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... The main reactions of glycerol metabolism are condensed in the glycerol-3-phosphate shuttle (Fig. 2), a cycle centred at two different isoforms of glycerol-3-phosphate dehydrogenase (GPD) (Dipple and McCabe, 2001;Green, 1936;Hopkinson et al., 1974;Lin, 1977;Mráček et al., 2013). For years, the physiological importance of this shuttle was underestimated. ...
... Biological relevance for glycerol metabolism: from basic aspects to male (in)fertility . The cross-talk between those metabolic pathways is centred on G3P, and it is referred as intermediary metabolism (Dulermo and Nicaud, 2011;Euler et al., 1937;Green, 1936;Lin, 1977;Mráček et al., 2013;Mugabo et al., 2016;Schlossman and Bell, 1977). Being at the crossroads of so many metabolic pathways, 70−90% of the whole body glycerol metabolism takes place in the liver (Peroni et al., 1995). ...
... One of the possible outcomes of the intermediary metabolism is the reversible dehydrogenation of G3P into dihydroxyacetone phosphate (DHAP) by the cytosol enzyme GPD (cGPDH or GPD1), having NAD + as electron acceptor (Green, 1936;Wu et al., 2015). DHAP is a substrate for both gluconeogenesis or glycolysis Wu et al., 2015). ...
Over the past decades, there have been several studies suggesting that semen quality is declining. Interestingly, these observations are paired with a significant increase in the number of individuals diagnosed with metabolic diseases, including obesity and diabetes mellitus. Hence, it is tempting to hypothesize that obesity and its associated comorbidities and risk factors (such as a hypercaloric diets) impair the homeostasis of the male reproductive health, with a possible direct effect on the testes. The blood and interstitial fluids of obese individuals usually have increased levels of glycerol, notably due to triglyceride and phospholipid catabolism and high fructose intake. Glycerol is metabolized via intermediary metabolism by a group of reactions centred at the glycerol-3-phosphate shuttle, which links the metabolic pathway of glucose, lipids and oxidative phosphorylation, illustrating its high relevance for biological systems. Glycerol enters and exits the cells by the action of specialized carriers, known as aquaglyceroporins, whose functional importance for male reproductive health has emerged in the last few years. Notably, glycerol has antispermatogenic properties. When present in high concentration in the testis, it causes blood-testis barrier disruption, impairing tubular fluid homeostasis. Nevertheless, glycerol metabolism in testicular cells remains a matter of debate. Herein we discuss previous and current research concerning the role of glycerol and its metabolism in testicular cells, and how it can influence testicular activity.
... It is hard to believe that the research on the mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) started already 75 years ago  and yet there are still many questions to be answered, both concerning the structure/function relationship and regulation of this enzyme. This becomes even more striking when we consider its simple structure. ...
... Studies of metabolic function of mGPDH started in 1936 by Green and coworkers [1,40,41] who found that this enzyme is present in variable amount in many mammalian tissues. The highest enzyme activity was found in mammalian BAT  and in insect flight muscles [43,44]. ...
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 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.
... tissues contain two types of ol-glycerophosphate dehydrogenases, a soluble diphosphopyridine nucleotide-linked enzyme (1) and a particulate enzyme which is linked to the respiratory chain and functions without the mediation of a readily dissociable coenzyme (2,3). ...
... The details of the extraction and partial purification of the dehydrogenase will be described in a subsequent paper. DISCUSSION The data presented on the effect of respiratory chain inhibitors on oc-glycero-P oxidation, the identical stimulation of a-glycero-P and of succinate oxidation by cytochrome c in cytochrome-deficient mitochondria, and the mutually competitive inhibition of succinic and ol-glycero-P oxidases, added to the evidence in the literature (3,20), strongly suggest that mammalian cr-glycero-P dehydrogenase is linked to O2 by way of the classical respiratory chain. The experiments cited and the demonstration of an antimycin A insensitive ol-glycero-P-fumarate reaction further suggest that either a common respiratory chain serves succinic and a-glycero-P dehydrogenases, at least between cytochrome b and 02, or that if the two enzymes are linked to separate cytochrome chains, the components must be very similar in sensitivity to inhibitors and a crossover must occur between the chains in the course of normal electron transport. ...
... The FAD-linked oxidation of R-glycerophosphate (Glp) 1 to DHAP occurs in at least three contexts. The first two are R-glycerophosphate dehydrogenases (GlpDs) from mito-chondria [mitoGlpD (1)(2)(3)(4)] and from bacteria [bactGlpD (5,6)], clearly homologous enzymes having sequences that are ca. ...
... The FAD-linked oxidation of R-glycerophosphate (Glp) 1 to DHAP occurs in at least three contexts. The first two are R-glycerophosphate dehydrogenases (GlpDs) from mito-chondria [mitoGlpD (1)(2)(3)(4)] and from bacteria [bactGlpD (5,6)], clearly homologous enzymes having sequences that are ca. 30-33% identical. ...
... There are 5 QDHs in animals that are linked to the mitochondrial ETC. These comprise Succinate dehydrogenase (also known as Complex II) [26, ; Electron-Transferring Flavoprotein (ETF) dehydrogenase ; Proline dehydrogenase ; Glycerol 3-phosphate dehydrogenase ; and Dihydroorotic acid dehydrogenase . We note that there are other quinone-coupled enzymes in animals including sulphide:quinone oxidoreductase (sqrd-1 in C. elegans ) as well as orthologues of D-and L-hydroxyglutarate dehydrogenase  (D2HGDH and L2HGDH; F45D5.12 and Y45G12B.3 respectively). ...
Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets in the anaerobic metabolic pathways STHs need to survive in their mammalian host. These require rhodoquinone (RQ), an electron carrier used by STHs and not their hosts. We identified 25 genes predicted to act in RQ-dependent metabolism including sensing hypoxia and RQ synthesis and found 9 are required. Since all 9 have mammalian orthologues, we used comparative genomics and structural modeling to identify those with active sites that differ between host and parasite. Together, we found 4 genes that are required for RQ-dependent metabolism and have different active sites. Finding these high confidence targets can open up in silico screens to identify species selective inhibitors of these enzymes as new anthelmintics.
... Thus, already in 1919, MEYERHOF found that glycerol phosphate was oxidized by muscle and liver tissues. This led to much further work and the discovery that animal tissues contain two different a-glycerophosphate dehydrogenases (GPDH), one of which is a particle bound flavoprotein (GREEN 1936), while the other is found in the cytosol and utilises NAD as a co-enzyme (v. EULER, ADLER and GUNTHER 1937). ...
The net replication method. Methods were developed for screening of glycerol non-utilizing mutants from Neurospora crassa. The strains utilized produce only mononucleate conidia (pe ft, microconidia). Some strains were inherently colonial, independent of temperature, others were of the cot (colonial, temperature-sensitive) type. Mutagen (ultraviolet light) treated conidia were spread on plates with medium containing glucose as the source of carbon, and overlaid with a disc of Terylene net. Outgrowing colonies were transferred with the nets to plates with glycerol as carbon. Replication was accomplished by a period of growth. Non-replicating colonies were isolated from the glucose plates and retested as presumptive glycerol non-utilizing mutants.
Glycerol non-utilizing mutants. One glycerol non-utilizing mutant, gly-u (234), appears otherwise physiologically normal and segregates as a nuclear gene in crosses. Another, gly-u (245), has a very long lag period (ca 2—3 weeks) on glycerol medium, but eventually adapts itself to growth on this carbon source. Photographs of replicated colonies as well as mutants are presented. The biochemical pathways of glycerol metabolism are outlined from work by various authors, and the present studies are discussed in this connection.
... succinate (3.7 X 10 -3 ~); cytochrome c (1 X 10-~ M); Cat12 (3 X 10 -a ~); A1CI~ (4.2 X 10 -5 ~t). (8).--Na a-glycerophosphate (3.7 X 10 -2 ~); cytochrome c (1 X 10-5 M). ...
The rates of activity of the dehydrogenase systems in Tetrahymena, which are concerned with carbohydrate oxidation, in descending order of activity are: lactic > isocitric > succinic = glucose > glucose-6-phosphate = 6-phosphogluconic = malic > glutamic = cytochrome linked α-glycerophosphate dehydrogenase. No evidence was obtained to indicate the presence of DPN linked α-glycerophosphate dehydrogenase.
... Cette protéine de 79kDa est une protéine membranaire extrinsèque dont le site actif est dirigé vers l'espace intermembranaire ( Figure 10) . Cette enzyme, qui est codée par le génome nucléaire, est surtout exprimée dans le tissu adipeux brun, le cerveau, les muscles striés squelettiques, le foie et les testicules (Green, 1936;Koza et al., 1996;Ohkawa et al., 1969). ...
Grâce à plusieurs ubiquinones oxydoréductases, de nombreuses voies métaboliques sont sous la dépendance de la chaîne respiratoire de mammifère (Béta-oxydation, synthèse des pyrimidines, navette redox, cycle de Krebs). Physiologiquement, la chaîne respiratoire dispose d’une multitude de substrats respiratoires pouvant alimenter simultanément ces oxydoréductases. Il a été émis l’hypothèse que l’organisation supramoléculaire (appelé supercomplexe) du complexe I de la chaîne respiratoire permettrait de favoriser et prioriser le flux d’électron provenant de ce complexe par rapport aux autres oxydoréductases. L’objectif de ce travail de thèse a été de déterminer la possible incidence fonctionnelle de cette organisation supramoléculaire. En particulier, nous avons développé de nouvelles stratégies expérimentales afin de déterminer si cette organisation supramoléculaire pouvait orchestrer l’approvisionnement en électron de la chaine respiratoire depuis les différentes ubiquinones oxydoréductases. La première partie de ce travail a caractérisé l’incidence fonctionnelle d’une désorganisation de l’organisation supramoléculaire de la chaine respiratoire consécutive à la perte du facteur d’assemblage COX7A2L. Cette étude a démontré que la perte des supercomplexes III2-IV de la chaîne respiratoire n’était associé à aucun défaut bioénergétique majeur affectant la respiration associée à l’oxydation du NADH ou du succinate. La seconde partie de ce travail de thèse a mis en évidence que les chaines respiratoires des mitochondries hépatiques et cardiaques privilégient l’oxydation du succinate au détriment du NADH. Cette observation démontre que l’organisation supramoléculaire du complexe I avec les autres constituants de la chaine respiratoire ne permet pas de favoriser l’oxydation du NADH. Notre travail a surtout permis de montrer que l’inhibition directe du complex II par l’oxaloacetate intramitochondrial pouvait être un mécanisme extrêmement réactif permettant de réorienter les flux métaboliques intramitochondriaux et d’orchestrer l’activité des complexe I et II de la chaine respiratoire afin de privilégier la réoxydation du NADH.
... In 2019, Nilsson and colleagues used in silico modeling to examine optimal metabolic pathways for ATP synthesis (Nilsson et al., 2019). They discovered that beyond ∼40% VO 2 max, mitochondrial energy flux utilizes the glycerolphosphate shuttle (Green, 1936;Mráček et al., 2013) to transport electrons directly to ubiquinone and entering at complex III, effectively bypassing complex I to avoid to "backpressure" of the complex I proton pump (Nilsson et al., 2019;Glancy et al., 2020). Notably, this is nearly the same workload where Vollestad reported that type II fibers recruitment begins (Vøllestad and Blom, 1985), which are less reliant on oxidative energy production and have greater state 3 respiration with glycerophosphate as a substrate (Willis and Jackman, 1994). ...
An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.
Nachdem von den repräsentativen Vereinigungen der Biochemiker und klinischen Chemiker Richtlinien für die Festlegung von Enzym-Einheiten
festgelegt wurden, wird empfohlen, in Zukunft auf „private“ Einheiten zu verzichten und als Aktivitäts-Einheit (U) „µMol/min/1000
ml“ zu verwenden. Die Umrechnungsfaktoren für die gebräuchlichen Methoden, insbesondere diejenigen, die sich des Optischen
Testes bedienen, werden angegeben. Grundsätzliche Probleme der Aktivitäts- und Bezugs-Einheiten bei biochemischen und diagnostischen
Enzym-Bestimmungen werden kurz diskutiert.
EinleitungDie von PN-Enzymen katalysierten ReaktionenModelluntersuchungen zum Mechanismus des WasserstofftransfersDie Bindung der PN an die Dehydrogenasen und die Stereochemie des WasserstofftransfersSchlussbetrachtung
Activities of the enzymes monoamine oxidase (EC 18.104.22.168), alpha-glycerophosphate dehydrogenase (EC 22.214.171.124) and cytochrome oxidase (EC 126.96.36.199) were determined in homogenates and in the mitochondrial fraction prepared from individual regions of pig brain. The variation in the activity of alpha-glycerophosphate dehydrogenase paralleled that of cytochrome oxidase, but this was not the case with monoamine oxidase. The differences in the activities of the enzymes among homogenates of the various regions of the brain persisted in mitochondria prepared from these homogenates. The purification of these three enzymes paralleled each other when mitochondria were prepared, suggesting that the three enzymes are bound to the same particles.
Die Wechselbeziehungen innerhalb des Netzwerks von Redox-Reaktionen, das sich über einen großen Teil der metabolisch wesentlichen Funktionen lebender Zellen erstreckt, werden erörtert. Anschließend an die Darlegung grundsätzlicher Gegebenheiten bei der Zerlegung der Brennstoffe werden einige Gruppen von Redox-Systemen in verschiedenen Räumen der Zellen und Gewebe in ihren Beziehungen zur Biosynthese, zur Bioenergetik und zur Zellatmung behandelt. Neuere Ergebnisse aus dem Arbeitskreis der Verfasser stehen dabei im Vordergrund. Die Beispiele zeigen, wie weitgehend die Entwicklung der Problematik der dynamischen Biochemie an den Fortschritt der Cytologie gebunden ist.
IntroductionCatabolic Process: Carbohydrate FermentationOxidative Breakdown of CarbohydrateThe Anabolic Process: Synthesis of CarbohydrateRegulatory MechanismsOrientation of Reactions (the Pasteur Effect)Summary
In this study frozen sections of avian striated muscles were incubated for mitochondrial -glycerophosphate dehydrogenase (-GPD) reaction, and the effect of menadione, phenazine methosulfate (PMS) or phenazine ethosulfate (PES) as intermediate electron acceptors was evaluated. Under histochemical conditions, PMS or PES-linked -GPD reaction was poor in the chicken posterior latissimus dorsi and chicken pectoralis muscles. However, PMS or PES-linked -GPD reaction was present characteristically in ths subsarcolemmal mitochondria of the broad white fibres of the pigeon pectoralis muscle only; the subsarcolemmal mitochondria of the narrow red fibres lacked such a reaction pattern. The above reaction pattern, however, differed when compared with the menadione-linked -GPD reaction. The present histochemical evidence suggests the existence of an inherent heterogeneity in the mitochondrial populations of the different avian striated muscle fibres studied.
1. The concentration of ATP in a lens brei is maintained when the brei is incubated in oxygen with alpha-glycerophosphate. Lack of alpha-glycerophosphate or incubation in nitrogen causes the concentration to decrease. alpha-Glycerophosphate has some effect under anaerobic conditions but this is not sufficient to account for the maintenance in oxygen. 2. Manometric experiments show that alpha-glycerophosphate enhances the respiration of lens preparations. This respiration can be further increased by the addition of ADP and is abolished by cyanide and antimycin. The inference from these experiments is that a mitochondrial system able to oxidize alpha-glycerophosphate is present, i.e. the particulate half of the alpha-glycerophosphate cycle. 3. More than the calculated proportion of NADH is used when limiting amounts of dihydroxyacetone phosphate are added to lens tissue in spectrophotometric experiments. Dihydroxyacetone phosphate is therefore regenerated and an alpha-glycerophosphate cycle is operative. 4. A preparation of a particulate alpha-glycerophosphate dehydrogenase that takes up oxygen with methylene blue as electron acceptor is described. 5. Methods for obtaining mitochondria from lens are compared, and a useful extraction medium is defined. 6. Mitochondria with activities of the same order of magnitude as those obtained from liver, with alpha-glycerophosphate and glutamate as substrates, are prepared from epithelium detached from the capsule; some respiratory control is observed.
Several strain-specific markers were found to be histochemically visualizable in parts of the central nervous system in allophenic mice. These markers therefore provide a new basis for mapping the normal developmental lineages of major parts of the nervous system, and for identifying the focus of mutant gene action in some neurological mutations. Cell strains in mosaic animals were visualized on the basis of a quantitative difference in β-galactosidase activity (Bgs-locus), in the Purkinje zone of the cerebellum, and in the hippocampal pyramidal zone of the cerebrum. The differential between strains was increased if the beige () mutation was included in the high-activity strain. (β-galactosidase is lysosomal, and enhanced visualization in beige results from its enlarged and aggregated lysosomes.) Purkinje cell-strain visualization was also obtained by an indirect fluorescent antibody technique, in sections treated with antisera containing antibodies against strain-type histocompatibility alloantigens, including H-2. The above markers reveal considerable interspersion of cells from separate lineages in short sequences of each genotype. Purkinje and pyramidal cells of the same brain sometimes differ appreciably in genotypic composition. The enzyme glucosephosphate isomerase was found histochemically to be localized in nerve fibers rather than cell bodies in the brain. However, it was prominent in the cell bodies of the spinal ganglia, so that biochemical determination of ganglion strain-types is possible by means of strain-specific isozymes (Gpi-1-locus). Individual ganglia contained both cell strains and thus are not individually derived as clones from the neural crest.
1. Es wurde in gewaschenen Erythrocyten von Gesunden der Gehalt an Aldolase, Trioseisomerase, Phosphoglyceraldehyddehydrogenase, Milchsäuredehydrogenase und Hämiglobinreduktasesystem bestimmt.
2. Die gefundenen Fermentaktivitäten wurden auf die Zellzahl, den Hämoglobingehalt und das Erythrocyteneinzelvolumen berechnet.
3. Unter Zugrundelegung der Umsatzzahlen für kristalline Fermente wurde der Anteil der einzelnen Gärungsfermentproteine am Gesamteiweiß und Nichthämoglobineiweiß der Erythrocyten bestimmt und mit den entsprechenden Daten des Muskels verglichen.
4. Im Erythrocyten und Reticulocyten wurden weder löslichel-α-Glycerophosphatdehydrogenase (Baranowski-Enzym) noch strukturgebundenel-α-Glycerophosphatdehydrogenase (Green-Enzym) gefunden.l-α-Glycerophosphat war ebenfalls nicht nachweisbar.
5. Die begrenzenden Reaktionsstufen der Glykolyse im Erythrocyten sind nach unseren Fermentuntersuchungen die Aldolase- und die oxydierende Gärungsfermentreaktion.
α-Glycerophosphate dehydrogenase was crystallized as rectangular plates, having the following properties: . The molecular weight has been estimated to be 173,000 g./mole.α-Glycerophosphate dehydrogenase was found to be relatively thermolabile, and is most stable at pH 5.7. Approximate Michaelis constants at pH 7.0 were found as follows: for α-glycerophosphate, 1.1 × 10−4M; for dihydroxyacetone phosphate, 4.6 × 104M; for DPN, 3.8 × 10−4M. The equilibrium constant for the oxidation of α-glycerophosphate was found to be 5.8 × 10−12M at 25 °C. All the properties studied indicate that within the pH range 7–8 the reaction favors formation of α-glycerophosphate.
The purification and partial resolution of the α-glycerophosphate oxidase of Streptococcus faecalis 10C1 is described. The purified enzyme oxidizes l-α-glycerophosphate, forming hydrogen peroxide and dihydroxyacetone phosphate. Flavine adenine dinucleotide is the prosthetic group. The enzyme has a Km for l-α-glycerophosphate of 4 × 10−3M and a pH optimum of 5.8. In addition to oxygen, ferricyanide and 2,6-dichlorophenolindophenol serve as electron acceptors for the enzyme, whereas methylene blue and cytochrome c are reduced at much slower rates. The purified enzyme is not markedly inhibited by metal-binding agents or sulfhydryl group inhibitors. Azide, acriflavine, and atebrin are inhibitory.
Effects of the substrate on the initial reaction velocity of nicotinamide adenine dinucleotide-linked l-glycerol 3-phosphate dehydrogenase purified from rabbit liver were studied.
The true Michaelis constants for all substrates were determined in 0.1 m tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.5) and were found to be 0.68, 0.19, 0.018, and 0.004 mm for l-glycerol 3-phosphate, nicotinamide adenine dinucleotide, dihydroxyacetone phosphate, and reduced nicotinamide adenine dinucleotide,
respectively. The true Michaelis constants measured in 0.1 m glycine-NaOH buffer (pH 10.0) were found to be 0.065 and 0.03 mm for l-glycerol 3-phosphate and nicotinamide adenine dinucleotide, respectively.
High concentration of all four substrates in the reaction mixture was found to be to some extent inhibitory.
At low concentrations of l-glycerol 3-phosphate or nicotinamide adenine dinucleotide, a plot of initial reaction velocity as a function of l-glycerol 3-phosphate or nicotinamide adenine dinucleotide concentration is sigmoidal. No sigmoidicity is seen in plotting
of initial reaction velocity as a function of either dihydroxyacetone phosphate or reduced nicotinamide adenine dinucleotide
under the conditions used. However, a plot of initial reaction velocity as a function of dihydroxyacetone phosphate concentration
is sigmoidal in the presence of l-glycerol 3-phosphate but is not sigmoidal in the presence of nicotinamide adenine dinucleotide. On the other hand, a plot
of initial reaction velocity as a function of reduced nicotinamide adenine dinucleotide concentration is sigmoidal in the
presence of nicotinamide adenine dinucleotide but is not sigmoidal in the presence of l-glycerol 3-phosphate. The results suggest that there are two allosteric sites, one for l-glycerol 3-phosphate and the other for nicotinamide adenine dinucleotide. The binding of l-glycerol 3-phosphate to its allosteric site lowers the Michaelis constant for l-glycerol 3-phosphate but increases the Michaelis constant for dihydroxyacetone phosphate. Similarly, the binding of nicotinamide
adenine dinucleotide to its allosteric site lowers the Michaelis constant for nicotinamide adenine dinucleotide but increases
the Michaelis constant for reduced nicotinamide adenine dinucleotide. Interestingly, the binding of l-glycerol 3-phosphate to its allosteric site does not change the Michaelis constant for either nicotinamide adenine dinucleotide
or its reduced form. Similarly, the binding of nicotinamide adenine dinucleotide to its allosteric site does not change the
Michaelis constant for either l-glycerol 3-phosphate or dihydroxyacetone phosphate. On the basis of the present study, it is not clear whether dihydroxyacetone
phosphate or reduced nicotinamide adenine dinucleotide will bind to the allosteric site for l-glycerol 3-phosphate or nicotinamide adenine dinucleotide, respectively.
A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM
d-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN3, 10 mM MgCl2, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 μm. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.
A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.
In this study frozen sections of avian striated muscles were incubated for mitochondrial alpha-glycerophosphate de hydrognease (alpha=GPD) reaction, and the effect of menadione, phenazine methosulfate (PMS) or phenazine ethosulfate (PES) as intermediate electron acceptors was evaluated. Under histochemical conditions, PMS or PES-linked alpha-GPD reaction was poor in the chicken posterior latissimus dorsi and chicken pectoralis muscles. However, PMS or PES-linked alpha-GPD reaction was present characteristically in the subsarcolemmal mitochondria of the "broad white" fibres of the pigeon pectoralis muscle only; the subsarcolemmal mitochondria of the narrow red fibres lacked such a reaction pattern. The above reaction pattern, however, differed when compared with the menadione-linked alpha-GPD reaction. The present histochemical evidence suggests the existence of an inherent heterogeneity in the mitochondrial populations of the different avian striated muscle fibres studied.
Die Funktion der Muskelmitochondrien bei der Energieversorgung des Muskels ist in der Abb. 1 illustriert. Die Verbrennung der Substrate durch das Dehydrogenase-system der Endoxydation und durch die Atmungskette führt in der oxydativen Phosphorylierung zur Speicherung der Verbrennungsenergie in der Phosphatbindungsenergie. Das Produkt der oxydativen Phosphorylierung ATP steht mit dem Kreatinsystem in Verbindung. Beide, das ATP und Kreatinphosphat, dienen zum Transport der Phosphatenergie von den Mitochondrien zum Verbrauch im extramitochondrialen Raum. Die Adeninnucleotide sind damit Transportmetabolite der Phosphatübertragung.
Coenzyme Q (CoQ, ubiquinone) is the electron‐carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH‐dehydrogenase (complex I, CI) and succinate dehydrogenase (complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron‐transferring flavoprotein dehydrogenase, mitochondrial glycerol‐3‐phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ‐linked metabolism depends on CoQ re‐oxidation by the mitochondrial complex III (cytochrome bc1 complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ‐linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ‐dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation.
The paper focuses on intermolecular interactions, particularly interactions between proteins and natural intermediates (small molecules). Molecules with a molecular weight of up to 1000 Da are free in cytoplasmic solution and form a pool of intermediates. Methods of computer modeling for prediction of protein-proteinaceous, protein-ligand, protein - a small molecule of interactions are presented. The program for modeling predicted biological activity in silico is Prediction of Activity Spectrum for Substances (PASS). In the Search Tool for Interacting Chemicals (STITCH) system, it is possible to identify potential protein interaction partners for small molecules. A review of the literature presents modern data on small molecules - metabolic switches, such as α-glycerophosphatedihydroxyacetone phosphate, pyruvate-lactate, oxaloacetate-malate. The molecules we study have different and multiple effects on metabolism and on intercellular interaction systems. Natural intermediates are at the intersection of metabolic pathways of metabolism of proteins, carbohydrates, lipids; they are signal molecules, participate in regulation of protein function, gene expression, enzyme activity. An increasing interest in deciphering protein-small molecule/metabolite interactions at the systemic level will lay a conceptual foundation that provides insight into complex epigenetic regulation under various environmental influences. A complete interplay, including a protein-small molecule interaction, will be crucial to eventually unraveling the complex relationships between the genotype and phenotype and to provide a deeper understanding of health and disease.
In diesem Kapitel werden wasserstoff-übertragende Enzyme besprochen, bei welchen die Substrat-spezifität insoweit nur teilweise bekannt ist, als wir wohl entweder die unter physiologischen Bedingungen fungierenden Donator-substrate oder in anderen Fällen die Akzeptor-substrate kennen, während wir über die entsprechenden Wasserstoff-akzeptoren bzw. -donatoren noch keine Klarheit haben. Wasserstoff-übertragende Enzyme, deren Akzeptor-spezifität noch unbekannt ist, werden hier als Dehydrogenasen bezeichnet, während bei unbekannter Donator-spezifität der Name Reductasen verwendet wird.
Die Fructose (oder nach der älteren Bezeichnung Lävulose) gehört zu den am längsten bekannten Zuckern. Sie wurde erstmals 1847 von Dubrunfaut
23 aus invertierten Rohrzuckerlösungen als Ca-Verbindung isoliert, und es zeigte sich in der Folge, daß dieser Zucker im Pflanzenreich außerordentlich weit verbreitet ist. Viel später (1885) wies Kiliani
52 mit Hilfe der Cyanhydrin- synthese nach, daß es sich um einen Ketozucker handelt. Für die Tierphysiologen und Mediziner hatte die Lävulose zunächst ausschließlich als Nährstoff Interesse. Sie kann sich in Nahrungsmitteln in freier Form, als Saccharose oder Oligosaccharid vorfinden*. In unserer Nahrung dürften die künstlich mit Rohrzucker gesüßten Speisen und Getränke die wichtigste Quelle darstellen. Als erster hat wohl PflüGer (1908) den einwandfreien Beweis geleistet, daß im tierischen Organismus Fructose in Glucose übergeht („Über die Fähigkeit der Leber, die Circularpolarisation zugeführter Zuckerstoffe umzukehren“)78. Er fütterte Hunde mit einer völlig kohlenhydratfreien Nahrung, der er reine Fructose zulegte. Die Hydrolyse des während der Fütterungsperiode deponierten Leberglykogens ergab polarimetrisch reine Glucose. Über den Weg, der zu dieser Umwandlung führt, konnten allerdings erst die späteren Forschungen Anhaltspunkte geben.
Es soll im folgenden nachzuweisen versucht werden, daß zwischen dem Gehirn und der Leber tatsächlich Beziehungen bestehen. Zunächst seien die klinischen Tatsachen vorgetragen, die diese Beziehung beweisen.
Energy required for vital function whether in the animal, the bacterium or the higher plant is obtained by burning fuel. In most plant tissues the fuel is sugar or one of its storage products (either di- or polysaccharides). In other instances it is fat, and in a few plants, protein storage products may be burned to provide energy. The energy available in these molecules, in the form of bond energy, is derived from the sun either directly or indirectly. For this energy to become available to the cell, a series of transformations must occur which, in the case of the sugars and their related products, and for at least part of the fat molecule, involves the formation of phosphorylated compounds. In short, if substrates are to be used as energy sources they must first undergo phosphorylation. This appears to be true for almost all instances of sugar oxidation. There are, however, a few isolated cases in some animal tissues and especially in bacteria where prior phosphorylation may not be required.
Between 1940 and 1970, pioneers in the new field of cell biology discovered the operative parts of cells and their contributions to cell life. Cell biology was a revolutionary science in its own right, but in this book, it also provides fuel for yet another revolution, one that focuses on the very conception of science itself. Laws have traditionally been regarded as the primary vehicle of explanation, but in the emerging philosophy of science it is mechanisms that do the explanatory work. William Bechtel emphasizes how mechanisms were discovered by cell biologists.
The present chapter discusses metal-containing flavoprotein dehydrogenases. Flavoproteins are involved in a large variety of key metabolic reactions in all forms of life. They catalyze over a potential span of several hundred millivolts oxidation–reduction reactions involving alkanes, alkenes, alcohols, aldehydes, ketones, inorganic and organic acids, amines, thiols, disulfides, quinones, nicotinamide-adenine dinucleotides, purines, pyrimidines, pteridines, and transition metal complexes. They can also catalyze one- and two-electron reduction of molecular oxygen. Many flavoproteins contain metal such as iron, molybdenum, and zinc. The combination of flavin and metal often serves to adjust electron transfer between single-electron and double-electron donors and acceptors. Multiple-electron reduction of an acceptor without detectable loss of intermediates is achieved by the device of having multiple flavins and metals in the same enzyme molecule. The chapter discusses the enzymes that are respiratory chain-linked NADH dehydrogenases, succinate dehydrogenases, L-glycerol-3-phosphate dehydrogenase, choline dehydrogenase, and L (+)-lactate.
This chapter discusses the transphosphorylation by phosphatases. The group-specific acid and alkaline phosphatases catalyze a direct transfer of the phosphate group from suitable “donors” (substrates) to certain "acceptors" to form new phosphate esters. The method of assay of transphosphorylation activity described is that of Morton. A donor (substrate) chosen so that the liberated molecule may be readily estimated is used at a high concentration and at the optimal pH for transfer (and hydrolysis). The percentage transfer is independent of the donor concentration, but the optimal pH for hydrolysis is a function of donor concentration. Hence the pH used is markedly suboptimal for hydrolysis of the ester formed by phosphate transfer. A specific enzymic method is employed for estimation of the synthesized ester that should always be less than 10–4 M concentration at the completion of the reaction. In this way the true initial rate of transphosphorylation is measured. The acceptor is used at the optimal concentration for transphosphorylation. The concomitant hydrolysis is measured by liberated inorganic P. The chapter also discusses the application of assay method to crude tissue preparations.
1. The "indirect" thoracic muscles of adult dipterous and hymenopterous insects consist of a unique type of muscle characterized by the presence of numerous spherical, intracytoplasmic bodies termed "sarcosomes." 2. When the muscle is teased or ground, the sarcosomes are liberated as a turbid suspension of bodies ranging from 1 to 4 µ in diameter. A method is described for the isolation of sarcosomes by a simple differential centrifugation. 3. The cytochemical, chemical, and enzymatic properties of sarcosomes were examined for the purpose of appraising their relation to the cytoplasmic bodies of other tissues. 4. Fresh sarcosomes are slowly but selectively stained by the mitochondrial reagents, Janus green B and pinacyanol. Fixed sarcosomes give a positive reaction with Regaud's mitochondrial stain. 5. Chemical analyses show that approximately 29 per cent of the dry weight of sarcosomes consists of lipids and 60 per cent of protein. Microbiological assay indicates the presence of about 1 gamma of riboflavin per milligram of nitrogen. These values resemble those reported for isolated mitochondria of vertebrate liver and kidney. 6. When examined spectroscopically the sarcosomes, like the vertebrate mitochondria, show a high titer of cytochromes a, b, and c. 7. The titer of cytochrome oxidase varies systematically with the adult age of the insect. A similar relation is observed for the enzyme catalase. 8. Isolated sarcosomes show significant titers of succinoxidase, α-glycerophosphate dehydrogenase, malic dehydrogenase, and pyruvic dehydrogenase. The following dehydrogenases could not be demonstrated: xanthine, phenylalanine, glycine, lactic, choline, glutamic, and alcohol. These results are compared with those previously reported for vertebrate mitochondria. 9. In view of their manifold points of biochemical similarity, it is concluded that the sarcosomes are the mitochondria of this highly specialized muscular tissue.
Kinetic studies of respiratory enzymes in mammalian brain showed that greatest respiratory activity of rat brain mitochondria was with α-glycerophosphate. α-Glycerophosphate was oxidized at a rate 50% greater than that obtained with succinate and several fold those obtained with other Krebs cycle intermediates. The oxidation of α-glycerophosphate was inhibited by ethylenediamine tetraacetate (Versene). Mg ions completely reversed this inhibition. The data suggest that the extraordinary rapid rate of α-glycerophosphate oxidation must be considered in the evaluation of brain metabolism.
Conidien von Fusarium decemcellulare (Brick) wurden verwendet, um das Wirken von anionoberflächenaktiven Substanzen auf die Metallaufnahme und das Keimvermögen metallbehandelter Pilzsporen zu prüfen. Die Untersuchungen haben gezeigt, daß die Sporen durch bestimmte anionaktive Stoffe (Na-laurylsulfat) mehr Metall aufnehmen und das Metall in der Spore durch Komplexbildner leichter sichtbar wird. Äthylurethan verhielt sich in seinem Wirken wie eine anionaktive Substanz und nicht wie ein spezifischer Enzymhemmstoff der Reduktasen.
The metabolism of glycerol and pyruvate, singly or together, in rat kidney slices in vitro has been studied under aerobic and anaerobic conditions.The aerobic data were in substantial agreement with the results obtained in similar experiments with rat liver slices which have been reported previously.The anaerobic results provided suggestive evidence that the dismutation of pyruvate furnished high-energy phosphates and hydrogen acceptors for the metabolism of glycerol. A possible explanation has been discussed.
1.1. In an attempt to clarify the structure of the active center of α-glycerol-3-phosphate dehydrogenase (EC 188.8.131.52), the behavior of the kinetics parameters of the enzyme as a function of pH was examined.2.2. The values of 6.61 found for the of the free enzyme and 6.72 for the of the enzyme-substrate complex suggested the presence of atleast one histidyl residue at the active center.3.3. Photo-oxidation of the enzyme in the presence of Rose Bengal as sensitizer inactivated the enzyme. This inactivation was dependent on the destruction of a group or groups having a of 184.108.40.206. Chemical modification of the enzyme with diazo-i-H-tetrazole, under conditions where neither bisazohistidine nor monoazo- or bisazotyrosine could be detected, showed that the conversion of 1.07 histidine to the monoazo product led to a 50% inactivation. When 1.77 monoazohistidine residues had been formed, the level of inactivation was 94%. Complete inactivation was attained when 2.14 histidines had been converted to the monoazoderivative.5.5. The results reported here strongly suggest that two histidine residues at the active center of the α-glycerolphosphate dehydrogenase are critical for the activity of this enzyme.
We isolated l-glycerol 3-phosphate: NAD oxidoreductase (EC 220.127.116.11) from the cytoplasm of rat skeletal muscle. The crystalline enzyme was homogeneous as judged by electrophoresis at pH 5.4, 7.0, and 8.6, by amino acid analysis, and by ion exchange column chromatography. It was free of at least high molecular weight contaminants on ultracentrifugation. The purest enzyme fraction obtained had a specific activity of 263 units per mg of protein, corresponding to the ability to catalyze the oxidation of 126 µmoles of NADH per min per mg of protein, under assay conditions in which the enzyme was not saturated with substrate. The corresponding molecular activity was 7,380 molecules of NADH oxidized per min per molecule of enzyme of molecular weight 58,300. The molar extinction coefficient at 280 mµ was 0.38 × 10⁵m⁻¹ cm⁻¹.
Amino acid analysis based on milligrams of protein hydrolyzed or on assumed unit residues showed 3 tyrosine, 2 tryptophan, 8 arginine, 9 cysteine or half-cystine, and 28 lysine plus arginine residues per minimal molecular weight, which was 29,150. Molecular weight estimation by gel filtration gave 63,000. Peptide mapping of the ¹⁴C-S-carboxymethylated enzyme showed 3 tyrosine, 1 or 2 tryptophan, 7 arginine, 7 to 11 ¹⁴C-S-carboxymethylcysteine, and 29 tryptic peptides. These chemical studies show that the enzyme has a molecular weight of 58,300 and is composed of two subunits that are indistinguishable by peptide mapping.
This molecular weight is not consistent with the accepted value for the crystalline rabbit muscle enzyme, even though these two enzymes are closely related in their electrophoretic properties, extinction coefficients expressed per gram, kinetic properties, ratios of amino acids, and, presumably, genetic origins. The molecular weight of the rat muscle enzyme is closer to that of the enzyme crystallized from bee thoraces (65,400).
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