Evaluation of liver function by co-administration methodology using 13C-labelled benzoic acid and hippuric acid coupled with nuclear magnetic resonance spectroscopy

The amount of hippuric acid synthesized and excreted in the urine after benzoic acid loading (hippuric acid test) is a useful index of liver function. However, the hippuric acid test gives erroneous results in the event of failure of renal excretory function. A new stable isotope co-administration methodology using nuclear magnetic resonance (NMR) spectroscopy has been developed to overcome this defect. [7-(13)C]Benzoic acid and [glycine carbonyl-13C]hippuric acid ([gly-13C]hippuric acid), each 0.4-0.6 mmol kg(-1) were simultaneously administered intravenously as probes to normal or liver-injured rats and the urine was analysed by 100 MHz 13C NMR spectroscopy. Consequently, urinary excretion of [7-(13)C]hippuric acid formed from [7-(13)C]benzoic acid and [gly-13C]hippuric acid was successfully traced with very simple and convenient procedures. The urinary excretion of [7-(13)C]hippuric acid indicated the combined functions of hippuric acid synthesis and renal excretion, whereas that of [gly-13C]hippuric acid was indicative of renal excretion of hippuric acid only. The heights of resonances for C7 of [7-(13)C]hippuric acid and the glycine carbonyl carbon of [gly-13C]hippuric acid were used to calculate the concentrations of labelled hippuric acids. [7-(13)C]Hippuric acid was excreted more slowly than [gly-13C]hippuric acid by both normal and liver-injured rats. The liver-injured rats excreted the labelled hippuric acids more slowly than the normal rats. The kinetic parameters were computed for the individual rats on the basis of Michaelis-Menten elimination for benzoic acid and first-order elimination for hippuric acid. The maximum rates of metabolism (Vmax) (4.8-5.8 micromol min(-1) kg(-1)) and the renal elimination rate constants of hippuric acid (Kre) (0.010-0.021 min(-1)) in the liver-injured rats were lower than those (Vmax 6.7-11.8 micromol min(-1) kg(-1); Kre 0.026-0.045 min(-1)) in the normal rats. These results have demonstrated that liver function can be evaluated from the Vmax value even though the renal function of hippuric acid excretion (Kre) is impaired. Thus the co-administration methodology is feasible and can remove the defect of the previous hippuric acid test. These results could form the basis for a more convenient and reliable hippuric acid test in man.     

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.     

2D 1H-NMR conformational study of phosphatidylserine diluted in perdeuterated dodecylphosphocholine micelles. Evidence for a pH-induced conformational transition

The conformation of phosphatidylserine (DMPS) diluted in perdeuterated dodecylphosphocholine micelles (DPC) has been investigated by 1D and 2D proton NMR spectroscopy. Chemical shift pH dependence showed that the pK relative to the serine carboxyl titration (3.4 +/- 0.05) was nearly identical to that measured in bilayers. Chemical shift and NOE data revealed that the phosphatidylserine molecule undergoes a conformational transition upon titration of the serine carboxyl group. The NOE network observed between the different parts of the molecule was sufficiently abundant to allow, in combination with molecular modeling methods, an assessment of the conformational changes. The conformational changes mainly involve the glycerol backbone, which is parallel to the whole molecule, that is, to the layer normal, at low pH and becomes perpendicular to the whole molecule at neutral pH. In both cases, the conformations are remarkably close to those observed for the crystal forms of zwitterionic and negatively charged phospholipids. Two-dimensional proton NMR study of phospholipids, diluted in perdeuterated DPC micelles, appears to be a simple and relevant method to obtain complete and direct information on their conformations in a model membrane-solution interface.     

1H NMR structure of an antifungal gamma-thionin protein SIalpha1: similarity to scorpion toxins

The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.     

Surface charge response of the phosphatidylcholine head group in bilayered micelles from phosphorus and deuterium nuclear magnetic resonance

Solid-state phosphorus (31P) and deuterium (2H) nuclear magnetic resonance (NMR) spectroscopy over the temperature range of 25-50 degreesC were used to investigate bilayered micelles (bicelles) composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in the presence of either the anionic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol (DMPG) or the cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). The 31P-NMR spectra demonstrate that bicellar structures form with DMPG/DMPC ratios ranging from 0 to 50/50 and with DMTAP/DMPC ratios from 0 to 40/60, while the overall concentration of DHPC remains constant. The formation of bicelles containing charged amphiphiles is contingent upon the presence of NaCl, with 50 mM NaCl being sufficient for bicelle formation at all concentrations of charged amphiphile investigated, while 150 mM NaCl affords better resolution of the various 31P-NMR resonance signals. The 2H-NMR spectra demonstrate that the quadrupolar splittings (Deltanu) of head group-deuterated DMPC change inversely as a function of the amount of negative versus positive charge present, and that the changes for deuterons on the alpha-carbon are opposite in sense to those for deuterons on the beta-carbon. This indicates that head group-deuterated phosphatidylcholine functions as a molecular voltmeter in bicelles in much the same fashion as it does in spherical vesicles.     

The orientation and dynamics of substance P in lipid environments

The membrane-associated conformation of substance P (RPKPQQFFGLM-NH2) has been previously proposed to be the NK1-receptor-active conformation. In this work, NMR methods are applied to explore the orientation and dynamics of substance P at lipid surfaces for which the peptide's three-dimensional structure had been previously determined. Here the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS) micelles has been found to cause sequence specific changes in the acid- and base-catalyzed amide proton exchange rates relative to the solution state values. On binding of substance P to SDS micelles, the FFG portion showed the largest decreases in the base-catalyzed amide exchange rates. Similar sequence-specific changes in substance P are observed in the presence of DPC micelles, albeit at much weaker levels due to fast exchange between free and bound forms of the peptide. These differences are attributed to the location of the amide protons either in the surface double layer (via electrostatic effect) or inserted into the polar head group region of the micelles (via low dielectric). The sequence-specific effects of micelle association were also observed in the homonuclear nonselective spin-lattice relaxation time; these, in combination with spin-spin relaxation times, were used to calculate correlation times for the backbone amide protons. These data combined with paramagnetic broadening observations on peptide protons in the presence of spin-labeled lipids yield a detailed model of the interaction of substance P with lipid surfaces.     

cDNA cloning and expression of a family of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase sequence homologs from Caenorhabditis elegans

The initiation of mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTase) (EC 2.4.1.41). By screening two mixed-stage Caenorhabditis elegans cDNA libraries, a total of 11 distinct sequence homologs of the ppGaNTase gene family were cloned, sequenced, and expressed as truncated recombinant proteins (gly-3, gly-4, gly-5a, gly-5b, gly-5c, gly-6a, gly-6b, gly-6c, gly-7, gly-8, and gly-9). All clones encoded type II membrane proteins that shared 60-80% amino acid sequence similarity with the catalytic domain of mammalian ppGaNTase enzymes. Two sets of cDNA clones (gly-5 and gly-6) contained variants that appeared to be produced by alternative message processing. gly-6c contained a reading frameshift and premature termination codon in the C-terminal lectin-like domain found in most other ppGaNTase proteins, and a second clone (gly-8) lacked the typical C-terminal region completely. Homogenates of nematodes and immunopurified preparations of the recombinant GLY proteins demonstrated that worms express functional ppGaNTase enzymes (GLY-3, GLY-4, GLY-5A, GLY-5B, and GLY-5C), which can O-glycosylate mammalian apomucin peptide sequences in vitro. In addition to demonstrating the existence of ppGaNTase enzymes in a nematode organism, the substantial diversity of these isoforms in C. elegans suggests that mucin O-glycosylation is catalyzed by a complex gene family, which is conserved among evolutionary-distinct organisms.     

The mammalian staufen protein localizes to the somatodendritic domain of cultured hippocampal neurons: implications for its involvement in mRNA transport

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1, 2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. Studies on mice with a null mutation in the GnT I gene have indicated that N-glycans play critical roles in mammalian morphogenesis. This paper presents studies on N-glycans during the development of the nematode Caenorhabditis elegans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13, and gly-14). All three cDNAs encode proteins (467, 449, and 437 amino acids, respectively) with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in both insect cells and transgenic worms showed that gly-12 and gly-14, but not gly-13, encode active GnT I. All three genes were expressed throughout worm development (embryo, larval stages L1-L4, and adult worms). The gly-12 and gly-13 promoters were expressed from embryogenesis to adulthood in many tissues. The gly-14 promoter was expressed only in gut cells from L1 to adult developmental stages. Transgenic worms that overexpress any one of the three genes show no obvious phenotypic defects. The data indicate that C. elegans is a suitable model for further study of the role of complex N-glycans in development.     

Compartmentation of lactate and glucose metabolism in C6 glioma cells. A 13c and 1H NMR study

13C and 1H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]L-lactate or [1-13C]D-glucose. Using 11 mM [3-13C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.     

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, Abeta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles. In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of Abeta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6. 0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for Abeta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of Abeta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.     

A new antitubercular mulinane diterpenoid from Azorella madreporica Clos

Bioactivity-guided fractionation of the petroleum ether extract of Azorella madreporica Clos has led to the isolation of the novel, antitubercular mulinane diterpenoid 1. The structure has been elucidated on the basis of its 1D and 2D NMR spectra and by comparison with mulinolic acid 2 and a dehydration product 3 obtained from 1. The MIC of 1 for growth inhibition of the H37Rv strain of Mycobacterium tuberculosis was determined as 20 microg/mL. LC-MS and NMR have suggested the presence of this new compound in four other species of Azorella.     

Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II

Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances. The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation. This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data. Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

The heme prosthetic group of lactoperoxidase. Structural characteristics of heme l and heme l-peptides

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic 1H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic 1H NMR experiments on the low spin bis(cyano)-Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydroxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Paramagnetic 1H NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(III) complexes of the isolated heme species provide information regarding peptide content. Sequence analyses of peptides released from two heme l-peptide species by base hydrolysis suggest that heme-protein ester linkages in lactoperoxidase occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. These results confirm the earlier reported structural proposal (Rae, T. D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).     

Solution structure of methionine-oxidized amyloid beta-peptide (1-40). Does oxidation affect conformational switching?

The solution structure of Abeta(1-40)Met(O), the methionine-oxidized form of amyloid beta-peptide Abeta(1-40), has been investigated by CD and NMR spectroscopy. Oxidation of Met35 may have implications in the aetiology of Alzheimer's disease. Circular dichroism experiments showed that whereas Abeta(1-40) and Abeta(1-40)Met(O) both adopt essentially random coil structures in water (pH 4) at micromolar concentrations, the former aggregates within several days while the latter is stable for at least 7 days under these conditions. This remarkable difference led us to determine the solution structure of Abeta(1-40)Met(O) using 1H NMR spectroscopy. In a water-SDS micelle medium needed to solubilize both peptides at the millimolar concentrations required to measure NMR spectra, chemical shift and NOE data for Abeta(1-40)Met(O) strongly suggest the presence of a helical region between residues 16 and 24. This is supported by slow H-D exchange of amide protons in this region and by structure calculations using simulated annealing with the program XPLOR. The remainder of the structure is relatively disordered. Our previously reported NMR data for Abeta(1-40) in the same solvent shows that helices are present over residues 15-24 (helix 1) and 28-36 (helix 2). Oxidation of Met35 thus causes a local and selective disruption of helix 2. In addition to this helix-coil rearrangement in aqueous micelles, the CD data show that oxidation inhibits a coil-to-beta-sheet transition in water. These significant structural rearrangements in the C-terminal region of Abeta may be important clues to the chemistry and biology of Abeta(1-40) and Abeta(1-42).     

Structure of complex cell wall polysaccharides isolated from Trichoderma and Hypocrea species

The structure of fungal polysaccharides isolated from the cell wall of Trichoderma reesei, T. koningii, and Hypocrea psychrophila, have been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysaccharides have an irregular structure, idealized as follows: [formula: see text] The proportions of the different side chains vary from a species to another, being n above some three times larger in H. psychrophila than in T. reesei or T. koningii.     

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata

BACKGROUND: Killer lymphocytes secrete perforin, a 67 kDa protein that initiates T-cell cytolysis following aggregation and pore formation in target membranes. The resulting pores cause a breakdown of the transmembrane osmotic gradient and allow other cytolytic mediators to enter the target cell and initiate apoptosis. The cytolytic domain resides within the first 34 residues of the amino terminus of perforin, with residues 1-19 being sufficient for cytolytic activity. RESULTS: The solution structure of a 22-residue synthetic peptide (P22), corresponding to the amino terminus of human perforin, has been determined using high resolution nuclear magnetic resonance spectroscopy in the presence and absence of perdeuterated detergent (SDS) micelles. In aqueous solution, P22 exists mainly in a random conformation. However, it adopts a hook-like structure at the carboxyl terminus in the presence of SDS micelles when the positively charged residues cluster to form a turn that provides a binding surface to the negatively charged sulfate headgroups. CONCLUSIONS: The strong electrostatic interaction between the cationic region of the P22 peptide and the lipid headgroups probably weakens the membrane, facilitating insertion of the relatively neutral/hydrophobic stretch of P22, and is representative of the initial step of the lytic pathway. The structural model described here is probably relevant to understanding the mechanisms of other cationic antimicrobial peptides.     

Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.     

Glucose and lactate metabolism in C6 glioma cells: evidence for the preferential utilization of lactate for cell oxidative metabolism

13C and 1H nuclear magnetic resonance spectroscopy (NMR) was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The 13C enrichment of cell metabolites was examined after a 4-h incubation in media containing 5.5 mM glucose and 11 mM lactate, each metabolite being alternatively labelled with either [1-13C]D-glucose or [3-13C]L-lactate. The results indicated that exogenous lactate was the major substrate for oxidative metabolism. They were consistent with the concept of the existence of 2 pools of both lactate and pyruvate, of which 1 pool was closely connected with exogenous lactate and oxidative metabolism, and the other pool was closely related to glycolysis and disconnected from oxidative metabolism. The molecular basis of this behaviour could be related to different locations for the lactate dehydrogenase isoenzymes, as suggested by their immunohistochemical labelling.     

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.