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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phylogenetic utility of the mitochondrial COI (cytochrome oxidase subunit I) and nuclear Gpdh (glycerol-3-phosphate dehydrogenase) genes was studied in the Drosophila melanogaster species group. The rate of substitution was higher in the COI gene than in the Gpdh gene. In addition, multiple substitutions, not only for transitional but also for transversional substitutions, occurred faster in the COI gene. None of the trees obtained using the COI gene supported the well-established monophyly of the ananassae subgroup. In addition, the incongruence length difference test, Templeton test, and partitioned Bremer support revealed that the trees based on the COI data are considerably different from those based on the Gpdh and the combined data set. Thus, the COI gene did not show good phylogenetic performance in the melanogaster group. The present analyses based on the Gpdh gene and the combined data set revealed that the ananassae subgroup branched off first in the melanogaster group followed by the montium subgroup and further by the melanogaster subgroup in contrast to the most recent phylogenetic hypothesis based on Amy multigenes.
Mol Phylogenet Evol 2001 Mar
PMID:Phylogenetic utility of mitochondrial COI and nuclear Gpdh genes in Drosophila. 1127 33

In a new experimental type 2 diabetic syndrome, a 40% reduction of pancreatic beta cells was observed by morphometric analysis. In diabetic islets, as compared to control islets, insulin release was decreased in response to high glucose but not to other stimuli, and total glucose oxidation and utilization were unchanged or slightly reduced. The extent of metabolic and functional impairment appeared proportional to the beta-cell loss. However, a substantial decrease was found in protein level and activity (by 77 and 60%, respectively, versus controls) of mitochondrial FAD-glycerophosphate dehydrogenase (mGDH), the key enzyme of the glycerophosphate shuttle. Interestingly, in diabetic islets, as recently reported for mGDH-deficient transgenic mice, definite functional alterations (mainly in response to D-glyceraldehyde) were only obtained upon pharmacological blockade of the second shuttle (i.e. malate-aspartate) responsible for mitochondrial transfer of reducing equivalents. In conclusion, in this diabetes model with reduction of beta-cell mass, the islets, despite decreased mGDH amount and activity, appear metabolically and functionally active in vitro, likely through the intervention of adaptive mechanisms, yet prone to failure in challenging situations.
Mol Cell Endocrinol 2001 Apr 25
PMID:Metabolic and functional studies on isolated islets in a new rat model of type 2 diabetes. 1132 16

We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC 1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD, this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues.
Mol Cell Biochem 2001 Apr
PMID:Survey of normal appearing mouse strain which lacks malic enzyme and Nad+-linked glycerol phosphate dehydrogenase: normal pancreatic beta cell function, but abnormal metabolite pattern in skeletal muscle. 1145 71

In terms of glucose sensing by pancreatic islet beta-cells, emphasis is currently placed on both the role of glucokinase, with negligible activity of low-Km hexokinase(s), and the prevalence of the oxidative over non-oxidative modality of glycolysis, a situation tentatively attributed, in part at least, to a low activity of lactate dehydrogenase. Conflicting information is available, however, on the activity of both low-Km hexokinase(s) and lactate dehydrogenase in purified beta-cell homogenates. This issue was reinvestigated, therefore, in two populations of purified rat islet beta-cells selected on the basis of their low (betaL) or high (betaH) content in reduced pyridine nucleotides. The size and protein content of betaH cells represented about twice that of betaL cells. Such was also the case for low-Km hexokinase(s), lactate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, glutamate dehydrogenase and glutamate-alanine and glutamate-aspartate transaminases. Whether in betaH or betaL cells, the activity of low-Km hexokinase(s) was at least as high as or higher than that of glucokinase. In both betaH and betaL, the activity of lactate dehydrogenase exceeded that required to catalyze the full reduction of glucose-derived pyruvate to L-lactate, as estimated from the rate of D-glucose phosphorylation under physiological conditions. These findings thus argue against a low expression of either low-Km hexokinase(s) or lactate dehydrogenase as major determinants of the glucose-sensing device in beta-cells.
Int J Mol Med 2001 Sep
PMID:Enzymic activities in two populations of purified rat islet beta-cells. 1149 57

Single-strand conformational polymorphism analysis of mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH) gene has revealed mutations in both the calcium- and FAD-binding domains of this enzyme in some diabetic patients. It was now investigated whether site-directed mutations in the FAD-binding domain of the mGDH gene may affect the mitochondrial anchoring and catalytic activity of the enzyme. COS-7 cells were transfected with plasmid cDNA coding for either wild-type or mutated human mGDH (G --> A substitutions at positions 352, 355, and 364 and A --> C substitution at position 390) fused, when required, at the N-terminus of green fluorescent protein. The activity of mGDH was measured by both radioisotopic ((3)HOH production from l-[2-(3)H]glycerol 3-phosphate) and colorimetric (iodoformazan formation) procedures. In cells transfected with the mGDHwt-EGFP or mGDHmut-EGFP constructs, the fused protein was found by confocal microscopy exclusively in the mitochondria, colocalized with a mitochondrial marker. In homogenates of COS-7 cells transfected with mGDHmut, however, the catalytic activity of the enzyme was decreased, this coinciding with low ratios between both the activities measured in the absence/presence of exogenous FAD and the results obtained by the colorimetric/radioisotopic procedure. Thus, although the present site-directed mutations of the mGDH gene failed to impair the mitochondrial anchoring of the enzyme, they led to catalytic defects that were, in some respect, comparable to those previously encountered in the lymphocytes or islets of type 2 diabetic patients.
Mol Genet Metab 2002 Feb
PMID:Site-directed mutations in the FAD-binding domain of glycerophosphate dehydrogenase: catalytic defects with preserved mitochondrial anchoring of the enzyme in transfected COS-7 cells. 1185 36

Regional differences in muscle fiber types and their glucose uptake in superficial, middle and deep regions of mouse gastrocnemius at rest were studied. The enzyme activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (alpha-GPDH) and myofibrillar ATPase were used for the classification of each muscle fiber type. For the evaluations of glucose uptake, 2-deoxyglucose (2-DG) microradioautography and expression of the glucose transporter 4 (GLUT4) protein were applied. In superficial region of mouse gastrocnemius, all fibers were IIx or IIb and were low for 2-DG uptake and GLUT4 expression. They were anaerobic and fast twitch. In middle region, fibers showing low 2-DG uptake and small amounts of GLUT-4 protein (IIx and IIb) dominated (80.4%). Most fibers were anaerobic and fast twitch. Others were type IIc and IIa. In deep region, most fibers (86.9%) showed high 2-DG uptake and large amounts of GLUT4 protein (I, IIc and IIa). They were oxidative and slow twitch.
Cell Mol Biol (Noisy-le-grand) 2001
PMID:Regional difference in muscle fiber type and glucose uptake of mouse gastrocnemius at rest. 1193 59

Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (Km) for dihydroxyacetone is 137.39 (+/- 25.56) microM whereas the Km for glycerol-3-phosphate is 468.66 (+/- 27.59) microM. The kinetic mechanism of purified enzyme is 'ordered Bi-Bi' and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35 degrees C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (+/- 1000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (+/- 2000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mM N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn2+ and by 2,4-dichlorophenoxyacetic acid.
Mol Cell Biochem 2002 Feb
PMID:Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis). 1195 53

Activities of enzymes associated with glycerol synthesis were compared in the liver of two osmerid fishes, the smelt (Osmerus mordax), which can accumulate high (400 mM) levels of glycerol and capelin (Mallotus villosus) that does not accumulate glycerol. Animals were sampled at approximately the same time of year and temperature thus negating potential seasonal effects. These species are closely related, reducing interpretative issues involving comparison between unrelated species. We found that key enzyme activities were elevated in the smelt relative to the non-glycerol accumulating capelin, namely enzymes involved with glycolysis (phosphofructose kinase-1 and aldolase), amino acid metabolism (aspartate aminotransferase and alanine aminotransferase), gluconeogenesis (phosphoenolpyruvate carboxykinase) and glycerol synthesis (glycerol-3-phosphate dehydrogenase). The enzyme profiles strongly support the hypothesis that smelt can synthesize glycerol by utilizing glycogen and amino acids as the carbon source and that they have increased capacity for metabolic flux through loci required for synthesis of the three carbon intermediate dihydroxyacetone phosphate and subsequently glycerol synthesis.
Comp Biochem Physiol A Mol Integr Physiol 2002 Jun
PMID:Comparison of liver enzymes in osmerid fishes: key differences between a glycerol accumulating species, rainbow smelt (Osmerus mordax), and a species that does not accumulate glycerol, capelin (Mallotus villosus). 1202 Jun 59

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.
J Biochem Mol Biol 2003 Mar 31
PMID:Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum. 1268 13

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.
J Mol Biol 2003 May 30
PMID:Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct. 1275 80


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