Inactivation of metabolic enzymes by photo-treatment with zinc meta N-methylpyridylporphyrin

Cell proliferation is notably dependent on energy supply and generation of reducing equivalents in the form of NADPH for reductive biosynthesis. Blockage of pathways generating energy and reducing equivalents has proved successful for cancer treatment. We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP4+) can act as photosensitizers, preventing cell proliferation and causing cell death in vitro. The present study demonstrates that upon illumination, ZnTM-3-PyP inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, NADP+ -linked isocitrate dehydrogenase, aconitase, and fumarase in adenocarcinoma LS174T cells. ZnTM-3-PyP4+ was significantly more effective than hematoporphyrin derivative (HpD) for inactivation of all enzymes, except aconitase and isocitrate dehydrogenase. Enzyme inactivation was accompanied by aggregation, presumably due to protein cross-linking of some of the enzymes tested. Inactivation of metabolic enzymes caused disruption of cancer cells' metabolism and is likely to be one of the major reasons for antiproliferative activity of ZnTM-3-PyP.     

Effect of lipophilicity of Mn (III) ortho N-alkylpyridyl- and diortho N,N′-diethylimidazolylporphyrins in two in-vitro models of oxygen and glucose deprivation-induced neuronal death

In vivo investigations have confirmed the beneficial effects of hydrophilic, cationic Mn(III) porphyrin-based catalytic antioxidants in different models of oxidative stress. Using a cell culture model of rat mixed neuronal/glial cells, this study investigated the effect of MnTnOct-2-PyP⁵⁺ on oxygen and glucose deprivation (OGD)-induced cell death as compared to the effects of widely studied hydrophilic analogues MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ and a standard compound, dizocilpine (MK-801). It was hypothesized that the octylpyridylporphyrin, MnTnOct-2-PyP⁵⁺, a lipophilic but equally potent antioxidant as the other two porphyrins, would be more efficacious in reducing OGD-induced cell death due to its higher bioavailability. Cell death was evaluated at 24 h using lactate dehydrogenase (LDH) release and propidium iodide staining. At concentrations from 3–100 µm, all three porphyrins reduced cell death as compared to cultures exposed to OGD alone, the effects depending upon the concentrations and type of treatment. To assess the effect of lipophilicity the additional experiments were performed using submicromolar concentrations of MnTnOct-2-PyP⁵⁺ in an organotypic hippocampal slice model of OGD with propidium iodide and Sytox staining. When compared to oxygen and glucose deprivation alone, concentrations of MnTnOct-2-PyP⁵⁺ as low as 0.01 µm significantly (p<0.001; power 1.0) reduced neuronal cells similar to control. This is the first in vitro study on the mammalian cells which indicates that MnTnOct-2-PyP⁵⁺ is up to 3000-fold more efficacious than equally potent hydrophilic analogues, due entirely to its increased bioavailability. Such remarkable increase in efficacy parallels 5.7-orders of magnitude increase in lipophilicity of MnTnOct-2-PyP⁵⁺ (log P=?0.77) when compared to MnTE-2-PyP⁵⁺ (log P_ow=?6.43), P_ow being partition coefficient between n-octanol and water.     

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant,ascorbate

Abstract

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O₂ ^·?, reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP⁵⁺, MnTnHex-2-PyP⁵⁺, and a meta isomer MnTnHex-3-PyP⁵⁺, which differ greatly with regard to their metal-centered reduction potential, E_1/2 (Mn^IIIP/Mn^IIP) and lipophilicity, were explored. Employing Mn^IIIP/Mn^IIP redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP⁵⁺ was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP⁵⁺ is most prone to oxidative degradation with H₂ , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.     

Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP⁺ was characterized in aqueous solutions at 36–47°C under treatment with low frequency (27 kHz, 60 W/cm²) and high frequency ultrasound (880 kHz, 1 W/cm²). To this end, we measured three effective first-order inactivation rate constants: thermal k _in ^* , total (thermal and ultrasonic) k _in, and ultrasonic k _in(US). The values of the constants were found to be higher for the free enzyme than for its complexes GPDH-GP and GPDH-NADP⁺ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (E _a) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (ΔH*, ΔS*, and ΔG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of E _a and ΔH* values in comparison with the corresponding values for thermal inactivation. The E _a, ΔH*, and ΔS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the ΔH* and ΔS* values was observed for all the inactivation processes for free GPDH and its complexes.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

IDH1 mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain

Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP + to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1^R132 or the homologous IDH2^R172. Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1^R132 and IDH2^R172 mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1^R132 and IDH2^R172 mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1^R132 or IDH2^R172 may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.     

Sucrose controls storage lipid breakdown on gene expression level in germinating yellow lupine (Lupinus luteus L.) seeds

This study revealed that cytosolic aconitase (ACO, EC 4.2.1.3) and isocitrate lyase (ICL, EC 4.1.3.1, marker of the glyoxylate cycle) are active in germinating protein seeds of yellow lupine. The glyoxylate cycle seems to function not only in the storage tissues of food-storage organs, but also in embryonic tissue of growing embryo axes. Sucrose (60 mM) added to the medium of in vitro culture of embryo axes and cotyledons decreased activity of lipase (LIP, EC 3.1.1.3) and activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). The opposite effect was caused by sucrose on activity of cytosolic ACO, ICL as well as NADP⁺-dependent (EC 1.1.1.42) and NAD⁺-dependent (EC 1.1.1.41) isocitrate dehydrogenase (NADP-IDH and NAD-IDH, respectively); activity of these enzymes was clearly stimulated by sucrose. Changes in the activity of LIP, ACO, NADP-IDH, and NAD-IDH caused by sucrose were based on modifications in gene expression because corresponding changes in the enzyme activities and in the mRNA levels were observed. The significance of cytosolic ACO and NADP-IDH in carbon flow from storage lipid to amino acids, as well as the peculiar features of storage lipid breakdown during germination of lupine seeds are discussed.     

The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key events in the development of glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma (ICC), and angioimmunoblastic T-cell lymphoma. They also cause D-2-hydroxyglutaric aciduria and Ollier and Maffucci syndromes. IDH1/2 mutations are associated with prolonged survival in glioma and in ICC, but not in AML. The reason for this is unknown. In their wild-type forms, IDH1 and IDH2 convert isocitrate and NADP⁺ to α-ketoglutarate (αKG) and NADPH. Missense mutations in the active sites of these enzymes induce a neo-enzymatic reaction wherein NADPH reduces αKG to D-2-hydroxyglutarate (D-2HG). The resulting D-2HG accumulation leads to hypoxia-inducible factor 1α degradation, and changes in epigenetics and extracellular matrix homeostasis. Such mutations also imply less NADPH production capacity. Each of these effects could play a role in cancer formation. Here, we provide an overview of the literature and discuss which downstream molecular effects are likely to be the drivers of the oncogenic and survival-prolonging properties of IDH1/2 mutations. We discuss interactions between mutant IDH1/2 inhibitors and conventional therapies. Understanding of the biochemical consequences of IDH1/2 mutations in oncogenesis and survival prolongation will yield valuable information for rational therapy design: it will tell us which oncogenic processes should be blocked and which “survivalogenic” effects should be retained.     

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via trans-hydrogenation of cytosolic NADPH into mitochondrial FADH₂ with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD⁺ and NADP⁺ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P₂ (used as source of dihydroxyacetone phosphate) and NADP⁺; the addition of citratein vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P₂ and NADP⁺.     

SOD-like activity of Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.     

Mn porphyrin-based SOD mimic,MnTnHex-2-PyP5+, and non-SOD mimic,MnTBAP3−, suppressed rat spinal cord ischemia/reperfusion injury via NF-κB pathways

Abstract

Herein we have demonstrated that both superoxide dismutase (SOD) mimic, cationic Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP⁵⁺), and non-SOD mimic, anionic Mn(III) meso-tetrakis(4-carboxylatophenyl)porphyrin (MnTBAP^3?), protect against oxidative stress caused by spinal cord ischemia/reperfusion via suppression of nuclear factor kappa B (NF-κB) pro-inflammatory pathways. Earlier reports showed that Mn(III) N-alkylpyridylporphyrins were able to prevent the DNA binding of NF-κB in an aqueous system, whereas MnTBAP^3? was not. Here, for the first time, in a complex in vivo system—animal model of spinal cord injury—a similar impact of MnTBAP^3?, at a dose identical to that of MnTnHex-2-PyP⁵⁺, was demonstrated in NF-κB downregulation. Rats were treated subcutaneously at 1.5 mg/kg starting at 30 min before ischemia/reperfusion, and then every 12 h afterward for either 48 h or 7 days. The anti-inflammatory effects of both Mn porphyrins (MnPs) were demonstrated in the spinal cord tissue at both 48 h and 7 days. The downregulation of NF-κB, a major pro-inflammatory signaling protein regulating astrocyte activation, was detected and found to correlate well with the suppression of astrogliosis (as glial fibrillary acidic protein) by both MnPs. The markers of oxidative stress, lipid peroxidation and protein carbonyl formation, were significantly reduced by MnPs. The favorable impact of both MnPs on motor neurons (Tarlov score and inclined plane test) was assessed. No major changes in glutathione peroxidase- and SOD-like activities were demonstrated, which implies that none of the MnPs acted as SOD mimic. Increasing amount of data on the reactivity of MnTBAP^3? with reactive nitrogen species (RNS) (.NO/HNO/ONOO^?) suggests that RNS/MnTBAP^3?-driven modification of NF-κB protein cysteines may be involved in its therapeutic effects. This differs from the therapeutic efficacy of MnTnHex-2-PyP⁵⁺ which presumably occurs via reactive oxygen species and relates to NF-κB thiol oxidation; the role of RNS cannot be excluded.     

Changes in insulin receptor,hexokinase and NADPH producing enzymes in Choroid plexus during experimental diabetes

The activities of insulin receptor and the enzymes hexokinase (EC 2.7.1.1) and NADP-dependent malic enzyme (EC1.1.1.40), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in rat choroid plexus in alloxan induced diabetes. A significant decrease was observed in the activities of all the enzymes except isocitrate dehydrogenase and also the choroid plexus insulin receptor activity was decreased. A reversal of the efect was observed with insulin administration to diabetic rats. It may be concluded that the enzymes of choroid plexus together with insulin receptor are directly controlled by-the concentration of insulin.     

Nickel-induced changes in carbon metabolism in wheat shoots

In this study, we analyzed the toxic effect of Ni during the development of wheat shoots. Typical developmental alterations in carbon metabolism-related parameters reflecting changes associated with the transition of the seedlings from heterotrophic to autotrophic metabolism were observed in the control shoots between the 1st and the 4th days. Adverse effects of 50 and 100 μM Ni became evident starting from the 4th day of growth on the metal-containing media. We found that Ni-induced stimulation of phosphoenolpyruvate carboxylase (PEPC) activity coincided with decrease in the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) level and with declines in net photosynthetic rate (P_N) and stomatal conductance (g_s). Application of Ni resulted in increased activities of several dehydrogenases: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (NADP-ICDH) and malate dehydrogenase (NADH-MDH). In contrast, the activities of malic enzymes (NADP-ME and NAD-ME) decreased due to Ni stress. Treatment with Ni led to accumulation of glucose and declined concentration of sucrose as well as considerable increases in concentrations of malic and citric acids. Our results indicate that Ni stress redirects the carbon metabolism of developing wheat shoots to provide carbon skeletons for synthesis of amino acids and organic acids as well as to supply reducing power to sustain normal metabolic processes and to support defense mechanisms against oxidative stress.     

The topology of superoxide production by complex III and glycerol 3-phosphate dehydrogenase in Drosophila mitochondria

The topology of superoxide generation by sn-glycerol 3-phosphate dehydrogenase and complex III in intact Drosophila mitochondria was studied using aconitase inactivation to measure superoxide production in the matrix, and hydrogen peroxide formation in the presence of superoxide dismutase to measure superoxide production from both sides of the membrane. Aconitase inactivation was calibrated using the known rate of matrix superoxide production from complex I. Glycerol phosphate dehydrogenase generated superoxide about equally to each side of the membrane, whereas centre o of complex III in the presence of antimycin A generated superoxide about 30% on the cytosolic side and 70% on the matrix side.     

Effect of Cadmium on Activities of Some Enzymes of Glycolysis and Pentose Phosphate Pathway in Pea

Activities of alcohol dehydrogenase, hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were significantly inhibited by cadmium in germinating pea (Pisum sativum L. cv. Bonneville) seeds. The effect was concentration dependent in the range of 0.25 to 1.0 mM CdCl₂. The magnitude of detrimental effect on these enzymes was reduced during later stage of germination (9 d) largely because of fall in the activities of these enzymes in the control seeds germinated in water. In vitro, activities of hexokinase, glucose-6-phosphate dehydrogenase, and alcohol dehydrogenase were inhibited at 0.5 mM Cd²⁺ in the reaction mixture by 62, 67, and 36 %, respectively, however, 6-phosphogluconate dehydrogenase was insensitive to Cd²⁺. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of the sodium/potassium ratio on glyceraldehyde-3-phosphate dehydrogenase interaction with red cell vesicles

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the ³¹P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K. (1977) Biochim. Biophys. Acta 464, 82–92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation change induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na⁺ + K⁺)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew ((1970) J. Physiol. London 207, 393–402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by ³¹P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na⁺ + K⁺)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.