidrogenase/genética , Humanos , Masculino , Camundongos , Fosforilação , Fosfotirosina/metabolismo , Fosfotirosina/fisiologia , Capacitação Espermática/genética , Motilidade dos Espermatozoides/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/enzimologia , Espermatozoides/fisiologia
7.
J Anim Sci ; 88(6): 1999-2008, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20154153

RESUMO

Our objectives were to isolate bovine stromal-vascular cells using explants and to determine media components that promote differentiation into mature adipocytes for studies of lipogenic enzyme regulation. Stromal-vascular cells were grown from explants and treated with differentiation media for 8 d after reaching confluence. Differentiation was assessed by measuring radiolabeled acetate incorporation into lipids, glycerol-3-phosphate dehydrogenase activity, and the mRNA expression of fatty acid binding protein-4, PPAR-gamma, and acetyl-CoA carboxylase-alpha (ACCalpha). After 8 d of differentiation, medium containing 10 microg/mL of insulin, 0.25 microM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 mM octanoate, and 2% Intralipid (Fisher Scientific, Suwanee, GA) produced greater acetate incorporation (P < 0.001) and glycerol-3-phosphate dehydrogenase activity (P < 0.001) compared with other media tested. This differentiation medium also increased mRNA expression of fatty acid binding protein-4, PPARgamma, and ACCalpha by 180-, 7-, and 3-fold, respectively, compared with undifferentiated control cells (P < 0.05). To further improve the differentiation protocol, the effects of Intralipid, rosiglitazone, and troglitazone were examined. Removal of 2% Intralipid did not improve any differentiation measures. Addition of rosiglitazone (1 microM), a PPAR-gamma agonist, increased acetate incorporation and ACCalpha mRNA (P < 0.01). Addition of troglitazone (5 microM), another PPAR-gamma agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest expression of ACCalpha mRNA (P < 0.01), but was not superior to medium that included rosiglitazone for any other differentiation measures. Cell-seeding density influences the cell divisions required to reach confluence, and increased plating density (2 x 10(4) cells/cm(2) vs. 6.7 x 10(3) cells/cm(2)) increased acetate incorporation by 100% (P < 0.001). Differentiating stromal-vascular cells in the presence of trans-10, cis-12 CLA inhibited differentiation of stromal-vascular cells into mature adipocytes, reducing radiolabeled acetate incorporation into lipids (P < 0.001), stearoyl-CoA desaturase-1 mRNA (P < 0.05) and protein abundance (P < 0.05), and ACCalpha protein abundance (P < 0.05). We have developed a method to differentiate primary bovine adipocytes, which will allow us to study the regulation of lipogenic enzymes by nutrient and endocrine factors.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo/fisiologia , Bovinos/fisiologia , Diferenciação Celular/fisiologia , Glicerolfosfato Desidrogenase/fisiologia , Ácidos Linoleicos Conjugados/fisiologia , Acetatos/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Adipócitos/citologia , Tecido Adiposo/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Cromanos/farmacologia , Emulsões Gordurosas Intravenosas/farmacologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Masculino , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Tiazolidinedionas/farmacologia , Troglitazona
8.
J Bacteriol ; 192(3): 779-85, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19966010

RESUMO

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.


Assuntos
Enterococcus faecalis/metabolismo , Glicerol/metabolismo , Aerobiose , Anaerobiose , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/fisiologia , Peróxido de Hidrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/fisiologia
9.
Biochim Biophys Acta ; 1787(11): 1309-16, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19413950

RESUMO

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.


Assuntos
Cálcio/fisiologia , Glicerolfosfato Desidrogenase/fisiologia , Isocitrato Desidrogenase/fisiologia , Complexo Cetoglutarato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Piruvato Desidrogenase (Lipoamida)-Fosfatase/fisiologia , Animais , Ativação Enzimática , Humanos
10.
Mol Membr Biol ; 24(4): 269-81, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17520483

RESUMO

Gut2, the mitochondrial glycerol-3-phosphate dehydrogenase, was previously shown to become preferentially labelled with photoactivatable phosphatidylcholine (PC), pointing to a functional relation between these molecules. In the present study we analyzed whether Gut2 functioning depends on the PC content of yeast cells, using PC biosynthetic mutants in which the PC content was lowered. PC depletion was found to reduce growth on glycerol and to increase glycerol excretion, both indicating that PC is needed for optimal Gut2 functioning in vivo. Using several in vitro approaches the nature of the dependence of Gut2 functioning on cellular PC contents was investigated. The results of these experiments suggest that it is unlikely that the effects observed in vivo are due to changes in cellular Gut2 content, in specific activity of Gut2 in isolated mitochondria, or in the membrane association of Gut2, upon lowering the PC level. The in vivo effects are more likely an indirect result of PC depletion-induced changes in the cellular context in which Gut2 functions, that are not manifested in the in vitro systems used.


Assuntos
Glicerolfosfato Desidrogenase/fisiologia , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Glicerol/metabolismo , Proteínas Mitocondriais , Fosfatidilcolinas/deficiência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
11.
Protein Sci ; 13(12): 3161-71, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15557260

RESUMO

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.


Assuntos
Archaeoglobus fulgidus/enzimologia , Glicerolfosfato Desidrogenase/química , Glicerolfosfato Desidrogenase/fisiologia , NADP/metabolismo , Sequência de Aminoácidos , Animais , Archaeoglobus fulgidus/genética , Sítios de Ligação , Cristalografia por Raios X , Leishmania mexicana/enzimologia , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência
12.
J Biol Chem ; 277(36): 32892-8, 2002 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-12093799

RESUMO

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is important for metabolism of glycerol phosphate for gluconeogenesis or energy production and has been implicated in thermogenesis induced by cold and thyroid hormone treatment. mGPD in combination with the cytosolic glycerol phosphate dehydrogenase (cGPD) is proposed to form the glycerol phosphate shuttle, catalyzing the interconversion of dihydroxyacetone phosphate and glycerol phosphate with net oxidation of cytosolic NADH. We made a targeted deletion in Gdm1 and produced mice lacking mGPD. On a C57BL/6J background these mice showed a 50% reduction in viability compared with wild-type littermates. Uncoupling protein-1 mRNA levels in brown adipose tissue did not differ between mGPD knockout and control pups, suggesting normal thermogenesis. Pups lacking mGPD had decreased liver ATP and slightly increased liver glycerol phosphate. In contrast, liver and muscle metabolites were normal in adult animals. Adult mGPD knockout animals had a normal cold tolerance, normal circadian rhythm in body temperature, and demonstrated a normal temperature increase in response to thyroid hormone. However, they were found to have a lower body mass index, a 40% reduction in the weight of white adipose tissue, and a slightly lower fasting blood glucose than controls. The phenotype may be secondary to consequences of the obligatory production of cytosolic NADH from glycerol metabolism in the mGPD knockout animal. We conclude that, although mGPD is not essential for thyroid thermogenesis, variations in its function affect viability and adiposity in mice.


Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Glicerolfosfato Desidrogenase/fisiologia , Mitocôndrias/enzimologia , Termogênese , Glândula Tireoide/fisiologia , Tecido Adiposo/citologia , Animais , Western Blotting , Peso Corporal , Citosol/enzimologia , Feminino , Vetores Genéticos , Genótipo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Modelos Genéticos , RNA Mensageiro/metabolismo , Análise de Sobrevida , Temperatura , Fatores de Tempo
13.
J Biol Chem ; 277(36): 32899-904, 2002 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-12093800

RESUMO

The activities of either the mitochondrial or cytosolic glycerol phosphate dehydrogenase (mGPD, cGPD) plus that of glycerol kinase are required for the use of glycerol in aerobic metabolism and gluconeogenesis. A knockout mouse lacking mGPD has reduced body weight and fertility but shows remarkably normal liver and muscle metabolite levels. The BALB/cHeA mouse strain, which lacks cGPD, breeds well and is phenotypically normal, although it demonstrates metabolite abnormalities in certain tissues. Crosses were made between these two strains, and mice were generated that lacked both dehydrogenases. These mice, although active and nursing well for several days, failed to grow, and usually died within the first week. Liver glycerol phosphate levels were elevated 30-fold, whereas liver ATP, ADP, and AMP levels were reduced by 30-40%. Plasma glycerol was elevated 30- to 50-fold to 30-50 mm, and urine glycerol exceeded 0.45 m (4% w/v). GPD-deficient mice were hypoglycemic, had a 50% increase in plasma free fatty acids, and developed ketonuria within the first day of life. Uncoupling protein-1 mRNA in brown adipose tissue was reduced 60%. These mice share some features of both glycerol kinase deficiency and hereditary fructose intolerance, suggesting the phenotype may be due to the combined effects of the loss of a gluconeogenic substrate, the osmotic effects of glycerol, and the metabolic effects of the accumulation of a phosphorylated metabolite.


Assuntos
Citosol/enzimologia , Glicerol/sangue , Glicerol/urina , Glicerolfosfato Desidrogenase/fisiologia , Hipoglicemia/etiologia , Cetose/etiologia , Mitocôndrias/enzimologia , Animais , Genótipo , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/genética , Hipoglicemia/metabolismo , Hipoglicemia/mortalidade , Cetose/metabolismo , Cetose/mortalidade , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Químicos , Análise de Sobrevida , Fatores de Tempo
14.
Metab Eng ; 3(4): 301-12, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11676566

RESUMO

Glycerol, one of the most important by-products of alcoholic fermentation, has positive effects on the sensory properties of fermented beverages. It was recently shown that the most direct approach for increasing glycerol formation is to overexpress GPD1, which encodes the glycerol-3-phosphate dehydrogenase (GPDH) isoform Gpd1p. We aimed to identify other steps in glycerol synthesis or transport that limit glycerol flux during glucose fermentation. We showed that the overexpression of GPD2, encoding the other isoform of glycerol-3-phosphate dehydrogenase (Gpd2p), is equally as effective as the overexpression of GPD1 in increasing glycerol production (3.3-fold increase compared to the wild-type strain) and has similar effects on yeast metabolism. In contrast, overexpression of GPP1, encoding glycerol 3-phosphatase (Gpp1p), did not enhance glycerol production. Strains that simultaneously overexpress GPD1 and GPP1 did not produce higher amounts of glycerol than a GPD1-overexpressing strain. These results demonstrate that GPDH, but not the glycerol 3-phosphatase, is rate-limiting for glycerol production. The channel protein Fps1p mediates glycerol export. It has recently been shown that mutants lacking a region in the N-terminal domain of Fps1p constitutively release glycerol. We showed that cells producing truncated Fps1p constructs during glucose fermentation compensate for glycerol loss by increasing glycerol production. Interestingly, the strain with a deregulated Fps1 glycerol channel had a different phenotype to the strain overexpressing GPD genes and showed poor growth during fermentation. Overexpression of GPD1 in this strain increased the amount of glycerol produced but led to a pronounced growth defect.


Assuntos
Glicerol/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia
15.
Mem. Inst. Oswaldo Cruz ; 95(5): 707-9, Sept.-Oct. 2000. tab
Artigo em Inglês | LILACS | ID: lil-267898

RESUMO

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.


Assuntos
Animais , Voo Animal/fisiologia , Glicerolfosfato Desidrogenase/metabolismo , Insetos Vetores/enzimologia , Músculos/enzimologia , Triatominae/enzimologia , Glicerolfosfato Desidrogenase/fisiologia , Panstrongylus/enzimologia , Triatoma/enzimologia
16.
Mem Inst Oswaldo Cruz ; 95(5): 707-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10998220

RESUMO

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.


Assuntos
Voo Animal/fisiologia , Glicerolfosfato Desidrogenase/metabolismo , Insetos Vetores/enzimologia , Músculos/enzimologia , Proteínas de Protozoários/metabolismo , Triatominae/enzimologia , Animais , Glicerolfosfato Desidrogenase/fisiologia , Panstrongylus/enzimologia , Proteínas de Protozoários/fisiologia , Triatoma/enzimologia
17.
J Biol Chem ; 275(3): 1587-93, 2000 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-10636849

RESUMO

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).


Assuntos
Glicerolfosfato Desidrogenase/genética , Ilhotas Pancreáticas/enzimologia , Ácido Amino-Oxiacético/farmacologia , Animais , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Glucose/farmacologia , Glicerolfosfato Desidrogenase/fisiologia , Insulina/metabolismo , Secreção de Insulina , Malatos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Potássio/metabolismo , Fatores de Tempo
18.
Biochem Genet ; 36(9-10): 329-50, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9919359

RESUMO

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.


Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/fisiologia , Fatores Etários , Animais , Fusão Gênica Artificial , Eletroforese em Acetato de Celulose , Modelos Biológicos , Deleção de Sequência , Transformação Genética , beta-Galactosidase/metabolismo
19.
Mol Biol Cell ; 8(9): 1665-75, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9307964

RESUMO

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.


Assuntos
Drosophila melanogaster/fisiologia , Voo Animal/fisiologia , Complexos Multienzimáticos/metabolismo , Músculos/enzimologia , Músculos/fisiologia , Animais , Drosophila melanogaster/enzimologia , Glicerolfosfato Desidrogenase/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Glicólise , Microscopia de Fluorescência , Complexos Multienzimáticos/fisiologia , Miofibrilas/enzimologia , Miofibrilas/fisiologia , Sarcômeros/enzimologia , Sarcômeros/fisiologia , Tórax
20.
Appl Environ Microbiol ; 62(10): 3894-6, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8837449

RESUMO

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.


Assuntos
Aldeído Redutase/metabolismo , Glicerolfosfato Desidrogenase/fisiologia , Saccharomyces cerevisiae/enzimologia , Xilose/metabolismo , Aldeído Redutase/genética , Anaerobiose , Biomassa , Reatores Biológicos , Fermentação , Genes Fúngicos/genética , Glucose/metabolismo , Oxirredução , Pichia/enzimologia , Saccharomyces cerevisiae/genética , Transformação Genética , Xilitol/biossíntese
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