Investigation of phosphatidylethanolamine bilayers by deuterium and phosphorus-31 nuclear magnetic resonance.

The motion of the ethanolamine head group in unsonicated lipid bilayers above and below the phase transition is studied by means of deuterium and phosphorus magnetic resonance. For this purpose, dipalmitoyl-3-sn-phosphatidylethanolamine is selectively deuterated at the two ethanolamine carbon atoms. The deuterium quadrupole splittings of the corresponding bilayer phases are measured at pH 5.5 as a function of temperature. In addition, the phosphorus-31 chemical shift anisotropies of planor-oriented and randomly dispersed samples of dipalmitoyl-3-sn-phosphatidylethanolamine are measured at pH 5.5 and 11 by applying a proton-decoupling field. The knowledge of the static chemical shift tensor (Kohler, S.J., and Klein, M.P. (1976), Biochemistry 15, 967) provides the basis for a quantitive analysis of the head-group motion. The nuclear magnetic resonance data are consistent with a model in which the ethanolamine group is rotating flat on the surface of the bilayer with rapid transitions occurring between two enantiomeric conformations.     

Comparisons of diisopropylfluorophosphate derivatives of chymotrypsin and chymotrypsinogen by phosphorus-31 nuclear magnetic resonance

The nature of the differences in the active sites of α-chymotrypsin and chymotrypsinogen has been investigated by phosphorus-31 NMR studies of their diisopropylfluorophosphate derivatives. The phosphorus-31 resonance of the modified zymogen occurs 2 ppm upfield from that for the enzyme. An even greater separation is seen between diisopropylphosphoryl-neo-chymotrypsinogen and -α-chymotrypsin. A plausible interpretation of the chemical shift differences is based on the known structures for α-chymotrypsin, chymotrypsinogen and diisopropylphosphoryl-trypsin.     

A sex-specific metabolite identified in a marine invertebrate utilizing phosphorus-31 nuclear magnetic resonance

Hormone level differences are generally accepted as the primary cause for sexual dimorphism in animal and human development. Levels of low molecular weight metabolites also differ between men and women in circulating amino acids, lipids and carbohydrates and within brain tissue. While investigating the metabolism of blue crab tissues using Phosphorus-31 Nuclear Magnetic Resonance, we discovered that only the male blue crab (Callinectes sapidus) contained a phosphorus compound with a chemical shift well separated from the expected phosphate compounds. Spectra obtained from male gills were readily differentiated from female gill spectra. Analysis from six years of data from male and female crabs documented that the sex-specificity of this metabolite was normal for this species. Microscopic analysis of male and female gills found no differences in their gill anatomy or the presence of parasites or bacteria that might produce this phosphorus compound. Analysis of a rare gynandromorph blue crab (laterally, half male and half female) proved that this sex-specificity was an intrinsic biochemical process and was not caused by any variations in the diet or habitat of male versus female crabs. The existence of a sex-specific metabolite is a previously unrecognized, but potentially significant biochemical phenomenon. An entire enzyme system has been synthesized and activated only in one sex. Unless blue crabs are a unique species, sex-specific metabolites are likely to be present in other animals. Would the presence or absence of a sex-specific metabolite affect an animal's development, anatomy and biochemistry?     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

Studies of yeast phenylalanine-accepting transfer ribonucleic acid backbone structure in solution by phosphorus-31 nuclear magnetic resonance spectroscopy.

Approximately 17 diester phosphates from the backbone structure of yeast tRNAPhe give rise to phosphorus resonances, which are resolved in its 31P NMR spectrum. To localize these diester phosphates within the tRNA structure, 31P NMR spectra of several chemically or enzymatically modified yeast tRNAPhe species were recorded. To this end selective modifications were performed in the anticodon, the DHU, and the T psi C loop. Modifications, performed in different loop regions, give rise to perturbation of different characteristic 31P resonances. The 31P spectra were correlated with the corresponding 1H NMR spectra of the ring N hydrogen-bonded protons and interpreted in view of the X-ray results obtained on yeast tRNAPhe. It is concluded that the diester phosphate groups, which experience an unusual shift, can be accounted for in the X-ray structure in terms of hydrogen-bonded phosphates groups and diester phosphates with a diester geometry, deviating from the normal double-helical conformation.     

Investigation of the mineral phases of bone by solid-state phosphorus-31 magic angle sample spinning nuclear magnetic resonance

Phosphorus-31 magic angle sample spinning NMR spectra have been employed to investigate the structure and composition of the mineral deposits in chicken bone. Three different pulse sequences, Bloch decay, cross-polarization, and dipolar suppression, were employed to obtain spectra from bone specimens of varying age. These were compared to the spectra obtained from a variety of crystalline and noncrystalline synthetic calcium phosphate solids used as reference standards. The results suggest that the most suitable model for the major solid calcium phosphate mineral phase in bone is a hydroxyapatite containing approximately 5-10% CO32- and approximately 5-10% HPO42- groups, the latter in a brushite-like configuration. From the NMR line shapes it was deduced that the fraction of HPO42- groups was highest in the youngest bone and decreased progressively with increasing age of the specimen.     

Active proton uptake by chromaffin granules: observation by amine distribution and phosphorus-31 nuclear magnetic resonance techniques.

The hydrogen ion activity within isolated chromaffin granules can be estimated from the distribution of the weak base methylamine and from phosphorus-31 nuclear magnetic resonance spectra of ATP contained in the granules. Following the addition of ATP to the external medium, the internal pH drops by 0.2 to 0.5 unit. This change occurs only in medium containing a permeant anion such as chloride and is abolished by an uncoupler of oxidative phosphorylation. These results indicate that the chromaffin granule membrane possess an electrogenic proton pump directed inward.     

A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear magnetic resonance.

A systematic phosphorus-31 nuclear magnetic resonance study of some nucleic acid constituents (6-N-(dimethyl)adenylyl-(3',5')-uridine and some nucleotide methyl esters) is presented. The temperature dependent phosphorus-31 chemical shifts were analyzed by standard thermodynamic procedures. It is shown that gt conformations about the P-O ester bonds have a lower free energy content relative to gg conformers.     

Head-group conformation in phospholipids: a phosphorus-31 nuclear magnetic resonance study of oriented monodomain dipalmitoylphosphatidylcholine bilayers

Angular-dependent 31P NMR spectra of oriented biaxial monodomain DPPC.H2O multilayers are employed to study head-group conformation in this phospholipid. The results indicate that the O-P-O plane of the phosphate, where the O's are the nonesterified oxygens of the phosphodiester, is tilted at 47 +/- 5 degrees with respect to the bilayer normal. This PO4 orientation could result in the choline moiety being extended parallel to the bilayer plane, and it will explain the breadth of the axially symmetric 31P powder spectrum observed for DPPC in excess water. This work is the first direct observation of this conformation for lecithins and it illustrates the utility of high-resolution solid-state NMR in structural studies of disordered systems.     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Quantitative hepatic phosphorus-31 magnetic resonance spectroscopy in compensated and decompensated cirrhosis

Few studies have examined the physiological/biochemical status of hepatocytes in patients with compensated and decompensated cirrhosis in situ. Phosphorus-31 magnetic resonance spectroscopy ((31)P MRS) is a noninvasive technique that permits direct assessments of tissue bioenergetics and phospholipid metabolism. Quantitative (31)P MRS was employed to document differences in the hepatic metabolite concentrations among patients with compensated and decompensated cirrhosis as well as healthy controls. All MRS examinations were performed on a 1.5-T General Electric Signa whole body scanner. The concentration of hepatic phosphorylated metabolites among patients with compensated cirrhosis (n = 7) was similar to that among healthy controls (n = 8). However, patients with decompensated cirrhosis (n = 6) had significantly lower levels of hepatic ATP compared with patients with compensated cirrhosis and healthy controls (P < 0.02 and P < 0.009, respectively) and a higher phosphomonoester/phosphodiester ratio than controls (P < 0.003). The results of this study indicate that metabolic disturbances in hepatic energy and phospholipid metabolism exist in patients with decompensated cirrhosis that are not present in patients with compensated cirrhosis or healthy controls. These findings provide new insights into the pathophysiology of hepatic decompensation.     

Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion. Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal. Subsequent addition of EDTA to the suspension restored the original spectrum. These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.     

Response of the headgroup of phosphatidylglycerol to membrane surface charge as studied by deuterium and phosphorus-31 nuclear magnetic resonance

The response to membrane surface charge of the glycerol headgroup of dimyristoyl-phosphatidylglycerol (DMPG) was investigated via deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy. The membrane surface charge was manipulated by adding various amounts of neutral dimyristoylphosphatidylcholine (DMPC) and/or positively charged didodecyldimethylammonium bromide (DDAB) to the negatively charged DMPG, selectively deuterated at the alpha and beta segments of its glycerol headgroup. The deuterium and phosphorus-31 nuclear magnetic resonance spectra were all characteristic of random dispersions of liquid-crystalline lipids in a bilayer configuration. Differential scanning calorimetry showed that all mixtures investigated exhibited gel to liquid-crystalline phase transitions below 35 degrees C. Measurements of the deuterium quadrupole splitting and of the phosphorus-31 chemical shift anisotropy lead to the following observations. (1) Dilution of the negative surface charge density by the addition of DMPC had little effect on the quadrupole splitting from either alpha- or beta-deuterated DMPG. (2) Direct cancellation of the negative surface charge density by addition of DDAB led to a progressive decrease in the quadrupole splitting measured from alpha-deuterated DMPG, while the quadrupole splitting measured from beta-deuterated DMPG increased. For alpha-deuterated DMPG addition of 0.3 mole fraction of DDAB resulted in the appearance of two distinct quadrupole splittings. No such effect was observed for beta-deuterated DMPG.     

连续提取和液体磷核磁共振表征太湖梅梁湾沉积物中磷的剖面分布

应用连续提取法(SMT)和液体磷核磁共振(³¹PNMR)技术研究了太湖北部梅梁湾沉积物中磷形态和组成的剖面变化。结果表明,铁/铝磷是沉积物中磷的主要形态,约占总磷含量的44.0-54.6%。总磷、无机磷、有机磷和铁/铝磷含量均随沉积深度增加呈降低趋势,至18cm以下略有增加,而钙磷却在柱样下部随沉积深度增加呈累积趋势。³¹PNMR显示,沉积物磷主要由正磷酸盐(72.0-99.2%)和磷酸单酯(0.8-25.9%)构成,磷酸二酯、膦酸盐和焦磷酸盐的相对含量非常低,分别为1.0%、0.4-1.0%和 0.1%。正磷酸盐含量在沉积物表层9cm内减少了65%,9cm以下波动变化,但总体呈降低趋势。这些特征表明沉积物中磷对梅梁湾上覆水体具有强烈的释放潜力,是太湖富营养化发生的重要因素。     

A phosphorus-31 nuclear magnetic resonance study on the complex of chicken gizzard myosin subfragment 1 with adenosine diphosphate

The complex of Mg-ADP with chicken gizzard myosin subfragment 1 (S1), obtained by the treatment with Staphylococcus aureus V8 proteinase, was observed with 31P NMR at various temperatures between 0 and 25°C. The signal of S1·ADP complex was observed at -2 to -3 ppm as a rather broad peak. As compared with the results for rabbit skeletal muscle S1·ADP complex (Tanokura M, Ebashi S: J Biochem 113: 19-21, 1993), the signal was assigned to -phosphate of ADP in the S1·ADP complex. The signal of the complex was so broad and weak that the dependences on temperature and magnetic field strength were not clear. The observation suggests the tight interaction of S1 with the phosphate moieties of ADP in the complex and the extremely anisotropic distribution of electrons around phosphorus nuclei.