In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.     

Characterization of oxidized nicotinamide adenine dinucleotide (NAD(+)) analogues using a high-pressure-liquid-chromatography-based NAD(+)-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD(+))-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD(+)) and products (adenosine 5'-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD(+) analogue, 2'-F-ribo-NAD(+), was a competing substrate or a competitive inhibitor for this toxin. This NAD(+) analogue was hydrolyzed at a rate of 0.2% that of NAD(+) yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD(+) analogue, epsilon-NAD(+), is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD(+) as determined using HPLC. The rate of hydrolysis of epsilon-NAD(+) calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD(+)-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD(+) and its analogues.     

Identification of diadenosine-triphosphate in mature bovine lenses

Mature bovine lenses contain 75-100 microM of a previously unidentified nucleoside polyphosphate. Using (31)P NMR spectroscopy we have identified this compound as diadenosine-5',5'-triphosphate. The accumulation of this compound in the lens may be a consequence of the high levels of activities of t-RNA synthetases during lens differentiation and growth. The function, if any, of this compound in the bovine lenses is presently unknown.     

Muscle pain after exercise is linked with an inorganic phosphate increase as shown by31P NMR

³¹P nuclear magnetic resonance (NMR) spectra were obtained from the forearm muscles of 5 subjects before and after performing a muscle stretching (eccentric) exercise routine. Spectra collected before and immediately after exercise showed normal resting phosphorylated metabolite levels and unchanged intracellular pH (pH_i). Measurements made on the day following exercise, when muscular pain was apparent, revealed an elevated inorganic phosphate level. No significant changes in other metabolites or pH_i were detected. This study gives the first indication of biochemical change following a form of exercise that is associated with considerable muscle pain and damage. The findings may help in understanding pathological processes resulting in pain and damage in muscle.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high-performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer. We found a good linear response for each NAD(+)-related compound. The limits of quantification for NAD(+) and related compounds range from 0.1 to 1 pmol. The extraction efficiency of NAD(+) and related compounds from mouse erythrocytes is between 84 and 114%. The coefficients of variation for the analyses are all less than 6%. Using our method, we measured, in a single analysis, the amounts of NMN, NAMN, NAD(+), and 5'AMP present in mouse erythrocytes. Additionally, this is the first report of a direct determination of the amounts of NMN and NAMN present in any type of cell. These results indicate that our method sensitively, specifically, and simultaneously measures cellular NAD(+) and related compounds.     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Formation of new larger ring cyclic triphosphenium ions in solution - A P NMR study

In a notable extension of previous work, we have shown that several diphosphanes will form larger (7- or 8-membered ring) cyclic triphosphenium ions (CTIs) in solution by reaction with PX₃ (X = Cl, Br or I), sometimes in the presence of SnCl₂ or SnBr₂. We have also formed new CTIs from two ferrocene derivatives. The ions are readily identifiable by ³¹P NMR solution-state spectroscopy, having large ¹J_P-P values between the middle ‘bare’ phosphorus atom P_A and the outer phosphorus atoms P_B (and P_C where the outer groups are inequivalent). In addition, some data for known CTIs, but with different counter-ions, are presented. Three of the new CTIs have been successfully protonated, two by both AlCl₃/^tBuCl and triflic acid, and one via the triflic acid route only.     

Synthesis, characterization and DNA binding properties of oligopyridine-ruthenium(II)-amino acid conjugates

The DNA-binding properties of a number of ruthenium oligopyridine complexes with conjugated amino acids having the general formulae [Ru(terpy)(4-COY-4'-Mebpy)(X)](n)(+), X=NO (n=3), X=Cl (n=1) and NO(2) (n=1) and Y=AlaCONH(2) and TrpCONH(2) are reported. The new complexes were spectroscopically characterized and their DNA-binding properties were studied by means of circular dichroism (CD), (23)Na and (31)P NMR spectroscopy. The results show that the chlorido complexes interact by coordination to the DNA bases with the conjugated amino acid able to provide an additional interaction with the DNA helix. In addition, electrostatic interactions between all studied complexes and the DNA polyanion were observed. The nitro complexes were found to be insignificant, affecting only the (31)P NMR signal, probably due to changes in the hydration sphere of the DNA close to the phosphates.     

Stereochemistry of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates

Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ₃₁P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arising from R _P and S _P diastereomers were reversed.     

Facile pyramidal inversion at phosphorus in [R3EP7W(CO)3] type complexes: An EXSY NMR study

A series of [R₃EP₇W(CO)₃]²⁻ complexes where (E = Si, Ge, Sn, Pb; R = alkyl, phenyl) were prepared from [P₇W(CO)₃]³⁻ and R₃EX reagents (X = Cl, Br) in dmf or CH₃CN solutions. The Pb derivatives were prepared at −50 °C and are not thermally stable. The compounds were characterized by ³¹P NMR spectroscopy and selected ESI-MS studies. All compounds undergo rapid inversion at the ER₃-bound phosphorus atom. The barriers to inversion were measured by way of 2D ³¹P EXSY experiments at various temperatures. The analysis showed very low barriers to pyramidal inversion (ΔG^‡ 10.3-13.5 kcal/mol) that were essentially enthalpic in origin. The activation barriers generally increased with increasing electronegativity of the E atom and the steric bulk of the ER₃ substituents. The latter was interpreted by way of a non-linear transition state.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Phosphorus fertilizer recovery from calcareous soils amended with humic and fulvic acids

Precipitation of Ca phosphates negatively affects recovery by plants of P fertilizer applied to calcareous soils, but organic matter slows the precipitation of poorly soluble Ca phosphates. To study the effect of high molecular weight organic compounds on the recovery of applied P, a mixture of humic and fulvic acids was applied to calcareous soils with different levels of salinity and Na saturation which were fertilized with 200 and 2000 mg P kg^–1 as NH₄H₂PO₄. Recovery was measured as the ratio of increment in Olsen P-to-applied P after 30, 60 and 150 days, and associated P forms were studied using sequential chemical fractionation and ³¹P NMR spectroscopy. Application of the humic-fulvic acid mixture (HFA) increased the amount of applied P recovered as Olsen P in all the soils except in one soil with the highest Na saturation. In soils with high Ca saturation and high Olsen P, recovery increased from < 15% in the absence of amendment to > 40% at a 5 g HFA kg^–1 amendment rate (30 days incubation and 200 mg P kg^–1 fertilizer rate). This is ascribed to inhibition of the precipitation of poorly soluble Ca phosphates, consistent with the sequential chemical extraction (reduction of the HCl extractable P) and P concentration in 0.01 M CaCl₂ (1:10 soil:solution ratio) extracts. ³¹P NMR spectra revealed that in non-amended samples, most spectral shifts were due to poorly soluble P compounds (carbonate apatite); on the other hand, at the 5 g HFA kg^–1 rate, significant amounts of amorphous Ca phosphate and dicalcium phosphate dihydrate (DCDP) were identified. The increase in the recovery of applied P due to HFA reveals a positive effect of the application of organic matter as soil amendments on the efficiency of P fertilizers and also explains that manures and other organic sources of P were more efficient increasing available P than inorganic P fertilizers in calcareous soils.     

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)₂ have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the ³¹P-{¹H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from ¹H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane     

31P and19F NMR studies of glycophorin-reconstituted membranes: Preferential interaction of glycophorin with phosphatidylserine

Summary Glycophorin A, a major glycoprotein of the erythrocyte membrane, has been incorporated into small unilamellar vesicles composed of a variety of pure and mixed phospholipids. Nuclear spin labels including³¹P and¹⁹F have been used at natural abundance or have been synthetically incorporated in lipids to act as probes of lipid-protein interaction. Interactions produce broadening of resonances in several cases and it can be used to demonstrate preferential interaction of certain lipids with glycophorin.³¹P and¹⁹F probes show a strong preferential interaction of glycophorin with phosphatidylserine over phosphatidylcholine. There is some evidence that interactions are more pronounced at the inner surface of the bilayer and these results are rationalized in terms of the asymmetric distribution of protein and lipid.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Core peptide (CP) is a unique peptide derived from thetransmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immuneresponse both in vitro and in animal models of Tcell mediated inflammation. The structure of CP, withsequence GLRILLLKV, is similar to the amphipathic regionof many peptides. Unlike antimicrobial peptides,however, which damage cell membranes, electron microscopyand propidium iodide exclusion assays on cell membranessuggest that CP does not create pores and may act byinterfering with signal transduction at the membranelevel. To investigate this effect further we report theresults of ³¹P and ²H solid-state NMRspectroscopy of CP on model membranes. As predicted,even at high concentrations of CP, the structure of modelmembranes was not significantly perturbed. Only at thevery high peptide-to-lipid molar ratio of 1:10significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of thelipid bilayer.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Relevance of the ability of fructose 1,6-bis(phosphate) to sequester ferrous but not ferric ions

The cytoprotective activity of F16BP has been documented in severe conditions such as convulsions, reperfusion injury, septic shock, diabetic complications, hypothermia-induced injury, UV-provoked skin damage and in other processes including apoptosis and excitotoxicity. F16BP shows very efficient cytoprotective activity in astroglial cells exposed to H₂O₂-provoked oxidative stress and during neuronal injury caused by hypoxic conditions. As most of the aforementioned processes involve iron activity-related conditions, we investigated the ferric and ferrous iron binding properties of F16BP under physiological conditions using ³¹P NMR and EPR spectroscopy. Our results indicate that cytoprotective F16BP activity is predominantly based on ferrous iron sequestration. ³¹P NMR spectroscopy of F16BP employing paramagnetic properties of iron clearly showed that F16BP forms stabile complexes with Fe²⁺ which was verified by EPR of another divalent cation—Mn²⁺. On the other hand, F16BP does not sequester ferric iron nor does it increase its redox activity as shown by ³¹P NMR and EPR spin-trapping. Therefore, F16BP may be beneficial in neurodegenerative and other conditions that are characterised by ferric iron stores and deposits.     

Oligonucleotide Analogues with Dialkyl Silyl Internucleoside Linkages

Abstract

Oligonucleotide analogues with dialkyl-silyl inter-nucleoside linkages were prepared by “one pot” reactions starting from dialkyl-dichloro- resp. chloro-dialkylamino-silane derivatives.