Noninvasive measurements of transmural myocardial metabolites using 3-D (31)P NMR spectroscopy

A completely noninvasive three-dimensional (3-D) static magnetic field magnitude spatially localized (31)P spectroscopy technique has been developed and applied to study the in vivo canine myocardium at 9.4 T. The technique incorporates both Fourier series windows and selective Fourier transform methods utilizing all three orthogonal gradients for 3-D phase encoding. The number of data acquisitions for each phase-encoding step was weighted according to the Fourier coefficients to define cylindrical voxels. Spatially localized (31)P spectra can be generated for voxels of desired location within the field of view as a postprocessing step. The quality of localization was first demonstrated by using a three-compartment phantom. The technique was then applied to in vivo canine models and yielded (31)P cardiac spectra with an excellent signal-to-noise ratio. The in vivo validation experiments, using an implanted 2-phosphoenolpyruvate-containing marker, demonstrated that the technique is capable of measuring at least two transmural layers of left ventricular myocardium representing the subepicardium and subendocardium.     

Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.     

Structure of metal-nucleotide complexes bound to creatine kinase: 31P NMR measurements using Mn(II) and Co(II)

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of synthetic peptides by (32P)ATP and cyclic GMP-stimulated protein kinase.

Cyclic GMP-stimulated protein kinase from pig lung has been shown to phosphorylate synthetic peptides. The rate of phosphorylation was about one order of magnitude higher than that for mixed histones at a comparable concentration, $\text{i.e.}$ 0.1 mM. The peptides represented sites, phosphorylatable by cyclic AMP-stimulated protein kinase, in pyruvate kinase type L from rat liver, calf thymus histone H2B and the α-subunit of rabbit muscle phosphorylase b kinase. The shortest pyruvate kinase peptide that could be phosphorylated at a significant rate by cyclic GMP-stimulated protein kinase was Arg-Arg-Ala-Ser-Val-Ala, which is one amino acid residue longer than the minimal substrate of cyclic AMP-stimulated protein kinase. The apparent K_m was 0.3 mM which is about 10 times higher than that with cyclic AMP-stimulated protein kinase. The K_m was only slightly decreased upon successive extension of the peptide in the N-terminal direction to Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Modification of the sequence showed the importance of two adjacent arginyl residues, and substitution of arginine for the C-terminal alanine abolished the measurable activity. Thus, it has been demonstrated that there are both differences and similarities in substrate specificity of the two protein kinases.     

Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.     

Lipid analysis of bronchoalveolar lavage fluid (BAL) by MALDI-TOF mass spectrometry and 31P NMR spectroscopy

Despite the high clinical relevance, only the cellular moiety of bronchoalveolar lavage (BAL) has been intensively investigated and is used for diagnosis purposes. On the other hand, the cell-free fluid is, by far, less characterized. Although this fluid represents a relatively simple mixture of only a few different phospholipids (mainly phosphatidylcholine, phosphatidylglycerol and cholesterol), methods for the routine analysis of these fluids are still lacking. In the present investigation we have applied, for the first time, MALDI-TOF mass spectrometry, as well as 31P NMR spectroscopy to the analysis of organic extracts of bronchoalveolar lavage fluids. BAL from different mammals (rat, minipig, rabbit and man) were investigated and, for means of comparison, organic extracts of lung tissue were also examined. Both applied methods provide fast and reliable information on the lipid composition of the bronchoalveolar lavage. However, despite of its comparably low sensitivity, 31P NMR spectroscopy detects all phospholipid species in a single experiment and with the same sensitivity, whereas MALDI-TOF fails in the detection of phosphatidylethanolamine in the presence of higher quantities of phosphatidylcholine. In contrast, MALDI-TOF mass spectrometry is more suitable for the detection of cholesterol and the determination of the fatty acid composition of the individual phospholipids, especially lysolipids. It will be shown that all BALs exhibit significant, species-dependent differences that mainly concern the content of phosphatidylglycerol and lyso-phosphatidylcholine. It is concluded that both methods are suitable tools in lipid research due to the (in comparison to alternative methods) simplicity of performance.     

Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy

The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.     

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.     

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.     

Phosphorylation of casein by mitochondrial protein kinase(s)

Summary Chromatin fractions (DNA, histones and nonhistone chromosomal proteins NHCP) have been isolated from human peripheral B and T lymphocytes using different methods and analyzed in order to identify their lipid content.While DNA and histone fractions do not reveal the presence of lipids, a 2% of phospholipids is present in the NHCP fraction. The phospholipids associated with NHCP present a constant relative ratio among sphingomyelin, phosphatidyl-choline and phosphatidyl-ethanolamine both in B and T lymphocytes, whichever are the extraction procedures employed.These findings are related to the possible derepressive role of phospholipids on DNA-dependent RNA synthesis.     

Phosphoglycerides and derivatives in Taenia crassiceps examined by 31P NMR spectroscopy

Examination of the larval stage of the tapeworm, Taenia crassiceps, by 31P NMR spectroscopy revealed the presence of a major phosphoglyceride component. However, using saturation transfer, no exchange between glycerophosphorylcholine and phosphoglyceride or any other NMR-detectable phosphorus metabolites was detected.     

Magic-angle spinning (31)P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

We report the results of a solid-state (31)P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at approximately 0, -7 and -21 ppm. These resonances are assigned to orthophosphate, terminal (alpha) phosphates of polyphosphates and bridging (beta) phosphates of polyphosphates, respectively. The average polyphosphate chain length is approximately 3.3 phosphates. Similar results were obtained with whole L. major promastigotes. (31)P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques.     

Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy

Addition of choline, ethanolamine, or hemicholinium-3 (a choline kinase inhibitor) to the perfusate of human breast cancer cells monitored by 31P NMR spectroscopy resulted in significant changes to phosphomonoester (PME) and phosphodiester (PDE) signals. These results enable us to assign the PMEs to phosphcholine (PC) and phosphoethanolamine (PE), the PDEs to glycerophosphorylcholine and glycerophosphorylethanolamine, and to define the pathways producing them. The PMEs are products of choline and ethanolamine kinases, the first steps in phospholipid synthesis; and the PDEs are substrates of glycerophosphorylcholine phosphodiesterase, the last step in phospholipid catabolism. Furthermore, PC and PE peaks are twice as intense in cells at log phase versus confluency. We also observed these signals in vivo in human colon and breast tumors grown in mice. Since PMEs are low in most nonproliferating tissues, they could form a basis for noninvasive diagnosis. Also, PE and PC are situated between the control enzymes of two major synthetic pathways and will allow noninvasive 31P NMR studies of these pathways in intact cells and in vivo.     

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.

Phosphorylation by PKA of a site unique to B-Raf kinase

The Raf kinases serve as central intermediates to relay signals from Ras to ERK. Cell-specific effects of these signals on growth, differentiation and survival can be observed due to the recruitment of different isoenzymes of the Raf family. The in vitro phosphorylation of a site unique to B-Raf (Ser429) has been proposed to be responsible for the negative regulation of the isoenzyme by Akt. Using phosphopetide mapping and site-directed mutagenesis we showed that Ser429 is phosphorylated upon cAMP elevation in PC12 cells and proposed that PKA is a major kinase phosphorylating the B-Raf-specific site in vivo.     

Observation of slow dynamic exchange processes in Ras protein crystals by 31P solid state NMR spectroscopy

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.