Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures

³¹P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, ECoATParginine ECoADPP-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289–293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-³¹P (-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-³¹P distance of 3.0 Å, representing direct coordination of Co(II) to -P(ATP), changes to 4.0 Å when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-³¹P vector implies an excursion of at least 1.0 Å for the itinerant phosphoryl group on the surface of the enzyme.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by1H and31P NMR

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by¹H and³¹P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pH_i). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pH_i normal. When the aglycemic hypoxia was carried out in media which had no added calcium (≈10 μM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltagegated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate. Dedication This article is dedicated to our friend and colleague Herman Bachelard. We wish to thank him for his comradeship, advice and support over many years. Our hope for him is a long and fruiful retirement and that he will remain active in the neurosciences for many years, even though the establishment has blown for “full time”.     

A CAUTIONARY NOTE ON THE USE OF THE 31P NMR SPECTROSCOPY IN STEREOCHEMICAL CORRELATION ANALYSIS

Stereoselectivity in condensation of protected ribonucleoside 3′-H-phosphonates with hydroxylic components was investigated using ³¹P NMR spectroscopy. The correlation between absolute configuration at the phosphorus center and the chemical shifts of the produced H-phosphonate diesters and the corresponding phosphorothioates, was studied.     

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.     

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.     

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.     

31P Nuclear magnetic resonance studies of ethanol inhibition in Zymomonas mobilis

Ethanol inhibition of glucose catabolism in Zymomonas mobilis was investigated using ³¹P NMR spectroscopy in vivo and of perchloric acid extracts from cell suspensions incubated with 0, 5 and 10% (w/v) ethanol. In vivo ³¹P NMR experiments revealed slower glucose utilization and decreased levels of nucleoside triphosphates in the presence of 10% ethanol as compared to controls. Using ³¹P NMR spectroscopy of perchloric acid extracts, intracellular accumulation of 3.4 mM 3-phosphoglycerate was found when 10% ethanol was present in the medium. No accumulation of this metabolite occurred in cells incubated with 0 and 5% ethanol. Enzyme assays confirmed that phosphoglycerate-mutase and enolase were inhibited 31 and 40%, respectively, in the presence of 10% ethanol in the test system. Therefore, under the conditions used the decrease in the fermentative activity of Z. mobilis at high ethanol concentrations is due to inhibition of phosphoglycerate-mutase and enolase.Abbreviation KDPG 2-keto-3-deoxy-6-phosphogluconate     

Influence of temperature on 31P NMR chemical shifts of phospholipids and their metabolites I. In chloroform-methanol-water

Spectral overlap of ³¹P NMR resonances and the lack of reproducibility in chemical shifts corresponding to phospholipids in organic solvents challenge the accuracy of band assignments and quantification. To alleviate these problems, the use of temperature coefficients is proposed. Changes in temperature enable the resolution of overlapped resonances and provide a facile approach for the computation of temperature coefficients. The coefficients were evaluated for various glycero- and sphingo-phospholipids. Their values suggest that differences in H-bonding between the phosphate and the head groups are responsible for the changes of chemical shift with temperature. Among parent phospholipids, and in addition to sphingomyelin, the smallest temperature coefficient values (closest to zero) were observed for phosphatidylcholine, phosphatidylglycerol, dihydrosphingomyelin, and cardiolipin. The highest values were exhibited by phospholipids with protonated head groups, such as phosphatidylserine and phosphatidylethanolamine. The lowest and, in fact, negative values were measured for phospholipids with an exposed phosphate group: phosphatidic acid, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Diacyl, alkyl-acyl, and alkenyl-acyl phospholipids with the same head group exhibited comparable coefficients but differed slightly in chemical shifts. Compared to their parent glycerophospholipids, all lyso analogs had greater temperature coefficients, possibly due to the presence of an extra OH capable of forming a H-bond with the phosphate group.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

Determination of the transmembrane proton gradient in the anaerobic bacterium Desulfovibrio desulfuricans by 31P nuclear magnetic resonance

The transmembrane proton gradient of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN has been determined by in vivo³¹P nuclear magnetic resonance (NMR) spectroscopy in the absence of dioxygen. At pH 7.0 in the medium (pH_ex) the intracellular pH (pH_in) was 7.5. By lowering pH_ex to 5.9 pH_in decreased to 7.1. At pH_ex greater than 7.7 the transmembrane proton gradient (pH) was zero. The uncouplers 3,3,4,5-tetrachlorosalicylanilide (TCS) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), or the permeant anion thiocyanate caused complete dissipation of pH.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - TCS 3,3,4,5-tetrachlorosalicylanilide - MOPS 3-(N-morpholino)-propanesulfonic acid - P _i inorganic phosphate - pH _in (pH_ex) intracellular (extracellular) pH - pH transmembrane proton gradient (pH_in-pH_ex) - electrochemical membrane potential - chemical shift in parts per million - NMR nuclear magnetic resonance     

Principles of multiparametric optimization for phospholipidomics by 31P NMR spectroscopy

Phospholipids have long been known to be the principal constituents of the bilayer matrix of cell membranes. While the main function of cell membranes is to provide physical separation between intracellular and extracellular compartments, further biological and biochemical functions for phospholipids have been identified more recently, notably in cell signaling, cell recognition and cell–cell interaction, but also in cell growth, electrical insulation of neurons and many other processes. Therefore, accurate and efficient determination of tissue phospholipid composition is essential for our understanding of biological tissue function. ³¹P NMR spectroscopy is a quantitative and fast method for analyzing phospholipid extracts from biological samples without prior separation. However, the number of phospholipid classes and subclasses that can be quantified separately and reliably in ³¹P NMR spectra of tissue extracts is critically dependent on a variety of experimental conditions. Until recently, little attention has been paid to the optimization of phospholipid ³¹P NMR spectra. This review surveys the basic physicochemical properties that determine the quality of phospholipid spectra, and describes an optimization strategy based on this assessment. Notably, the following experimental parameters need to be controlled for systematic optimization: (1) extract concentration, (2) concentration of chelating agent, (3) pH value of the aqueous component of the solvent system, and (4) temperature of the NMR measurement. We conclude that a multiparametric optimization approach is crucial to obtaining highly predictable and reproducible ³¹P NMR spectra of phospholipids.     

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by31P NMR

Summary The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe³¹P NMR. The broad. asymmetric lineshape of the³¹P NMR spectrum of isolated brush-border vesicles demostrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18mm), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, watersoluble fatty acids do not, suggesting a difference in the absorption mechanisms.     

Respirational activity of Chlorella fusca monitored by in vivo P-31 NMR

Energy metabolism during dark respiration of the green alga Chlorella fusca was investigated by ³¹P NMR spectroscopy. The kinetics of the transition from anaerobic to aerobic conditions (and vice versa) was followed with a temporal resolution of 16 s. This transition is accompanied by a shift of the cytoplasmic pH from 6.8 to 7.4, while the vacuolar pH remains constant. Simultaneously, an increase in the concentration of nucleoside-triphosphates and a decrease in the concentration of cytoplasmic orthophosphate take place, as well as the formation of mobile polyphosphates. The concentration of ATP and P _i reach steady-state levels within 30 s. Upon the reverse transition, from aerobic to anaerobic conditions, steady-state concentrations are obtained only after 3 min.     

Na+-H+ exchange inhibition at reperfusion is cardioprotective during myocardial ischemia-reperfusion; 31P NMR studies

To help resolve the controversy as to whether or not Na⁺-H⁺ exchange is functioning during reperfusion of the ischemic myocardium we assessed the effects of dimethylamiloride (DMA, an amiloride analogue possessing selectivity for inhibition of the Na⁺-H⁺ exchanger) on cardiac function and intracellular pH during ischemia-reperfusion. Studies were performed in the presence of bicarbonate (modified Krebs-Henseleit buffer) or in the nominal absence of bicarbonate (HEPES buffer) in order to determine if similar cardioprotection and effects on intracellular pH were observed in the presence and absence of bicarbonate dependent transport processes. Isovolumic rat hearts were perfused in the Langendorff mode at a constant pressure of 80 mm Hg and subjected to 28 min total global ischemia at 37°C. Intracellular pH was determined from the pH dependent shift of the inorganic phosphate peak in ³¹P nuclear magnetic resonance spectra. DMA (20 µM) was infused for either 2.5 min before ischemia, for the initial 5 min of reperfusion, or at both time intervals. DMA had no effect on the intracellular pH during ischemia. Intracellular pH returned to pre-ischemic levels within 2.5 min of reperfusion in bicarbonate buffer. This normalization of pH was slower in HEPES perfusate. In both bicarbonate and HEPES perfused hearts all drug dosing regimens caused a significant increase in the recovery of mechanical function after reperfusion and slowed the recovery of intracellular pH during reperfusion. These results suggest that the Na⁺-H⁺ exchanger is activated during reperfusion of the ischemic myocardium, that this activation of the exchanger contributes to ischemia-reperfusion induced cardiac dysfunction and that administration of an inhibitor of Na⁺-H⁺ exchange at reperfusion significantly attenuates the deleterious effects of exchanger activation.     

A 31P NMR study of the interaction of amphibian antimicrobialpeptides with the membranes of live bacteria

Summary Amphibian skin is a rich source of peptides that are specific to pathogens and act by disrupting bacterial membranes. Three antimicrobial peptides were isolated from the skin glands of Australian tree frogs,Litoria caerulea andLitoria genimaculata. NMR spectroscopy was used to observe changes induced by these peptides in the³¹P resonances of bacterial membranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics disrupted the membranes ofBacillus cereus andStaphylococcus epidermidis (Gram-positive), leading to an increase in the isotropic³¹P NMR signal. Caerin 4.1, a narrow-spectrum antibiotic, however, did not affect the³¹P spectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents on the membranes of live bacteria.     

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]-Nuclear magnetic resonance spin lattice relaxation in lecithin reverse micelles

[³¹P] -Nuclear magnetic resonance (NMR) spin lattice relaxation times (T₁) have been measured for lecithin-nonpolar solvent-water as a function of added water for three solvents, namely, benzene, carbon tetrachloride and cyclohexane. In benzene and carbon tetrachloride systems, where spherical reverse micelles are formed, [³¹P]-NMR T₁, values increase linearly with added water. However, in cyclohexane, the trends in the [³¹P]-T₁ values indicate very different micellisation processes. Even at the lowest concentration of added water, the [³¹P]-T₁ values in this solvent are substantially larger than the corresponding values in benzene and carbon tetrachloride, which is attributed to the intramolecular chlorinephosphate interaction being the weakest in cyclohexane. At a higher water content of six mols of water per mol of lecithin in cyclohexane solvent, the [³¹P]-T₁ values show a sharp decrease indicating a sudden change in the dynamics of the phosphate group, and this confirms the on set of ‘reverse micelle-to-liquid crystalline’ phase transition observed in this system by other spectroscopic and physical techniques.     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide