Low Levels Of NAD Research Articles

Abstract TP36: Angiotensin II Induced Hypertension Increases the Brain Expression and Enzymatic Activity of CD38

Introduction: CD38 is an ectoenzyme that was shown to mediate the age-dependent decrease in NAD + levels. We have shown that CD38 expression and enzymatic activity are increased in the brain of spontaneously hypertensive stroke-prone rats, a model of human cerebral small vessel disease. Therefore, we hypothesized that hypertension (HTN) induced by angiotensin II (ANG-II) infusion leads to an increase in CD38 expression and enzymatic activity. Methods: Male C57BL/6J mice (10-13) weeks of age underwent subcutaneous infusion of ANG-II (600 ng/kg/min) or saline for a 2-week duration through implanted osmotic pumps. CD38 expression was measured by immunofluorescence and Western Blotting. CD38 enzymatic activity was assessed in the brain homogenate by measuring the NAD + analogue conversion to the fluorescent product (ε-ADPR). Dihydroethidium, diaminofluorescein, JZL1707 sensor, and immunohistochemistry were used to measure the levels of superoxide, nitric oxide (NO), NAD(P)H, and endothelial nitric oxide synthase (eNOS) in the brain, respectively. Results: Ang-II infusion caused a slow rise of blood pressure leading to HTN. The brain of ANG-II infused mice showed 48% increase in the expression (P=0.013) and 73% increase in enzymatic activity (P=0.044) of CD38 compared to control mice. The expression of CD38 localized mainly to the endothelial cells in the ANG-II infused mice brains. The brains of ANG-II infused mice also had 26% lower levels of NAD(P)H (P<0.0001), 226% higher superoxide (P<0.0001), 32% lower NO (P<0.0001) and 30% lower eNOS levels (P=0.003) compared to control mice. Conclusions: ANG-II induced HTN increases CD38 expression and enzymatic activity with oxidative stress and lower levels of NAD(P)H. These results suggest a potential role of elevated CD38 enzymatic activity in the pathogenesis of cerebrovascular dysfunction induced by ANG-II as well as that seen in other models of HTN.

Propyl gallate postharvest treatment improves the storability of longans by regulating the metabolisms of respiratory and disease-resistance substances

The influences of 0.5 mM propyl gallate, a ROS scavenger, on the metabolisms of respiratory and disease-resistance substances in ‘Fuyan’ longan fruit during storage at 15 °C were explored. Compared to the control longan, propyl gallate-treated longan displayed a higher rate of commercially acceptable fruit, the lower indices of pulp breakdown and fruit disease, and a lower rate of fruit weight loss. Additionally, propyl gallate-treated longan exhibited a lower rate of fruit respiration, the lower activities of SDH, PGI, CCO, AAO, PPO and AOX, and the lower levels of NAD and NADH, but the higher activities of NADK and G-6-PDH + 6-PGDH, and the higher levels of NADP and NADPH. Furthermore, propyl gallate-treated longan manifested the higher activities of CHI, GLU, PAL, POD, CAD, C4H and 4-CL, and the higher contents of lignin. These data indicated that, propyl gallate-improved the storability of longan was related to the fact that propyl gallate reduced respiration and increased disease resistance of longan through decreasing the respiratory pathways of EMP and TCA cycle, and raising PPP respiratory pathway, as well as through increasing the metabolism of disease-resistance substances. Above findings suggest that 0.5 mM propyl gallate has potential application in prolonging the shelf-life and restraining the fruit spoilage of postharvest fresh longan.

NAMPT INHIBITION AND INCREASED NAD-BIOAVAILABILITY ATENUATE LIVER DAMAGE IN CCl4-INDUCED MICE CHRONIC LIVER DISEASE

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme on the NAD + salvage biosynthetic pathway and a cytokine regulator with an important role in inflammation and fibrogenesis modulation. Use of FK866 (NAMPT inhibitor) has been proposed as a treatment on inflammatory diseases and cancer. However, FK866-induced depletion of NAD may also cause major impairment of the redox and bioenergetics homeostasis of the cell within the liver, thus limiting a favorable outcome for Chronic Liver Disease (CLD), as low NAD levels have been associated with higher Oxidative Stress and increased metabolic risk. The aim of this study was to evaluate the effects of NAMPT inhibition and concomitant NAD restoration on experimental CLD in vivo. NAMPT inhibition was evaluated within a CCl 4 -induced CLD model on male BALB/c mice and a mild improved outcome was observed on the histological and biochemical features. NAD restoration strategy was accomplished by the concomitant administration of its precursor, NMN, resulting in significant improve on the histological analysis; lower inflammatory infiltrate and fibrosis were measured by image analysis on digitalized micrographies. Lower levels of Direct Bilirubin were also observed. NAMPT inhibition and adequate NAD restoration were confirmed by a colorimetric assay of NADH and NAD + and biochemical features were measured by routinary Liver Function Tests. Silymarin was used as a hepatoprotective control. This study shows that NAMPT inhibition concomitant to NAD restoration significantly attenuate experimental liver damage. The authors declare that there is no conflict of interest.

Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators

NAD+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

Abstract 1164: A novel hypoxia-inducible factor-1 inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth

Abstract HIF-1 is associated with poor patient prognosis and therapeutic resistance of cancer. We have developed a novel hypoxia-inducible factor (HIF)-1 inhibitor, IDF-11774, as a clinical candidate for cancer therapy. Under hypoxic condition, IDF-11774 inhibited the accumulation of HIF-1α in vitro and in vivo in colorectal carcinoma HCT116 cells. IDF-11774 suppressed the angiogenesis of cancer cells by reducing the expression of HIF-1 target genes. Moreover, IDF-11774 reduced glucose uptake, leading sensitizing cell growth on low glucose condition. IDF-11774 also decreased the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Metabolic profile of IDF-11774-treated cells revealed low levels of NAD+, lactate, and intermediates in glycolysis and tricarboxylic acid (TCA) cycle. More importantly, we observed elevated AMP and diminished ATP level, leading high AMP/ATP ratio. Apparently, phosphorylation of AMPK increased, leading inhibition of mTOR signaling in cells. In vivo xenograft assays demonstrated that IDF11774 has significant anti-cancer efficacy by targeting cancer metabolism in mouse models containing the KRAS, PTEN or VHL mutation, which often occurs in many malignant cancers. Collectively, IDF-11774 suppresses the hypoxia-induced HIF-1α accumulation, thereby repressing tumor growth by regulating cancer metabolism. Citation Format: Misun Won, Hyun Seung Ban, Kyeong Lee, Hongsub Lee, Bo-Kyung Kim, Hwan Mook Kim, Ravi Naik, Song-Kyu Park, Joon-Tae Park, Inhyub Kim, Miso Nam, Geum-Sook Hwang. A novel hypoxia-inducible factor-1 inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1164. doi:10.1158/1538-7445.AM2017-1164

The novel hypoxia-inducible factor-1\u03b1 inhibitor IDF-11774 regulates cancer metabolism, thereby suppressing tumor growth

HIF-1 is associated with poor prognoses and therapeutic resistance in cancer patients. We previously developed a novel hypoxia-inducible factor (HIF)-1 inhibitor, IDF-11774, a clinical candidate for cancer therapy. We also reported that IDF-1174 inhibited HSP70 chaperone activity and suppressed accumulation of HIF-1α. In this study, IDF-11774 inhibited the accumulation of HIF-1α in vitro and in vivo in colorectal carcinoma HCT116 cells under hypoxic conditions. Moreover, IDF-11774 treatment suppressed angiogenesis of cancer cells by reducing the expression of HIF-1 target genes, reduced glucose uptake, thereby sensitizing cells to growth under low glucose conditions, and decreased the extracellular acidification rate (ECAR) and oxygen consumption rate of cancer cells. Metabolic profiling of IDF-11774-treated cells revealed low levels of NAD+, NADP+, and lactate, as well as of intermediates in glycolysis and the tricarboxylic acid cycle. In addition, we observed elevated AMP and diminished ATP levels, resulting in a high AMP/ATP ratio. The level of AMP-activated protein kinase phosphorylation also increased, leading to inhibition of mTOR signaling in treated cells. In vivo xenograft assays demonstrated that IDF-11774 exhibited substantial anticancer efficacy in mouse models containing KRAS, PTEN, or VHL mutations, which often occur in malignant cancers. Collectively, our data indicate that IDF-11774 suppressed hypoxia-induced HIF-1α accumulation and repressed tumor growth by targeting energy production-related cancer metabolism.

The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase

A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, Kd < 3 μM) over saturated and unsaturated fatty acids. Unusually for a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for all the substrates tested though product formation was observed for many. For nearly all substrates the preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with certain reactions proceeding at ≥ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty acid hydroxylation close to the ω-terminus.

Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles

BackgroundTwo self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified. MethodsPhylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system. ResultsKrac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively. ConclusionKrac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids. General significanceIn this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity.

Mechanisms involved in the inhibition of glycolysis by cyanide and antimycin A in Candida albicans and its reversal by hydrogen peroxide. A common feature in Candida species.

In Candida albicans, cyanide and antimycin A inhibited K(+) transport, not only with ethanol-O2 as the substrate, but also with glucose. The reason for this was that they inhibited not only respiration, but also fermentation, decreasing ATP production. Measurements of oxygen levels in cell suspensions allowed identification of the electron pathways involved. NADH fluorescence levels increased in the presence of the inhibitors, indirectly indicating lower levels of NAD(+) and so pointing to glyceraldehyde-3-phosphate dehydrogenase as the limiting step responsible for the inhibition of glycolysis, which was confirmed by the levels of glycolytic intermediaries. The cyanide effect could be reversed by hydrogen peroxide, mainly due to an activity by which H2O2 can be reduced by electrons flowing from NADH through a pathway that can be inhibited by antimycin A, and appears to be a cytochrome c peroxidase. Therefore, the inhibition of glycolysis by the respiratory inhibitors seems to be due to the decreased availability of NAD(+), resulting in a decreased activity of glyceraldehyde-3-phosphate dehydrogenase. Compartmentalization of pyridine nucleotides in favor of the mitochondria can contribute to explaining the low fermentation capacity of C. albicans. Similar results were obtained with three C. albicans strains, Candida dubliniensis and, to a lower degree, Candida parapsilosis.

Changes in Oxidative Damage, Inflammation and [NAD(H)] with Age in Cerebrospinal Fluid

An extensive body of evidence indicates that oxidative stress and inflammation play a central role in the degenerative changes of systemic tissues in aging. However a comparatively limited amount of data is available to verify whether these processes also contribute to normal aging within the brain. High levels of oxidative damage results in key cellular changes including a reduction in available nicotinamide adenine dinucleotide (NAD+), an essential molecule required for a number of vital cellular processes including DNA repair, immune signaling and epigenetic processing. In this study we quantified changes in [NAD(H)] and markers of inflammation and oxidative damage (F2-isoprostanes, 8-OHdG, total antioxidant capacity) in the cerebrospinal fluid (CSF) of healthy humans across a wide age range (24–91 years). CSF was collected from consenting patients who required a spinal tap for the administration of anesthetic. CSF of participants aged >45 years was found to contain increased levels of lipid peroxidation (F2-isoprostanes) (p = 0.04) and inflammation (IL-6) (p = 0.00) and decreased levels of both total antioxidant capacity (p = 0.00) and NAD(H) (p = 0.05), compared to their younger counterparts. A positive association was also observed between plasma [NAD(H)] and CSF NAD(H) levels (p = 0.03). Further analysis of the data identified a relationship between alcohol intake and CSF [NAD(H)] and markers of inflammation. The CSF of participants who consumed >1 standard drink of alcohol per day contained lower levels of NAD(H) compared to those who consumed no alcohol (p<0.05). An increase in CSF IL-6 was observed in participants who reported drinking >0–1 (p<0.05) and >1 (p<0.05) standard alcoholic drinks per day compared to those who did not drink alcohol. Taken together these data suggest a progressive age associated increase in oxidative damage, inflammation and reduced [NAD(H)] in the brain which may be exacerbated by alcohol intake.

Comparative Analysis of the Mitochondrial Physiology of Pancreatic β Cells.

The mitochondrial metabolism of β cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/β cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/β cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the β cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized β cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO2 evolution. This may be linked with lower levels of NAD+, which is required as a co-factor for CO2 producing reactions of the TCA cycle. β cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the β cell mitochondrial metabolism.

Inhibition of tissue repair by spironolactone: role of mineralocorticoids in fibrous tissue formation.

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 microg/h via osmotic minipump) rats were similar (3.33 +/- 0.44 g vs. 3.70 +/- 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 +/- 0.22 g and 1.27 +/- 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 +/- 0.08 vs. 7.5 +/- 2.0 microg/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 +/- 2.1 microg/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 +/- 0.4 g vs. 3.00 +/- 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11beta-HSD2. Spironolactone (p < 0.05) significantly reduced 11beta-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.

Enhanced Activity of a Ca-Calmodulin-Dependent Isozyme of NAD Kinase Caused by Gibberellic Acid in Photodormant Lettuce Seeds

Summary We tested the hypothesis that GA3, like red light (Zhang et al., 1994), enhances activity of a Ca-calmodulin- dependent isozyme of NAD kinase and causes lower levels of NAD+ and higher levels of NADP+ in half-seeds of Grand Rapids lettuce (Lactuca sativa L.). Half-seeds containing hypocotyl/radicle sections were imbibed for 1 h in H2O or 560 μM GA3, treated with far-red light, then kept in darkness for various times up to 17 h. NAD kinase activity from crude extracts was measured, and activities of a partially purified Ca-calmodulin-independent and a Ca-calmodulin-dependent isozyme were determined. In all experiments kinase activity increased with time after treatment. GA3 enhanced activity in crude extracts and of the Ca-calmodulin-dependent isozyme 30 min after irradiation, but it had no effect on the Ca-calmodulin-independent isozyme at any time period. GA3 caused increased amounts of NADP+ and NADPH and decreased amounts of NAD+, all effects significant 30 min after irradiation. Although these results might be explained partially by GA3-enhanced formation of Ca-calmodulin in vivo, it appears that GA3 additionally or alternatively induced formation of the Ca-calmodulin-dependent isozyme of NAD kinase. These effects of GA3 are similar to those of red light, and they support the hypothesis that Pfr somehow promotes synthesis of a gibberellin as it overcomes photodormancy of the seeds.

Substrate Channeling of NADH in Mitochondrial Redox Processes

This chapter discusses substrate channeling of NADH in mitochondrial redox processes. With purified complex I, even low levels of NAD or NADH decreased its associations with most dehydrogenases to below detectable limits. Therefore, a test of substrate channeling required the enzyme buffering method, which does not need stable enzyme–enzyme complexes. The enzyme buffering test was first applied to enzymes, and NADH channeling. A factor that might enhance enzyme associations during the steady-state reaction compared to the equilibrium association in the absence of reaction intermediates is favorable kinetic constants. It is likely that enzyme associations in vivo , especially in mitochondrial matrix, are very much more extensive than those in vitro . This is because of the enormously higher enzyme concentrations in vivo (individually and collectively) than are practical to study in vitro , and to the multiplicity of interactions among different macromolecules not simultaneously present in vitro . NADH channeling should also be considered in the context of data on enzyme associations and channeling by other metabolites within mitochondria. Thus, both products of the malate dehydrogenase reaction, NADH and oxaloacetate, are likely to be channeled. This might be important in circumventing the highly unfavorable equilibrium and forward rate of this reaction.

Fate of ecto‐NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.

Fate of ecto-NAD + glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages

In order to gain a better understanding of the role of ecto-NAD + glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized or mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ectoenzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD + glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD + glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [ 3H]-galactosylation indicated that NAD + glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD + glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.

Theophylline reduces poly(ADP-ribose) synthetase from chick embryo liver nuclei

The effect of theophylline on poly(ADP-ribosyl)ation was investigated. The poly(ADP-ribose) synthetase activity in vitro was markedly reduced in the liver nuclei prepared from theophylline-treated chick embryo. This reduction was not due to the enzyme inhibition by theophylline contamination in the nuclear fraction. The hydroxyapatite column chromatographic analysis of [ 3H]adenosine-labelled poly(ADP-ribose) molecules formed in vivo revealed that the in vivo formation of poly(ADP-ribose) molecules was also decreased by theophylline administration. The theophylline-induced reduction of poly(ADP-ribose) synthesis was not due to either low NAD levels or to a decrease in the chain length of the poly(ADP-ribose) molecule, rather this reduction was derived from a decrease in the number of poly(ADP-ribose) molecules. Possible mechanisms related to reduction of poly(ADP-ribose) synthesis in vivo are discussed.

The use of bacterial luciferase and a liquid scintillation spectrometer to assay the enzymatic synthesis of NAD+

Summary Low levels of NAD+ and the NAD+ synthesizing enzyme ATP:NMN adenylyltransferase can be measured with standard scintillation counting equipment in an assay based on quantitating the light emitted by bacterial luciferase in response to NADH. This assay is 100 to 1000 times more sensitive than the conventional assay based on absorbance at 340 nm. It has been particularly useful for following the purification of ATP/NMN adenylyltransferase, since the small amounts of available enzyme could be assayed in dilute fractions from gel filtration columns or in fractions from polyacrylamide gels.

Metabolic interaction of ethanol and sorbitol in relation to redox state of liver cytosol in hypo-, hyper-, and euthyroid rats

Previous investigations have shown that sorbitol delays the rate of ethanol oxidation and conversely that ethanol inhibits the oxidation of sorbitol. These mutual effects have furthermore been found to be enhanced in hypothyroid rats and attenuated in hyperthyroid ones. In the present paper, these effects are demonstrated to be positively correlated with corresponding changes in the redox state of the liver cytosol. Simultaneous oxidation of ethanol and sorbitol elevated the hepatic lactate/pyruvate ratio in vivo from 18 to 71 in hypothyroid rats, from 16 to 53 in euthyroid ones, and from 23 to 33 in hyperthyroid animals. The increases were of the same order when ethanol was metabolized alone. The lactate/pyruvate ratio doubled when sorbitol was given to hypothyroid rats but tended to decrease in hyperthyroid rats. Experiments with liver homogenate indicated no competition between ethanol and sorbitol for the corresponding enzymes, alcohol and sorbitol dehydrogenase. NADH was found to inhibit competitively the rate of oxidation of ethanol and sorbitol in vitro at low levels of NAD +, but calculations showed that the degree of inhibition would probably be of minor importance in vivo. The mechanism by which ethanol and sorbitol inhibit each other's oxidation is suggested to be competition for free NAD +.