Α-glycerophosphate Dehydrogenase Research Articles

Letters to the Editor

Letters to the Editor

Simple mendelian inheritance at a locus coding for alpha-glycerophosphate dehydrogenase in brown trout (Salmo trutta).

Simple mendelian inheritance at a locus coding for alpha-glycerophosphate dehydrogenase in brown trout (Salmo trutta).

Prolactin inhibition at the end of lactation programs for a central hypothyroidism in adult rat

Malnutrition during lactation is associated with hypoprolactinemia and failure in milk production. Adult rats whose mothers were malnourished presented higher body weight and serum tri-iodothyronine (T(3)). Maternal hypoprolactinemia at the end of lactation caused higher body weight in adult life, suggesting an association between maternal prolactin (PRL) level and programming of the offspring's adult body weight. Here, we studied the consequences of the maternal PRL inhibition at the end of lactation by bromocriptine (BRO) injection, a dopaminergic agonist, upon serum TSH and thyroid hormones, thyroid iodide uptake, liver mitochondrial alpha-glycerophosphate dehydrogenase (mGPD), liver and pituitary de-iodinase activities (D1 and/or D2), and in vitro post-TRH TSH release in the adult offspring. Wistar lactating rats were divided into BRO - injected with 1 mg/twice a day, daily for the last 3 days of lactation, and C - control, saline-injected with the same frequency. At 180 days of age, the offspring were injected with (125)I i.p. and after 2 h, they were killed. Adult animals whose mothers were treated with BRO at the end of lactation presented lower serum TSH (-51%), T(3) (-23%), and thyroxine (-21%), lower thyroid (125)I uptake (-41%), liver mGPD (-55%), and pituitary D2 (-51%) activities, without changes in the in vitro post-TRH TSH release. We show that maternal PRL suppression at the end of lactation programs a hypometabolic state in adulthood, in part due to a thyroid hypofunction, caused by a central hypothyroidism, probably due to decreased TRH secretion. We suggest that PRL during lactation can regulate the hypothalamus-pituitary-thyroid axis and programs its function.

Structure of α-Glycerophosphate Oxidase from Streptococcus sp.: A Template for the Mitochondrial α-Glycerophosphate Dehydrogenase,

The FAD-dependent alpha-glycerophosphate oxidase (GlpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the GlpO sequence is 30-43% identical to those of the alpha-glycerophosphate dehydrogenases (GlpDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. GlpO (GlpODelta, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 A resolution. Using the GlpODelta structure as a search model, we have also determined the intact GlpO structure, as refined at 2.4 A resolution. The first two domains of the GlpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GlpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact GlpO corresponds to a segment of missing electron density that links the substrate-binding domain to a betabetaalpha element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5-sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431-OG are hydrogen bonded to FAD-O2alpha in GlpODelta, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from Glp) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and d-amino acid oxidase-d-alanine complexes demonstrate that Arg346 of GlpODelta is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between GlpO and the bacterial and mitochondrial GlpDs is also discussed.

Heated Oligonucleotide Ligation Assay (HOLA): An Affordable Single Nucleotide Polymorphism Assay

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Hepatic deiodinase activity is dispensable for the maintenance of normal circulating thyroid hormone levels in mice

Thyroid hormone (TH) homeostasis depends on peripheral activation and inactivation of iodothyronines by selenoenzymes of the deiodinase (Dio) family. We genetically inactivated hepatic selenoenzyme expression, including Dio1, in order to determine the contribution of hepatic Dio to circulating TH levels. Serum levels of TSH, total T 4, and total T 3 were not different from controls. We measured Dio1 and Dio2 in kidney, skeletal muscle, heart, brown adipose tissue, and brain, but did not find compensatory up-regulation in these tissues. Finally, we determined expression in the liver of the following T 3 target genes: Spot14, α-glycerophosphate dehydrogenase (αGPD), and malic enzyme (ME). On the transcript level, both Spot14 and αGPD were reduced in Dio-deficient liver to about 60–70% of controls. However, mRNA and activity of ME were significantly increased in the same mice. Together, our results indicate that hepatic Dio1 activity is not absolutely required to sustain the euthyroid state in mice.

Skeletal muscle bioenergetics: a comparative study of mitochondria isolated from pigeon pectoralis, rat soleus, rat biceps brachii, pig biceps femoris and human quadriceps

The metabolism of mitochondria isolated from five functionally different skeletal muscles is compared. Data for a single ectothermic preparation are also reported. The mitochondria were prepared in yields of 44±7% from 50 to 100 mg muscle. The muscle content of mitochondrial protein ranged between 2 and 40 g kg −1. Twelve specific activities of key enzymes and metabolic systems were determined, 10 of these in functional assays with respiratory measurements. The specific activities of glutamate dehydrogenase, α-glycerophosphate dehydrogenase, and exo-NADH oxidase differed considerably among muscle sources. Seven specific activities, including very central reactions, showed low among-muscle variation. The activity of ATP synthesis, for instance, was 1.0–1.3 mmol min −1 g −1 mitochondrial protein, 25 °C. In vitro data were extrapolated to in vivo conditions of the muscles. The calculated rates of respiration and ATP synthesis were in accordance with reported tissue activities. Pigeon pectoralis mitochondria showed a unique cytochrome spectrum and a respiratory chain activity that might effect simultaneous carbohydrate and fatty acid respiration. In mitochondria from the other muscles, the respiratory chain activity balanced the carbohydrate oxidation capacity. In all muscles, the respiratory capacity exceeds that needed for oxidative phosphorylation. This may secure maximal mitochondrial ATP synthesis during maximal work rates and high cellular [Ca 2+].

Adaptations in metabolic capacity of rat soleus after paralysis.

To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., alpha-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.

Speciation and mitochondrial DNA diversification of the diving beetles Agabus bipustulatus and A. wollastoni (Coleoptera, Dytiscidae) within Macaronesia

Diversification of populations of two morphologically similar diving beetles within the Agabus tristis group, A. wollastoni and A. bipustulatus, was investigated, with partial mtDNA cytochrome b (Cyt b) sequences, allozymes and landmark-based morphometrics. The Madeiran endemic A. wollastoni was collected from 11 localities. Population genetic and morphological variation was compared to Scandinavian localities of the widespread west Palearctic A. bipustulatus, recorded also from the Azores. Agabus wollastoni and European A. bipustulatus specimens representing eight and 13 localities respectively, were used in evaluating their phylogenetic relationship. Maximum parsimony analysis of the Cyt b sequences showed that both the A. bipustulatus and A. wollastoni specimens form well-supported monophyletic groups. Three lines of evidence suggest that Agabus wollastoni has speciated through a few founders: (1) a well-supported mtDNA line; (2) the mean heterozygosity of A. wollastoni is lower when compared to A. bipustulatus on the mainland; and (3) several uncommon alleles of A. bipustulatus are missing in A. wollastoni. The Azorean A. bipustulatus population was drastically affected by the colonization event, since several loci have become fixed with a resulting lower mean heterozygosity. The colonization was relatively recent, as the mtDNA lineage represented in the Azores is deeply nested within the A. bipustulatus clade. Population structure shows moderate inbreeding of A. wollastoni, and extensive substructuring at all localities with moderate gene-flow between them. Morphological variation in A. wollastoni showed significant differentiation among several populations. Island colonizations, population structure of A. wollastoni, and an observed pattern of variation of the α-glycerophosphate dehydrogenase locus are discussed. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79, 653–666.

Marrow stromal cell recovery after radiation-induced aplasia in mice

Purpose : The identification of fibroblast-like cells of the marrow stroma by means of alkaline phosphatase (ALP) cytochemistry reveals delicate ALP-positive structures interspersed among haematopoietic cells and arranged in a loosely meshed network. These cells are often referred to as 'reticular' cells and the network they form is known as the 'ALP network'. The purpose was to analyse the evolution of this ALP network in relation to haemopoietic regeneration after whole-body irradiation. Materials and methods : The total surface occupied by ALP-positive processes revealed by means of ALP cytochemistry was expressed as a ratio of the total marrow area. ALP-positive cells were counted using nuclei as the defining unit. Cell proliferation was analysed by the detection of bromodeoxyuridine (BrdU) incorporation. Fat cells were identified by oil red O staining and α-glycerophosphate dehydrogenase (α-GPDH) activity. Results : The ALP network and ALP-positive cell number began to increase 24 h after 4-Gy irradiation to reach a maximum after 72 h, when the bone marrow was almost completely empty of haemopoietic cells. This increase was in advance of haemopoietic recovery and was not due to cell proliferation. A decrease in the ALP network occurred in parallel with an increase in haemopoiesis and was accompanied by a transient increase in fat cells on day 7. Conclusions : These data indicate that the recovery of the ALP network, which is partially due to the recruitment of ALP- positive cells, occurs in advance of the haemopoietic recovery and that the equilibrium between fat cells and ALP-positive cells seems to be controlled by haemopoietic cells.

Isoenzyme variation in the leaf-cutting ants Acromyrmex heyeri and Acromyrmex striatus (Hymenoptera, formicidae)

This is the first study of isoenzyme variability in the leaf-cutting ants (Myrmicinae, Attini) Acromyrmex heyeri (Forel, 1899) and A. striatus (Roger, 1863) which are common throughout the southern Brazilian state of Rio Grande do Sul. We studied the alloenzyme variability of malate dehydrogenase (MDH), α-glycerophosphate dehydrogenase (α-GPDH) and amylase (AMY) in 97 colonies of A. heyeri and 103 colonies of A. striatus. Five loci were found for these enzyme systems, one locus (Amy-1) being monomorphic in both species and four loci (Mdh-1, α-Gpdh-1, Amy-2, and Amy-4) being polymorphic. For each species there were exclusive alleles for the Mdh-1 and Amy-2 loci and differences were also found in the allele frequencies for the other polymorphic loci. Ontogenetically different gene activity was detected for the MDH and α-GPDH systems, with between-caste differences, probably related to flight activity, also being found for α-GPDH.

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and α-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([γ- 32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1- 32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [γ- 32P]ATP, and the radioactive product in the supernatant, [1- 32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.

A alpha-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes.

alpha-glycerophosphate dehydrogenase (alpha-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

Polimorfismo enzimático em populações de Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae)

Them aim scope of this study is to characterize the enzymatic polymorphism found in the Melipona quadrifasciata Lepeletier, 1936 populations from Ribeirao Preto, Sao Paulo and Espirito Santo, Brazil and its hybrids. Samples from each colony (about 52) were prepared for starch gel electrophoresis in order to investigate the genetic variation of the following enzimes: esterase (EST), isocitrate dehydrogenase (IDH), malic enzyme (ME), phosphoglucomutase (PGM), superoxide desmutase (SOD), α-glycerophosphate dehydrogenase (αPGD), malate dehydrogenase (MDH), leucine aminopeptidase (LAP), hexokinase (HK) and phosphoglucose isomerase (PGI). The analysis showed that LAP and HK did not show enzymatic activity and EST showed two alleles(est-sand and est-f) while all the others were shown to be monomorphic. The allele EST-S showed a frequency of 82,6%.

Microautoradiographic studies of glucose uptake in skeletal muscle fibers at rest

In the present study, differences in glucose uptake by muscle fibers in deep, middle, and superficial regions of the gastrocnemius were studied at rest by 2-deoxyglucose (2-DG) microautoradiography. Expression of the glucose transporter 4 (GLUT-4) protein, an isoform of the glucose transporter family, was analyzed as well. These data were compared with the activity of succinate dehydrogenase, a marker of oxidative metabolism, a-glycerophosphate dehydrogenase, an indicator of the glycolytic capacity, and myofibrillar ATPase. In the deep regions of the muscle, most fibers (86.9%) showed high 2-DG uptake and large amounts of GLUT-4 protein, whereas in the superficial regions, all fibers showed low 2-DG uptake and GLUT-4 expression. In the middle regions, fibers dominated (80.4%) showed low 2-DG uptake and small amounts of GLUT-4 protein. Analysis of metabolic properties revealed that most fibers in the deep region were oxidative and showed the highest 2-DG uptake; in the superficial region, the fibers were anaerobic and showed the lowest 2-DG uptake. In the middle region, most fibers were of the anaerobic and fast twitch type. It is concluded that 2-DG uptake correlates with GLUT-4 expression in the plasma membrane of type I and IIx fibers rather than with oxidative enzyme activity.

The activity of antioxidant enzymes and the content of uncoupling protein-1 in the brown adipose tissue of hypothyroid rats: comparison with effects of iopanoic acid

The activity of antioxidant enzymes, copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT), as well as that of the mitochondrial FAD-dependent a-glycerophosphate dehydrogenase (a-GPD) in the rat interscapular brown adipose tissue (IBAT) were studied after the treatment with methimazole (MMI) for three weeks or with iopanoic acid (IOP) for five days. Besides, the mitochondrial concentration of uncoupling protein-1 (UCP-1) and the activity of catecholamine degrading enzyme monoamine oxidase (MAO) in the IBAT as well as the activity of the catecholamine synthesizing enzyme, dopamine b-hydroxylase (DBH) in rat serum were examined. Judging by the significantly enhanced level of serum DBH, which is an index of sympathetic activity, and that of IBAT MAO, the increase in MnSOD and CAT activities in the IBAT of hypothyroid (MMI-treated) rats seems to be due to elevated activity of sympathetic nervous system (SNS). However, CuZnSOD activity is not affected by SNS. On the contrary, IOP, which is a potent inhibitor of T4 deiodination into T3 producing "local" hypothyroidism, did not change either SNS activity or activities of IBAT antioxidant enzyme. However, both treatments significantly decreased IBAT UCP-1 content and a-GPD activity suggesting that the optimal T3 concentration in the IBAT is necessary for maintaining basal levels of these key mitochondrial parameters.

Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp.

As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claiborne, A. (1998) J. Biol. Chem. 273, 23812-23822], the flavoprotein alpha-glycerophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50-52 residue insert that is completely absent in the homologous alpha-glycerophosphate dehydrogenases. On limited proteolysis with trypsin, the GlpO from Streptococcus sp. (m = 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 40 and 23 kDa. The combined application of sequence and mass spectrometric analyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1-Lys368; 40.5 kDa), while the 23-kDa band represents a C-terminal fragment (Ala405-Lys607; 22.9 kDa). Hence, limited proteolysis in effect excises most of the GlpO insert (Ser355-Lys404), indicating that this represents a flexible region on the protein surface. The active-site and other spectroscopic properties of the enzyme, including both flavin and tryptophan fluorescence spectra, titration behavior with both dithionite and sulfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] approximately 10 microM) demonstrate that the single major catalytic defect in the nicked enzyme corresponds to a 20-fold increase in K(m)(Glp); the basis for this altered kinetic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopped-flow experiments. These results indicate that the flexible surface region represented by elements of the GlpO insert plays an important role in mediating efficient flavin reduction.

Vitamin B 12-induced alterations in activities of α-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B 12 (0.25, 0.5, 1, 2 and 4 μg/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-α-glycerophosphate dehydrogenase (α-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The α-GPD activity increased at doses of 0.5, 1, 2 and 4 μg/g vitamin B 12. A dose of 0.5 μg/g vitamin B 12 induced less activity than higher doses. ME activity increased with 1, 2 and 4 μg/g of vitamin B 12/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B 12 administration. Cycloheximide treatment inhibited the vitamin B 12-induced increase in α-GPD and ME activity. Thus, vitamin B 12 is involved in the induction of some enzymes in fish brain.

Evolution of overwintering strategies in Eurasian species of the Drosophila obscura species group

Abstract The phylogenetic relationship of Eurasian species of the Drosophila obscura species group remains ambiguous in spite of intensive analyses based on morphology, allozymes and DNA sequences. The present analysis based on sequence data for cytochrome oxidase subunit I (COI) and α-glycerophosphate dehydrogenase ( Gpdh ) suggests that the phylogenetic position of D. alpina is also ambiguous. These ambiguities have been considered to be attributable to rapid phyletic radiation in this group at an early stage of its evolution. Overwintering strategies are diversified among these species D. alpina and D. subsilvestris pass the winter in pupal diapause, D. bifasciata and D. obscura in reproductive diapause, and D. subobscura and D. guanche without entering diapause. This diversity may also suggest rapid radiation at an early phase of adaptations to temperate climates. On the other hand, adult tolerance of cold was closely related to overwintering strategy and distribution D. obscura and D. bifasciata with reproductive diapause were very tolerant D. alpina and D. subsilvestris which pass the winter in pupal diapause were less tolerant D. subobscura having no diapause was moderately tolerant and D. guanche occurring in the Canary Islands was rather susceptible. Tolerance of high temperature at the preimaginal stages seemed to be also associated with overwintering strategy; i.e. lower in the species with pupal diapause than in those with reproductive diapause or without diapause mechanism.

Evolution of overwintering strategies in Eurasian species of the Drosophila obscura species group

The phylogenetic relationship of Eurasian species of the Drosophila obscura species group remains ambiguous in spite of intensive analyses based on morphology, allozymes and DNA sequences. The present analysis based on sequence data for cytochrome oxidase subunit I (COI) and α-glycerophosphate dehydrogenase (Gpdh) suggests that the phylogenetic position of D. alpina is also ambiguous. These ambiguities have been considered to be attributable to rapid phyletic radiation in this group at an early stage of its evolution. Overwintering strategies are diversified among these speciesD. alpina and D. subsilvestris pass the winter in pupal diapause, D. bifasciata and D. obscura in reproductive diapause, and D. subobscura and D. guanche without entering diapause. This diversity may also suggest rapid radiation at an early phase of adaptations to temperate climates. On the other hand, adult tolerance of cold was closely related to overwintering strategy and distribution D. obscura and D. bifasciata with reproductive diapause were very tolerant D. alpina and D. subsilvestris which pass the winter in pupal diapause were less tolerant D. subobscura having no diapause was moderately tolerant and D. guanche occurring in the Canary Islands was rather susceptible. Tolerance of high temperature at the preimaginal stages seemed to be also associated with overwintering strategy; i.e. lower in the species with pupal diapause than in those with reproductive diapause or without diapause mechanism.