A 3-aminobenzamide-resistant labeled protein in [P]NAD-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [³²P]NAD⁺. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [³²P]NAD⁺ was shown to be Mg²⁺-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [α-³²P]ATP, [γ-³²P]ATP and [³²P]orthophosphate, but not from [³H]NAD⁺ or [¹⁴C]NAD⁺. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [³²P]NAD⁺. This phosphorylation appears to occur via an Mg²⁺-dependent conversion of NAD⁺ to AMP with the eventual utilization of the α-phosphate for phosphorylation of the 72 000 molecular weight protein.     

NAD+-induced inhibition of phosphate transport in canine renal brush-border membranes Mediation through a process other than or in addition to NAD+ hydrolysis

We previously demonstrated inhibition of Na⁺-dependent ³²P_i transport in canine renal brush-border membranes in association with NAD⁺-induced ADP ribosylation of membrane protein(s) and postulated that NAD⁺ inhibits P_i transport across the brush-border membrane via ADP ribosylation. Recently it was shown that incubation of rat brush-border membrane with NAD⁺ resulted in release of P_i which was prevented by EDTA. It was proposed that NAD⁺-mediated inhibition of ³²P_i transport might occur through this mechanism. To determine whether NAD⁺ inhibited ³²P_i transport by a mechanism other than or in addition to release of P_i, we compared Na⁺-dependent ³²P_i counterflow in brush-border membrane equilibrated with P_i or with P_i generated from NAD⁺. Release of P_i from NAD⁺ incubated with brush-border membrane was confirmed. The increased uptake of ³²P_i which was demonstrated in brush-border membrane equilibrated with P_i was not measured when intravesicular P_i was generated from a concentration of NAD⁺ which effected ADP-ribosylation of brush border membranes (100 μM NAD⁺). In contrast, increased uptake of ³²P_i was demonstrated when intravesicular P_i was generated from 1 μM NAD⁺ which did not effect ADP ribosylation. Mg²⁺-dependent ADP ribosylation of brush-border membrane incubated with NAD⁺ was demonstrated which persisted during the time interval of ³²P_i uptake measurements. Our findings are compatible with the hypothesis that NAD⁺-induced ADP ribosylation of brush-border membrane protein(s) results in inhibition of P_i transport across the membrane in vivo. EDTA may act to prevent this inhibition in brush-border membrane by chelation of Mg²⁺ and decreased ADP ribosylation.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

CONTROL OF THE REDOX STATE OF THE PYRIDINE NUCLEOTIDES IN THE RAT CEREBRAL CORTEX. EFFECT OF ELECTROSHOCK-INDUCED SEIZURES

—The concentrations of most of the intermediates of glycolysis and of the tricarboxylic acid cycle were determined in the cerebral cortex of rats, frozen 10 s after the induction of a generalized seizure by electroshock. The apparent equilibrium constant for the combined glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactic dehydrogenase reactions, i.e. K_app= [Lactate] [3-Phosphoglycerate] [ATP]/[Pyruvate] [Glyceraldehyde-3-phosphate] [ADP] [HPO²₄], was evaluated and found to be similar to the value reported for the in vitro system at pH 7. During an estimated 4–5-fold increase in glycolytic flux imposed by the seizure, this system remained close to equilibrium. In control cortex the components of the aldolase reaction were deviated 80-fold from equilibrium but shifted slightly toward equilibrium during the seizure. The components of the aspartate aminotransferase reaction were maintained in equilibrium in both the control and the seizure states. Of 4 reactions used to assess the cytoplasmic and mitochondrial redox states, only the lactic dehydrogenase reaction was considered reliable in the acutely changing situation of the seizure, and yielded a calculated decrease in the cytoplasmic [NAD⁺]/[NADH] ratio. This change, coupled with an observed decrease in the [ATP]/[ADP] [HPO²₄] ratio during the seizure, supports the concept that in brain, as in liver (Krebs & Veech , 1969), the phosphate potential determines the redox state of the tissue.     

A simple, rapid preparation ofα-[32P]- labelled adenosine diphosphate

Hexokinase (EC 2.7.1.1) will convert commercially available α-[³²P]-labelled ATP into α-[³²P]-labelled ADP. A simple, rapid isolation procedure for the α-[³²P]-labelled ADP is described and this synthetic method can be used for the preparation of other α-[³²P]-labelled nucleoside diphosphates.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

MonoADP-Ribosylation of the NAD+-Dependent Alcohol Dehydrogenase from Entamoeba histolytica

The human parasite Entamoeba histolytica is an amitochondrial protozoan whose metabolism depends on glucose fermentation. Among the metabolic enzymes absolutely required for amoeba growth is the NAD⁺-dependent alcohol dehydrogenase (EhADH2). The polymeric form of EhADH2 was sedimented at 160,000g, and in this fraction we observed [³²P]-labeling of a 96-kDa protein under mono-ADP-ribosylation conditions with [³²P]NAD⁺. The [³²P]-labeled protein had the same molecular weight as the EhADH2 monomer. Because of the importance of monoADP-ribosylation in the regulation of many physiological processes, the aim of this study was to determine whether EhADH2 is ADP-ribosylated, and what would be the consequence of this modification on its alcohol and aldehyde dehydrogenase enzymatic activities. This study describes the ADP-ribosylation of EhADH2. This modification did not have an effect on the enzymatic activities, but it may regulate other functions of EhADH2.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Autophosphorylation of brain microtubule protein: evidence for endogenous protein kinase/phosphoprotein phosphatase cycling and multiple phosphorylation of a microtubule associated protein

Brain microtubule protein, prepared by two types of recycling methods, undergoes “flash” phosphorylation in the presence of [γ-³²p]ATP through sequential action of protein kinase and phosphoprotein phosphatase present in microtubule protein. SDS electrophoretic analysis indicates that MAP₁, tau protein, and tubulin are poorly phosphorylated, and MAP₂ is the major site of phosphorylation. To improve [³²P]phosphoprotein stability in the presence of the kinase/phosphatase cycle, 3′,5′-cyclicAMP, orthophosphate, or fluoride ion may be added. After separation from tubulin by phosphocellulose chromatography, the MAP fraction exhibits autophosphorylation. Finally, the maximal extent of autophosphorylation is observed with an ATP regenerating system using ADP, [³²P]acetyl-P, and bacterial acetate kinase; this results in the incorporation of 3–4 phosphoryl groups per MAP₂ subunit.     

Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H₂-driven production of biodegradable polymers and hydrocarbons. H₂ oxidation by R. eutropha takes place in the presence of O₂ and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H₂ utilization. The so-called soluble hydrogenase (SH) couples reversibly H₂ oxidation with the reduction of NAD⁺ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD⁺ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD⁺] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD⁺] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD⁺] ratios represents a novel and sensitive tool to determine the redox state of cells.     

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation

Incubating rat fat cell membranes with [³²P]NAD⁺ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42 000, 46 000 and 48 000. Partial proteolytic peptide maps of the M_r = 46 000 and 48 0000 toxin-specific substrates generated by elastase, α-chymotypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the M_r = 42 000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the M_r = 42 000 target displayed a number of peptides which were absent from the maps generated from either the M_r = 46 000 or 48 000 targets. These data suggest that the M_r = 46 000 and 48 000 substrates are closely related proteins, however the relationship between the M_r = 42 000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [³²P]NAD⁺     

Analysis of adenine nucleotides at the picomole level with 32P-phosphoenolpyruvate and pyruvate kinase.

A new sensitive method for adenine nucleotide analysis is described. The key reaction is the phosphorylation of ADP by [³²P]PEP in a reaction catalyzed by pyruvate kinase, with the extent of transfer of ³²P to ADP being determined by adsorbing the nucleotides onto charcoal. The nonadenine nucleoside diphosphates which also react in the pyruvate kinase reaction are corrected for by determining the ³²P retained in the nucleotide fraction after a second incubation with hexokinase and excess glucose. ATP is determined as ADP, after it is quantitatively converted by hexokinase in the presence of excess glucose. Similarly, AMP is analyzed by its conversion to ADP in an incubation with excess ATP and adenylate kinase. The sensitivity of the method for ADP and ATP is 0.05–0.5 pmoles while for AMP it is 5 pmoles.     

Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome. Adenosine triphosphate formation coupled with electron flow between ascorbate and ferricyanide.

(1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP-³²P_j exchange. The vesicles so formed exhibit proton uptake upon addition of Mg²⁺-ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP-³²P_i exchange by valinomycin + K⁺ reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys. 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K⁺ may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP-³²P_i exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [³²P]ATP formation from ADP and ³²P_i is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.