Deuterium nuclear magnetic resonance studies of bile salt/phosphatidylcholine mixed micelles

Mixed micelles of deoxycholate (DOC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) have been prepared in which the POPC was specifically deuterated in the 2-, 6-, 10-, or 16-position of the palmitoyl chain or in the N-methyl position of the choline head group. The deuterium nuclear magnetic resonance (2H NMR) spectrum of each of these specifically deuterated mixed micelles consists of a singlet whose line width depends upon the position of deuteration. Spin-spin relaxation times indicate a gradient of mobility along the POPC palmitoyl chain in the mixed micelle, with a large increase in mobility on going from the 10- to the 16-position. Spin-lattice relaxation times (T1's) demonstrate a similar gradient of mobility. Both trends in NMR relaxation behavior are consistent with a bilayer arrangement for the solubilized POPC. 2H T1 times for DOC/POPC micelles are significantly shorter than those measured in other bilayer systems, indicating unusually tight phospholipid acyl chain packing in the mixed micelle.     

Deuterium magnetic resonance studies of phospholipid bilayers

L-α-dipalmitoyl lecithin is selectively deuterated at two different chain positions. The residual quadrupole splittings of the corresponding phospholipid bilayers are measured by means of deuterium magnetic resonance and evaluated in terms of the segmental order parameters. The results are briefly compared with other esr and nmr investigations of lipid bilayers.     

Selective binding of polymyxin B to negatively charged lipid monolayers

It is demonstrated by direct measurement of surface radioactivity that the cationic polypeptide antibiotic polymyxin B is specifically adsorbed to negatively charged lipid monolayers. The latter attracted the following amounts of the biologically active $\text{mono}\text{-N[}{}^{14}\text{C]}\text{acetylpolymyxin B}$ derivative (PX): lipid A from Proteus mirabilis, 0.17; phosphatidic acid, 0.12; phosphatidylglycerol and phosphatidylserine, 0.11; dicetylphosphate, 0.107; sulfoquinovosyldiglyceride, 0.104; phosphatidylinositol and cardiolipin, 0.095; and phosphatidylethanolamine, 0.017 μg/cm². Adsorption of PX to phosphatidylcholine, monogalactosyldiglyceride and stearylamine was almost or completely zero. Total lipids from Escherichia coli adsorbed 0.057 in comparison to 0.051 μg PX/cm² of an artificial mixture of phosphatidylethanolamine/phosphatidylglycerol/cardiolipin in the proportions 75 : 25 : 5. The concentration of the surface active PX at the air/water interphase was 0.091 μg/cm². These saturation surface concentrations of PXat lipid monolayers were reached at 1 μg/ml bulk concentrations in 2 mM NaCl/1 mM Tris · HCl, pH 7.2. They decreased with decreasing surface charge density of the adsorbing monolayer. In an experiment with cardiolipin/phosphatidylethanolamine mixtures it was shown that two molecules of cardiolipin induced adsorption of one molecule PX giving a 1 : 1 ratio with regard to positive and negative charges. This could be due to a similar charge density of about one charge per 40–50 Ų in PX and lipid bilayers composed of phospholipids. The electrostatic PX-lipid interaction was severely inhibited by 10^?2 and 10^?1 M Ca²⁺ and Na⁺, respectively. It is discussed that the specificity of PX against Gram-negative bacteria is caused by the occurrence of lipid A, phosphatidylglycerol and cardiolipin at the cell surface of these microorganisms.     

Proton nuclear magnetic resonance studies on the iodide binding by horseradish peroxidase

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.     

Deuterium nuclear magnetic resonance investigation of dimyristoyllecithin--dipalmitoyllecithin and dimyristoyllecithin--cholesterol mixtures

Deuterium nuclear magnetic resonance (NMR) spectra of 1,2-dimyristoyl-3-sn-phosphatidylcholines (DMPCs) specifically deuterated in the 2-chain at one of positions 2', 3', 6', or 14' have been obtained by the quadrupole-echo Fourier transform method at 34.1 MHz (corresponding to a magnetic field strength of 5.2 T) or the pure material as a function of temperature, and in the presence of either 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) or cholesterol as a function of temperature and composition. The results with pure DMPC and DMPC--DPPC mixtures indicate that a sharp, intense deuterium resonance is characteristic of fluid-phase lipids, whereas a broad resonance is characteristic of solid-phase lipids. There is shown to be good agreement between the deuterium NMR derived DMPC--DPPC phase diagram and that derived by using other techniques. The deuterium NMR results obtained with the DMPC--cholesterol system are not interpreted in terms of a phase diagram. They do indicate, however, that the transition breadth is increased considerably and the temperature at which the lipid chains "solidify" is depressed by the addition of cholesterol to the DMPC bilayer. The particular nature of the increase and the depression is found to be dependent on where the label is located on the lipid.     

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.     

Proton and phosphorus nuclear magnetic resonance studies of an oligothymidylate covalently linked to an acridine derivative and of its binding to complementary sequences

An oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3'-phosphate [(Tp)4(CH2)5Acr] was synthesized. Its conformation in solution was investigated by proton magnetic resonance. Both intramolecular interactions between the acridine dye and thymines and intermolecular interactions were demonstrated. Both proton and phosphorus magnetic resonances were used to study the specific interaction of (Tp)4(CH2)5Acr with poly(rA) and (Ap)3A. The results were compared to those obtained when the acridine-containing substituent was replaced by an ethyl group attached to the 3'-phosphate of the oligothymidylate. The acridine dye strongly stabilized the complexes formed with both poly(rA) and (Ap)3A. Upfield shifts of both adenine and acridine proton resonances were observed in the complexes. These results were ascribed to an intercalation of the acridine ring between A X T base pairs of the duplex structure formed by the oligothymidylate with its complementary oligoadenylate sequence. An analysis of proton and phosphorus chemical shifts as well as measurements of T1 relaxation times at different temperatures allowed us to propose several structures for the complexes formed by (Tp)4(CH2)5Acr with its complementary sequence.     

Deuterium nuclear magnetic resonance studies on the plasmalogens and the glycerol acetals of plasmalogens of Clostridium butyricum and Clostridium beijerinckii

Deuterium nuclear magnetic resonance was used to investigate the structure of different lipid fractions isolated from the anaerobic bacteria Clostridium butyricum and Clostridium beijerinckii. The fractions isolated from C. butyricum were (1) phosphatidylethanolamine/plasmenylethanolamine and (2) the glycerol acetal of plasmenylethanolamine, and from C. beijerinckii similar fractions containing principally (1) phosphatidyl-N-monomethylethanolamine, along with its plasmalogen, and (2) the glycerol acetal of this plasmalogen were isolated. The third fraction from both species consisted largely of the acidic lipids phosphatidylglycerol and cardiolipin along with plasmalogen forms of these lipids. Palmitic acid with deuterium labels at C-2, C-3, or C-4 or oleic acid with deuterium labels at C-2 and C-9,10 was added to the growth medium and incorporated to various extents in the lipid fractions. Biochemical analysis showed that palmitic acid and oleic acid were preferentially bound to the sn-2 and sn-1 positions, respectively, of the glycerol backbone when both fatty acids were added to the medium. From the 2H NMR spectra, the hydrocarbon chain ordering near the lipid-water interface could be determined and appeared to be similar for all three lipid fractions. The deuterium quadrupole splitting and order parameter were low at the C-2 segment and increased by almost a factor of 2 at positions C-3 and C-4 for cells fed with deuterated palmitic acid along with unlabeled oleic acid. These results agree with previous findings on pure diacyl lipids in which the sn-2 chain was found to adopt a bent conformation at the carbon segment C-2. However, two unusual quadrupole splittings could be detected for the plasmalogens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic resonance studies on the binding of etomidate to lipid bilayers

Physicochemical studies on the binding of etomidate, a fast acting anaesthetic, with lipid bilayers have been carried out. ESR spin labeling studies indicate that the gel to liquid crystalline phase transition of dipalmitoyl phosphatidyl choline (DPPC) vesicles retains its cooperative nature on incorporation of the anaesthetic. For a 5:1 lipid to drug molar ratio, the phase transition occurs at an unusually lower temperature than those observed with other drug-DPPC systems. Results of 13C NMR and 1H NOE experiments suggest that the drug molecules reside in the close proximity of the terminal of hydrocarbon chains of the lipid molecules. 31P NMR and Electron Microscopic experiments indicate that the presence of etomidate alters the normal lamellar structure of DPPC vesicles into hexagonal (HII) type. Based on these observations, a model for drug-lipid binding has been proposed.     

Proton and iodine-127 nuclear magnetic resonance studies on the binding of iodide by lactoperoxidase

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.     

Nucleotide binding to tubulin-investigations by nuclear magnetic resonance spectroscopy

In an attempt to distinguish between the interaction of GTP and ATP with tubulin dimer, high-resolution ¹H- and ³¹P-NMR experiments have been carried out on the nucleotides in the presence of tubulin. The location of the ATP binding sites on the protein in relation to the GTP sites is still not clear. Using NMR spectroscopy, we have tried to address this question. Evidence for the existence of a site labelled as X-site and another site (labelled as L-site for both the nucleotides on tubulin has been obtained. It is suggested that this X-site is possibly the putative E-site. In order to gain further insight into the nature of these sites, the Mg(II at the N-site has been replaced by Mn(II and the paramagnetic effect of Mn(II on the linewidth of the proton resonances of tubulin-bound ATP and GTP has been studied. The results show that the L-site nucleotide is closer to the N-site metal ion compared to the X-site nucleotide. On the basis of these results, it is suggested that the L-site of ATP is distinct from the L-site of GTP while the X-site of both the nucleotides seems to be same. By using the paramagnetic effect of the metal ion, Mn(II), at the N-site on the relaxation rates of tubulin-bound ATP at L-site, distances of the protons of the base, sugar and phosphorous nuclei of the phosphorous moiety of ATP, from the N-site metal ion have been mapped. The base protons are 2 0.7–1 nm distant from the N-site metal ion, while the protons of the sugar are 2 0.8-1 nm from this metal ion site. On the other hand, the phosphorous nuclei of the phosphate groups are somewhat nearer (2 0.4–0.5 nm from the N-site metal ion.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP+-specific isocitrate dehydrogenase

The interaction of the 2'-phosphate-containing nucleotides (NADP+, NADPH, 2'-phosphoadenosine 5'-diphosphoribose, and adenosine 2',5'-bisphosphate) with NADP+ -specific isocitrate dehydrogenase was studied by using 31P NMR spectroscopy. The separate resonances corresponding to free and bound nucleotides, characteristic for slow exchange of nuclei on the NMR time scale, were observed in the spectra of the enzyme (obtained in the presence of excess ligand) with NADP+ and NADPH in the absence and presence of Mg2+ and with 2'-phosphoadenosine 5'-diphosphoribose in the absence of metal or in the presence of the substrate magnesium isocitrate. The position of the 31P resonance of the bound 2'-phosphate group in these spectra is invariant (delta = 6) in the pH range 5-8, indicating that the pK of this group is much lower in the complexes with the enzyme than that (pK = 6.13) in the free nucleotides. The additional downfield shift of this resonance by 1.8 ppm beyond that (delta = 4.22) of the dianionic form of the 2'-phosphate in free nucleotides suggests interaction with a positively charged group(s) and/or distortion of P-O-P angles as the result of binding to the enzyme. A single resonance of 2'-phosphate was observed in the spectrum of the enzyme complex with 2'-phosphoadenosine 5'-diphosphoribose in the presence of Mg2+, with the chemical shift dependent on the nucleotide to enzyme ratio, characteristic for the fast exchange situation. Addition of metal does not perturb the environment of the 2'-phosphate in the complexes of NADP+ and NADPH with isocitrate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance studies on the kinetics of tryptic fragments of calmodulin upon calcium binding

The kinetics of the Ca2+-dependent conformational change of the tryptic fragments F12 (residues 1-75) and F34 (residues 78-148) of calmodulin were studied by 1H-NMR. Resonances of two phenylalanines, 16 (or 19) and 65 (or 68), N epsilon, N epsilon, N epsilon-trimethyllysine-115 and tyrosine-138 were examined by the saturation-transfer technique or computer-aided line-shape simulation to obtain the rate of the conformational exchange between the Ca2+-free form and the Ca2+-bound form. The rates for F12 and F34 in the presence of 0.2 M KCl at 22 degrees C were 300-500 s-1 and 3-10 s-1, respectively. Activation parameters are as follows: Delta H not equal to = 11(+/- 2) kcal X M-1 and delta S not equal to = -9(+/- 5) cal X K-1 X M-1 for F12, and delta H not equal to = 16(+/- 2) kcal X M-1 and delta S not equal to = -2(+/- 5) cal X K-1 X M-1 for F34. These kinetic data for the conformational exchange are in agreement with those of Ca2+ dissociation from the binding sites obtained by 43Ca-NMR and stopped-flow fluorescence studies.