Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine

The stoichiometric palmitoyllysophosphatidylcholine (lysoPC)/gramicidin (4:1, mol/mol) lamellar complex (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141-144) is a useful model system to investigate the various aspects of lipid protein interactions. To study the effect of gramicidin on local order and motion of 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) we employed 31P and 2H nuclear magnetic resonance (NMR) using selectively deuterated lysoPC's and we compared the results to those obtained for lysoPC in bilayers with cholesterol (1:1, mol/mol) and dipalmitoylphosphatidylcholine (DPPC) (1:4, mol/mol). 2H-NMR experiments on acyl chain deuterated lysoPC showed similar quadrupole splittings in the liquid crystalline state for the lysoPC/DPPC and the lysoPC/gramicidin samples. In the lysoPC/cholesterol sample an increase of the quadrupole splitting was found. T1 measurements showed that gramicidin decreases the lysoPC acyl chain motion, especially at the C12 position. In the lysoPC/cholesterol sample an increase of motion was observed as compared to lysoPC in fluid bilayers of DPPC. 31P-NMR and 2-H-NMR measurements of lysoPC, deuterated at the alpha- and beta-position of the choline moiety, indicated an increase in headgroup flexibility in all samples as compared to the parent compound DPPC. In addition, a change in headgroup conformation was observed. The alpha- and beta-segments in all samples exhibited concerted motion. It was found that also in the polar headgroup gramicidin induces a decrease of the rate of motion.     

Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver

In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.     

Ketogenesis in the living rat followed by 13C-NMR spectroscopy. Infusion of [1,3-13C]octanoate

13C-NMR spectroscopy was used as a noninvasive approach to study the metabolism of [1,3-13C]octanoate in rat liver. Using a properly adjusted surface coil a liver selection of better than 90% was achieved in the intact animal without abdominal surgery. After infusion of [1,3-13C]octanoate via the jugular vein different patterns of metabolites were observed depending on the physiological state of the rat. In the fasted animal, the major metabolites were those of the Krebs cycle while in the diabetic animal ketogenic end products were predominant. As a fatty acid of medium chain length octanoate is imported into the inner mitochondrial space without control by the carnitine acyl transferase system. Hence, the metabolic differences observed between diabetic and fasted rats result from an intramitochondrial control mechanism. The in vivo 13C-NMR results therefore support previous biochemical in vitro studies which concluded that a major control of ketone body production occurs in the inner mitochondrial space, presumably via the redox potential of the liver. As an unexpected result, 13C-NMR provides evidence for the transitory esterification of the infused 13C-labeled octanoic acid. The corresponding 13C-NMR chemical shifts are typical for glycerides.     

2H-NMR studies on ether lipid-rich bacterial membranes: deuterium order profile of Clostridium butyricum

Palmitic acid specifically deuterated at different carbon atoms, has been incorporated biosynthetically into the membrane lipids of Clostridium butyricum. The lipids of this organism are rich in plasmalogens and their glycerol acetals and exhibit an unusual fatty acyl and alkenyl chain distribution with saturated chains mainly at the sn-2 position and unsaturated chains at the sn-1 position. The ordering of the deuterated hydrocarbon chains in whole cells was measured with deuterium nuclear magnetic resonance and was compared to the order profiles of isolated cell membranes and membranes formed from the total phospholipid extract. The shape of the order profiles was similar for all three membranes, but the absolute values of the order profiles in whole cells and isolated membranes were lower than those of the liposomal lipids. The order profiles have the same characteristic shape as those found for the lamellar liquid-crystalline phases of synthetic diacylphospholipids.     

Interaction of melittin with phosphatidylcholine membranes. Binding isotherm and lipid head-group conformation

The binding of melittin to nonsonicated bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with an ultracentrifugation assay and with 2H and 31P nuclear magnetic resonance. Melittin binding could best be described by a partition equilibrium with Kp = (2.1 +/- 0.2) X 10(3) M-1, measuring the binding isotherm in the concentration range of 0-100 microM melittin and taking into account electrostatic effects by means of the Gouy-Chapman theory. This partition coefficient is smaller than that deduced for small sonicated vesicles and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium magnetic resonance revealed a conformational change of the phosphocholine head group upon melittin binding. The quadrupole splittings of the alpha and beta segments of the choline head group varied linearly with the amount of bound melittin but in opposite directions; i.e., the alpha splitting decreased, and the beta splitting increased. This conformational change is not specific to melittin but is a response of the phosphocholine head group to positive membrane surface charges in general. Quantitatively, melittin is one of the most efficient head-group modulators, the efficiency per unit charge comparable to that of charged local anesthetics or hydrophobic ions.     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Hydration of Escherichia coli lipids. Deuterium T1 relaxation time studies of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine in H2O/2H2O mixtures (9:1, v/v) were investigated with 2H-NMR. Comparison of the 2H2O spin lattice relaxation time (T1) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H2O systems. Two distinct hydration regions could be discerned in the T1 relaxation time profile. (1) A minimum of 11-16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of T1 approximately equal to 90 ms. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with T1 = 500 ms and hydration water with T1 = 80-120 ms. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

13C NMR for the assessment of human brain glucose metabolism in vivo

Proton-decoupled 13C NMR spectra of the human head were obtained during hyperglycemic glucose clamping using intravenous infusions of [1-13C]glucose in normal volunteers. In addition to 13C signals of mobile lipids, a variety of new metabolite resonances could be resolved for the first time in the human brain. At an enrichment level of 20% [1-13C]glucose, the signals of alpha- and beta-glucose at 92.7 and 96.6 ppm, respectively, could be detected in the human brain after only an infusion period of 15 min. The spatial localization of the different regions of interest was confirmed by 13C NMR spectroscopic imaging with a time resolution of 9 min. Increasing the enrichment level to 99% [1-13C]glucose not only improved the time resolution but allowed the detection of metabolic breakdown products of [1-13C]glucose. The time course of 13C label incorporation into the C2, C3, and C4 resonances of glutamate/glutamine and into lactate could be recorded in the human brain. These results suggest the possibility of obtaining time-resolved, spatially selective, and chemically specific information on the human body.