Α-glycerophosphate Dehydrogenase Research Articles

Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by α-lipoamide dehydrogenase flavin ( E m 7.4 = −283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme ( E m 7.4 = −52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, α-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.

A high affinity Ca 2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Addition of Ca 2+ (0.01–1 mM) to a standard Trypanosoma rhodesiense Mg 2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg 2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca 2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm −3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked α-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca 2+-ATPase activity, using 200 nM free Ca 2+, of 90 and 21 mU mg −1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca 2+-ATPase, in addition to having no Mg 2+ requirement, was inhibited by submicromolar levels of Mg 2+. The Ca 2+-ATPase was found to have a K 0.5 − 128 ± 22 nM free Ca 2+, the response to increasing Ca 2+ concentration displaying an extremely high degree of co-operativity (Hill number ( n H) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K 0.5 = 6.2 ± 0.6 μM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites ( n H = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V 2O 8 3−) having any effect: 47% inhibition at 5.0 μM. The Ca 2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 μM) and trifluoperazine (50 μM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca 2+ in bloodstream T. rhodesiense.

The presence of α-glycerophosphate dehydrogenase (NAD +-linked) and adenylate kinase as core and integral membrane enzymes respectively in the glycosomes of Trypanosoma rhodesiense

A subcellular fraction enriched 12 times in glycosomes (NAD +-linked α-glycerophosphate dehydrogenase) and devoid of detectable contamination from other subcellular components, was prepared from bloodstream Trypanosoma rhodesiense. Using a method employing exposure to toluene as a means of studying normally latent glycosomal enzymes, and phospholipase A 2 as a membrane probe, the association of adenylate kinase and α-glycerophosphate dehydrogenase with the glycosome was studied. The normally latent glycerophosphate dehydrogenase (NAD + linked), it is proposed, is an intraglycosomal enzyme having no membrane association, but bound to the core by weak ionic linkages. As such it is possible to release the enzyme from permeable (toluene treated) glycosomes using Cl −, with a resulting 4-fold increase in the K m for dihydroxyacetone phosphate. The presence of Cl − also stimulates an increase in specific activity, but this is observed before any release of enzyme. In contrast adenylate kinase, a non-latent glycosomal enzyme, is clearly membrane associated, the use of phospholipase A 2 revealing an absolute dependence on phospholipid for activity. Restoration of activity appears to specifically require phosphatidyl choline and to be co-operative in nature ( n H = 1.56). It is proposed that adenylate kinase is an integral glycosomal membrane enzyme, probably affecting the control of intra-glycosomal ADP/ATP levels.

Biochemical studies on fatty liver. Part X. Induction of fatty liver by 2-acetylaminofluorene and its mechanism.

The induction and mechanism of fatty liver in the rat by the synthetic carcinogen 2-acetylaminofluorene (AAF) were investigated.The induction of this fatty liver was dose and time dependent, being gradually increased by the intake of a 0.05% AAF diet for 3 weeks. The AAF dosage was found to increase the activity of drug-metabolizing enzymes (p-nitroanisol demethylase and aniline hydroxylase) and to decrease the activity of pyruvate kinase and α-glycerophosphate dehydrogenase. The AAF dosage had no effect on the incorporation of [l-14C]acetate into the lipid fraction during in vitro incubation of liver slices. The supplement of adenine to the AAF diet had no effect on the accumulation of liver lipid.It is suggested from the result of treatment with Triton WR-1339 that a block in the secretion of triglyceride from the liver is a major cause of the induction of fatty liver by AAF.

Sensitivity of malic enzyme and α-glycerophosphate dehydrogenase of human adipose tissue to L-triiodothyronine

Sensitivity of malic enzyme and α-glycerophosphate dehydrogenase of human adipose tissue to L-triiodothyronine

A sensitive, continuous spectrophotometric method for assaying α-glycerophosphate dehydrogenase: Activation by menadione

By use of a more sensitive method than standard assays, it is demonstrated that α-glycerophosphate dehydrogenase activity can be monitored continuously in rat liver mitochondria; maximum activity was obtained by sonication in the presence of Triton X-100. Vitamin K 3 (menadione) seems to enhance the activity of the enzyme. The assay in the presence of menadione is linear over a much greater mitochondrial concentration (up to 250 μg of protein in the reaction mixture).

Genetics of Glossina morsitans morsitans (Diptera: Glossinidae). IX. Definition of linkage group III and further mapping of linkage groups I and II

In Glossina morsitans morsitans Westwood, linkage group III is defined as the autosome carrying the locus Mdh (malic dehydrogenase), the only locus so far identified in this linkage group. The locus αGpd.2 (α-glycerophosphate dehydrogenase) was located 45 map units (MU) to the left of Xo (xanthine oxidase) and the loci Est.1 and Est.2 (loci for two esterases found in the thorax) were mapped approximately 5–10 MU to the right of Ao (aldehyde oxidase) in linkage group II. The location of G6pd (glucose-6-phosphate dehydrogenase) has been confirmed to be approximately 37 MU to the left of oc (ocra body color) in linkage group I and it was shown that this region of the X chromosome does not involve the large paracentric inversion found in the Handeni line. A genetic map for 12 loci in the three linkage groups found in G. m. morsitans is presented.Key words: Diptera, Glossina, mapping, inversion, isozymes.

Histochemical localization of dehydrogenases in the cercaria and excysted metacercaria of Echinostoma revolutum (Trematoda)

1. 1. Histochemical studies for twelve dehydrogenases were done on cercariae and in vitro excysted metacercariae of Echinostoma revolutum. 2. 2. Strong to slightly positive activity was demonstrated at various sites for 6-phosphogluconate dehydrogenase (6-PGDH), glucose-6-phosphate dehydrogenase (G-6-PDH), α-glycerophosphate dehydrogenase (α-GPDH), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GLDH), NADH diaphorase (NADHD) and NADPH diaphorase (NADPHD). 3. 3. No activity was observed for alcohol dehydrogenase (ALDH) and β-hydroxybutyrate dehydrogenase (β-HBDH). 4. 4. With the exception of G-6-PDH, reactions were more intense in the cercaria than the excysted metacercaria, suggesting that cercariae require more energy than metacercariae.

Histochemical characteristics of the tensor tympani muscle in relation to the medial gastrocnemius muscle of the cat.

Serial 10-micron cryostat cross-sections of the tensor tympani muscle and of the medial gastrocnemius muscle from adult domestic cats were incubated for myofibrillar ATPase, NADH tetrazolium reductase (NADH-TR), succinic dehydrogenase (SDH), malate dehydrogenase (MDH) and menadione-linked alpha-glycerophosphate dehydrogenase (alpha GPDH). The optical density of individual tensor tympani and gastrocnemius muscle fibres after different incubation procedures was measured photometrically. The absorbance values of the tensor tympani fibres were related to the values of the type I, type IIA and type IIB fibres of the gastrocnemius muscle. Only two different types of fibre could be demonstrated in the tensor tympani, one type resembling the type I and another resembling the type IIA of the gastrocnemius muscle. The findings are discussed in relation to other, recent immunohistochemical studies on cat tensor tympani muscle fibres.

Inhibitory influence of thiourea on brain of singi fish ( Heteropneustes fossilis bloch) and subsequent recovery by l-triiodothyronine

Immersion of Singi fish in thiourea-containing medium (1 mg/ml) for 45 days significantly decreased the cranio-somatic index, weight of different parts of brain, viz. cerebrum, cerebellum, midbrain and medulla oblongata, and also protein and RNA contents of these different regions. The DNA content of these substructures remained unchanged. In other sets of experiment, thiourea treatment for 33 days caused reduction in mitochondrial cytochrome-linked α-glycerophosphate dehydrogenase (EC 1.1.99.5) activity, and total protein and RNA contents of whole brain, while the amount of mitochondrial protein and total DNA content of whole brain did not undergo significant variation. A single injection of l-triiodothyronine (0.5 μg/g) enhanced this enzyme activity, mitochondrial protein amount and total protein and RNA contents of whole brain of thiourea-treated fish to almost such levels as obtained by l-triiodothyronine injection in normal (control) Singi fish within 3 days. The CSI in normal fish increased by triiodothyronine injection. In thiourea-treated fish, the reduction of CSI was restored to just control level by triiodothyronine. The results are, therefore, discussed as additional supportive evidence of the responsiveness of fish brain to thyroid hormone.

Characterization of succinate dehydrogenase and α-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of α-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected α-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that α-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

Effects of short- and long-term thyroidectomy on mitochondrial and nuclear activity in adult rat brain

We compared subcellular activities in brain and liver at various times after thyroidectomy. Male S.D. rats were used on days 5, 10 or 60 after surgery. Mitochondrial properties were estimated by determining the respective activities of oxidative phosphorylation, succinate oxidase, succinate and β-hydroxybutyrate cytochrome c reductase and α-glycerophosphate dehydrogenase. Nuclear activity was estimated by measuring the RNA polymerase I activity. In brain, RNA polymerase I activity already declined at 5 days after thyroidectomy, whereas mitochondrial respiratory enzymes decreased significantly only after 60 days. In liver, nuclear RNA polymerase I and mitochondrial enzyme activities were observed to drop simultaneously by the 5th day after thyroid removal. On the other hand, daily T3 s.c. injections, 0.25 μg/100 g B.W., were given for 10 days to rats immediately after thyroidectomy (10 days Tx) or to chronically hypothyroid rats (60 days Hth). Hormonal treatment either maintained or restored subcellular activities to their normal level, both in brain and liver. These data suggest that the metabolic properties of brain mitochondria are sensitive to thyroid hormones, but that the brain needs less iodothyronines than other organs. The fast reduction of RNA polymerase I by thyroidectomy and its subsequent restoration by T 3 suggest that the nuclear activity greatly depends on thyroid status.

Electrophoretic study of enzymes from cereal aphid populations. I. Electrophoretic techniques and staining systems for characterising isoenzymes from six species of cereal aphids (Hemiptera: Aphididae)

AbstractOne–dimensional slab polyacrylamide gel electrophoretic techniques, staining systems and isoenzyme banding patterns for 14 soluble enzymes separated from crude homogenates of individuals of six species of cereal aphids (Sitobion avenae (F.), S. fragariae (Wlk.), Metopolophium dirhodum (Wlk.), M. festucae (Theo.), Rhopalosiphum padi (L.) and R. maidis (Fitch)) are described. The value of the techniques and banding patterns to taxonomic and population genetic studies of these and other aphid species are briefly discussed. With the six species, it was possible to separate the different genera as well as individual species within genera. The enzymes found to be most useful for inter–generic and/or -specific separations were adenylate kinase (AK), esterase (EST), glucose-6-phosphate dehydrogenase (G-6-PDH), hexokinase (HK), malate dehydrogenase (MDH), peptidase (PEP), phosphatase (PHOS), phosphoglucomutase (PMG), 6-phosphogluconate dehydrogenase (6-PGD) and sorbitol dehydrogenase (SORDH), whilst glutamate oxaloacetate transaminase (GOT), α-glycerophosphate dehydrogenase (α-GPD), malic enzyme (ME) and peroxidase (POD) were of relatively little taxonomic use. There were no banding pattern differences between the various morphs of S. avenae (first to fourth-instar nymphs, apterous and alate adults using the 14 enzymes.

Histochemical localization of glycolytic enzymes, alcohol and secondary-alcohol dehydrogenases in the testes of buffaloes, goats and rams

SUMMARYGlyceraldehyde-3-phosphate dehydrogenase (G-3-PDH), αglycerophosphate dehydrogenase (αGPDH), lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and secondary-alcohol dehydrogenase (SADH) were histochemically located in the testes of buffaloes, goats and rams. Two forms of αGPDH were observed: (i) NAD-dependent or cytoplasmic αglycero-phosphate dehydrogenase (αGPDHC) and (ii) NAD-independent or mitochondrial αglycerophosphate dehydrogenase (α GPDHM). The basic pattern of distribution of the various enzymes was similar in the three species; species-specific variations were observed but cell-specific variations were more pronounced. The main activities of G-3-PDH and αGPDHM were observed in the seminiferous tubules; interstitial tissue showed moderate (G-3-PDH) and trace to weak (αGPDHM) activity. In contrast, αGPDHC activity was more marked in the interstitial tissue and less in the seminiferous tubules, especially in the mature germinal elements. LDH and ADH respectively showed strong and moderate activities in the interstitial tissue and seminiferous tubules. SADH was noticeable only in the interstitial tissue of buffalo. The activities of all enzymes other than αGPDHC increased during spermiogenesis. The physiological significance of the results is discussed in relation to the carbohydrate metabolism of the testis.

GENETIC LOCALIZATION AND BIOCHEMICAL CHARACTERIZATION OF A TRANS-ACTING REGULATORY EFFECT IN DROSOPHILA

A region-specific, trans-acting regulatory gene that alters in vivo protein levels of alpha-glycerophosphate dehydrogenase (alpha-GPDH) has been mapped to position 55.4 on the third chromosome of Drosophila melanogaster. The gene has been found to affect the in vivo stability of alpha-GPDH in adult thoracic tissue but has no effect on alpha-GPDH levels in the abdomen. Although no other thoracic proteins were found to be influenced by the locus, it appears to modify the level of one additional abdominal protein. The action of the gene over development and its possible mode of control are discussed.

Electrophoretic Analysis of Isoenzymes of Acholeplasma Species

Thirteen Acholeplasma strains representing three named species, Acholeplasma laidlawii, Acholeplasma granularum, and Acholeplasma oculi, were examined for the presence of 30 enzymes by horizontal starch gel electrophoresis. A total of 29 enzymes were demonstrated. Twelve of these enzymes have not been reported previously in acholeplasmas (alcohol dehydrogenase, aldolase, alkaline phosphatase, arginase, arginine deiminase, carbarnyl phosphokinase, galactose dehydrogenase, galactose-6-phosphate dehydrogenase, glutamate dehydrogenase [nicotinamide adenine dinucleotide], glutamate dehydrogenase [nicotinamide adenine dinucleotide phosphate], α-glycerophosphate dehydrogenase, and xanthine dehydrogenase). An average group cluster analysis was carried out on the basis of estimates of dissimilarity coefficients. This analysis distinguished among the strains tested, was of some help in identification, and may also be useful in epidemiological investigations.

Increased lipid content in the rat axotomized superior cervical ganglion

Transection of postganglionic nerves of the rat superior cervical ganglion results in significant increases in the concentration of lipid as well as the total content of lipid in the ganglion. The purpose of this work was to characterize the effect of axotomy on the major classes of lipid in the ganglion and to examine possible relationships among increases in lipid content and changes in steady-state concentrations of metabolites utilized in lipid biosynthesis. Major classes of lipids in axotomized and contralateral intact ganglia were compared using the Folch extraction and partitioning scheme. Analyses of lipids were carried out 7 days after axotomy because total lipid content was increased maximally at that time. The increase in lipid content in axotomized ganglia was related primarily to an increase in phospholipids. Amounts of gangliosides, cerebrosides, cholesterol, and proteolipid protein did not change significantly after axotomy. The elevation of phospholipid content was accompanied by increases in steady-state concentrations of α-glycerophosphate and decreases in Coenzyme A in the ganglion; however, concentrations of acetyl CoA and long-chain acyl CoA compounds did not change significantly in axotomized ganglia. Increases in α-glycerophosphate do not appear to arise from enhanced utilization of glucose because α-glycerophosphate dehydrogenase activity was decreased in axotomized ganglia and the incorporation of [U- 14C] glucose into lipids in explants of the axotomized ganglia in vitro was not elevated. In contrast, incorporation of [U- 14C] glycerol into lipids of explants from axotomized ganglia was increased in vitro. The results suggest that utilization of extracellular glycerol for phospholipid biosynthesis may be enhanced in the rat's axotomized superior cervical ganglion.

Body energy balance and food intake: a neuroendocrine regulatory mechanism.

Body energy balance and food intake: a neuroendocrine regulatory mechanism.

A change in the affinity of larval α-glycerophosphate dehydrogenase in relation to the activity of the heat shock genes of Drosophila hydei

After activation of the heat shock genes in Drosophila hydei salivary glands, the K m of α-glycerophosphate dehydrogenase (E.C.1.1.1.8., α-GPDH) for α-glycerophosphate decreases, whereas the app. V m remains the same. Although RNA and protein synthesis are required for this change to occur in vivo, this decrease is not due to de novo synthesis of the enzyme: a similar change in K m can be produced in vitro when extracts from control glands are mixed with extracts from heat-shocked cells. The in vitro reaction is probably due to a deacetylation of the enzyme, since it can be prevented by eserine sulphate and acetyl CoA but not by ATP or a trypsin inhibitor. The activation of the heat shock genes is thus accompanied by the appearance of a factor capable of modifying α-GPDH activity. Alpha-glycerophosphate levels in induced and control glands are determined. The larval α-GPDH has been purified 1150-fold by affinity chromatography. The purified enzyme has a subunit mol. wt of 32,000 and thus differs from other known heat shock proteins. The K m of the enzyme purified from extracts of control or induced glands is the same as that measured in crude extracts, the decrease in app. K m found is therefore a property of the enzyme itself and not due to some contaminant present in the crude extract.

Effect of l-triiodothyronine on the mitochondrial α-glycerophosphate dehydrogenase activity, mitochondrial and total protein contents of brain of Singi fish ( Heteropneustes fossilis bloch)

A single injection of various doses (0.25, 0.5, 5, 20 and 50 μg/g) of l-triiodothyronine increased the mitochondrial cytochrome-linked α-glycerophosphate dehydrogenase (EC 1.1.99.5) activity and mitochondrial protein content of brain of Singi fish on the 3rd day. l-Triiodothyronine at the dose of 0.1 μg/g did not alter the α-glycerophosphate dehydrogenase activity and mitochondrial protein content of brain. The total protein content of the brain also increased on the 3rd day with 0.5 μg of l-triiodothyronine per g. Increased enzyme activity followed a dose-response relationship of a non-linear fashion. The enhancement of the α-glycerophosphate dehydrogenase activity of fish brain with a dose of 0.5 μg/g was found from the 1st day and it reached to a maximum level from the 3rd to the 5th day. The enzyme activity then sharply declined on the 6th or 7th day. Cycloheximide inhibited the l-triiodothyronine-induced increase in the α-glycerophosphate dehydrogenase activity, mitochondrial and total protein content of fish brain. The present study thus reveals the responsiveness of fish brain to thyroid hormone, and α-glycerophosphate dehydrogenase activity can be taken as a biochemical indices for the expression of thyroid hormone action in fish brain.