Indirect observation by 13C NMR spectroscopy of a novel CO2 fixation pathway in methanogens.

High-field carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the isotopic dilution of doubly carbon-13-labeled precursors for 2,3-cyclopyrophosphoglycerate, a novel primary metabolite that occurs in certain methanogens. A unique carbon dioxide fixation pathway that gives rise to asymmetric labeling of acetyl coenzyme A has been demonstrated in Methanobacterium thermoautotrophicum. The effect of selected metabolic inhibitors on the labeled species in the pathway has been examined by NMR. These techniques establish a general, sensitive method for the delineation of convergent biosynthetic pathways.

[Seperation and structure elucidation of alkaloids from Chinese drug buzhaye, Folium Microcos].

From the chloroform extracts of the dried Folium Microcos, four compounds were isolated by using repeated column chromatography on silica gel and recrystallization and their structures were elucidated by physicochemical properties and UV, MS and NMR, separately. They are N-methyl-6alpha-(deca-1', 3', 5'-trienyl)-3beta-methoxy-2beta-methylpiperidine, 6-(deca-1', 3', 5'-trienyl)-3-methoxy-2-methylpiperidine, N-methyl-6-(deca-1', 3', 5'-trienyl)-2, 3-dimethylpiperidine and N-methyl-6-(deca-1', 3', 5'-trienyl)-2-methylpiperidine, named as micropiperidine A, micropiperidine B, micropiperidine C and micropiperidine D, respectively. The latter three are new compounds.

15N-NMR studies of Methanobacterium thermoautotrophicum: comparison of assimilation of different nitrogen sources.

Methanobacterium thermoautotrophicum can utilize glutamine and urea as well as ammonia as the sole nitrogen source during growth on H2 and CO2. High-field 15N-NMR has been used to compare the assimilation of these different nitrogen sources by this organism. The 15N-NMR spectra of extracts of cells grown in media containing [delta-15N]glutamine as the nitrogen source show that the glutamine amide nitrogen is rapidly converted to glutamate. The 15N-NMR spectra of cell extracts from cells grown on [15N]urea show a marked increase in the labeling of the alpha-NH2 of glutamate concurrent with a decrease in the urea resonance. These two nitrogen sources do not show the metabolic shift to alanine as the major resonance in stationary phase as is seen with 15NH4Cl. This behavior is discussed in terms of the enzymes of nitrogen metabolism.

Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics.

For many of the enzymes that utilize or produce diacylglycerols, detergent mixed micelles are often used in assay systems to solubilize the lipophilic substrates or products. The assumption is often made that the diacylglycerol (DAG) is solubilized and well mixed throughout the population of micelles during the time course of the assay. In the present work the partitioning and exchange dynamics of diacylglycerols (from dihexanoyl-DAG to didecanoyl-DAG) in a variety of detergent micelles have been studied by NMR and fluorescence methods. In all detergents, the longer the DAG chain lengths, the more detergent is required for solubilization. However, efficiency of solubilization varies tremendously with Triton X-100 the most efficient (i.e. the least detergent is required), and deoxycholate the least efficient in solubilizing DAG. The mixing and exchange dynamics of pyrene-labeled DAG molecules in these micelles (measured by stopped-flow fluorescence) were fastest for Triton X-100 and slowest with charged bile salt micelles. Of the detergent systems characterized, Triton X-100 appears to be the optimal detergent for use in assays of enzymes that interact with DAG (beta-octylglucoside and diheptanoylphosphatidylcholine have good exchange dynamics, but higher amounts of these detergents are needed to solubilize DAG). Bile salt micelles provide the least solubilization and the slowest exchange kinetics (so slow that this could be a significant problem in some enzyme assays). This information on DAG behavior in micelles is discussed with respect to assays of an enzyme that generates DAG as product (phospholipase C) and one that uses DAG as substrate (DAG kinase). Although slow exchange of DAG occurs in some micelle systems, this does not appear to be a rate-limiting step in the kinetics for either of these enzymes.

Vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus.

Monomeric vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus, much better than other oxyanions (e.g., phosphate or iodate). The apparent efficiency of inhibition depends on the substrate aggregate structure. The measured inhibition constant with respect to monomeric phosphatidylcholine substrate is 0.21 mM under conditions where the K(m) is 0.12 mM; for micellar substrate the apparent Ki appears much lower and in fact tracks the apparent K(m) which decreases 10-fold. Vanadate inhibition is removed by addition of exogenous diacylglycerol, which by itself is an inhibitor. In contrast to its effect with monomeric or micellar substrate, vanadate does not strongly inhibit the PLC-catalyzed hydrolysis of small unilamellar vesicles of phosphatidylcholine. These results are interpreted in terms of the surface binding of the enzyme. Because of its ability to mimic the transition state of phosphate ester hydrolysis vanadate is also used to investigate the constraints on the occurrence of strained cyclic intermediates in phospholipid hydrolysis by PLC.

Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover.

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.

Correlation of 19F-NMR spectra of halothane in rat tumor and non-tumor tissues with membrane alterations.

The membrane environments in normal and tumor rat tissue and the effect of hyperthermia thereon are studied with 19F-NMR spectroscopy of the general anesthetic halothane. Normal and tumor cell types are clearly differentiated by the halothane resonance. A hydrophobic environment prominent in tumor tissue is more sensitive to heat treatment than the corresponding environments of normal cells. Studies of extracted lipids suggest that this may be due in part to the considerable difference in lipid temperature response which exists between normal and kidney tumor cells.

Charged detergents enhance the activity of phospholipase C (Bacillus cereus) towards micellar short-chain phosphatidylcholine.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus) activity toward diheptanoylphosphatidylcholine is increased 50-100% by low concentrations of both positively and negatively charged detergents. Zwitterionic and nonionic detergents have no such activating effect. This charged detergent activation requires an interface, since comparable detergent concentrations have no effect on the hydrolysis rate of monomeric dihexanoylphosphatidylcholine. From NMR and diacylglycerol solubility studies it is suggested that activation results from detergent interacting with diacylglycerol to accelerate product release from the enzyme.

Beta-aminoglutaric acid is a major soluble component of Methanococcus thermolithotrophicus.

13C- and 15N-NMR spectroscopy have been used to identify beta-aminoglutaric acid (beta-glutamic) as a major soluble component of the thermophilic, autotrophic marine methanogen Methanococcus thermolithotrophicus. This rare, non-protein amino acid has been recognized as a major dissolved free amino acid in marine sediments, but the microorganism responsible for its production has not previously been identified. The concentration of beta-aminoglutarate (beta-glutamate) is about one half that of free alpha-glutamate and increases (relative to the alpha-isomer) as cells enter the stationary phase. Analysis of the 13C label distribution in a 13CO2-pulse/12CO2-chase experiment shows that label enters the beta-aminoglutarate pool after it has decayed from other small soluble molecules. This implies that beta-aminoglutarate is a catabolic product of the cells. Preliminary biosynthesis studies with labeled precursors indicate that only a single acetate moiety is incorporated in this unusual compound. This information is used to suggest possible biosynthetic pathways.

31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanobacteria strains.

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.

Multinuclear NMR studies of the trp-repressor.

The binding of the corepressor, L-tryptophan, to the Escherichia coli trp-aporepressor in solution has been examined by 13C- and 19F-NMR spectroscopy. The binding of a number of tryptophan analogues have been studied by equilibrium dialysis. Evidence is presented that support the crystallographic studies (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. and Sigler, P. B. (1985) Nature 317, 782-786) that Val-58 is within the ring currents of the bound tryptophan and also close in space to the indole 5'-position, on the basis of heteronuclear 19F(1H)-NOE experiments. The tryptophan carboxylate is in hydrogen-bonding distance to a highly positively charged residue, probably Arg-54 and this bond strengthens on formation of the trp-repressor-DNA complex.

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl.

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.

Effect of vesicle composition and curvature on the dissociation of phosphatidic acid in small unilamellar vesicles--a 31P-NMR study.

Sonicated small unilamellar vesicles (SUVs) containing phosphatidic acid (PA) give two PA 31P-NMR resonances corresponding to PA molecules in the inner and outer leaflets of the bilayer. This NMR differentiation between the two monolayers is not due to a pH gradient across the membrane but instead reflects differential packing in the inner and outer leaflets imposed by the highly curved SUV surface. The apparent pKa of the outer-leaflet PA increases with decreasing surface curvature and with increasing PA content. The estimated relationship between the apparent pKa of the outer-leaflet PA headgroup and vesicle curvature may provide a qualitative probe for effects related to surface curvature in these model-membrane systems.

Observation of tyrosine-O-phosphate in Drosophila melanogaster larvae by 31P-NMR spectroscopy.

31P-NMR spectra of intact larvae and pupae of Drosophila melanogaster have been obtained at 109.3 MHz. A major resonance in these samples has been identified as tyrosine-O-phosphate. Its chemical shift reflects the hemolymph plasma pH. Upon disruption of the organisms (necessary for chemical analyses of tyrosine-O-phosphate), phosphatases rapidly hydrolyze this phosphate ester, generating inorganic phosphate and free tyrosine.

Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum.

When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest. Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS. There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids. Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more 'fluid' membrane. The suggestion is made that these membrane changes may be maladaptive to the performance of C. thermocellum.

The fluorinated anesthetic halothane as a potential NMR biologic probe.

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid.

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications.

The structural features of a phosphatidylcholine molecule important for binding to phospholipase C (Bacillus cereus) have been examined using kinetic analyses of a series of short-chain phosphatidylcholines and analogues. Lipids examined had varying chain lengths, methyl branched chains, phenyl alkanoate chains, and a single fatty acyl chain (lysophosphatidylcholines). A comparison of Vmax and Km for monomolecularly dispersed dibutyroyl-, dihexanoyl- and diheptanoylphosphatidylcholine indicates that the length of the fatty acyl chains must be at least six carbons for efficient binding of the phosphatidylcholine to the enzyme. Enzymatic rates of hydrolysis for pure short-chain phosphatidylcholine micelles of different chain lengths or detergent mixed micelles with comparable concentrations of short- and long-chain phosphatidylcholines show no dependence on substrate chain length greater than six carbons. Methyl branching of short-chain phosphatidylcholines only inhibits phospholipase C activity when the methyl group is adjacent to the carbonyl (e.g., di(2-methyl)hexanoylphosphatidylcholine). In a similar fashion, phosphatidylcholines with phenylalkanoate chains become poor substrates when the phenyl group is near the acyl linkage. As the phenyl group is moved from C-4 to C-2 a large increase in the micellar apparent Km is observed. Chain specificity (sn-1 and/or sn-2 ester linkages) for binding is not absolute, since phospholipase C will hydrolyze micellar short-chain lysophosphatidylcholines at rates one tenth of phosphatidylcholines. In contrast, substitution of ester linkages with ether moieties yields phosphatidylcholine analogues which are even poorer substrates and not good inhibitors of phospholipase C. These results suggest that the carbonyl group and its immediate environment are important for phospholipid interacting with this water-soluble lipolytic enzyme.

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus.

Two phospholipase D (PLD) enzymes with both hydrolase and transferase activities were isolated from Streptomyces chromofuscus. There were substantial differences in the kinetic properties of the two PLD enzymes towards monomeric, micellar, and vesicle substrates. The most striking difference was that the higher molecular weight enzyme (PLD57 approximately 57 kDa) could be activated allosterically with a low mole fraction of phosphatidic acid (PA) incorporated into a PC bilayer (Geng et al., J. Biol. Chem. 273 (1998) 12195-12202). PLD42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4-6-fold higher Vmax toward PC substrates than PLD57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD42/20 were cleavage products of PLD57. The larger component included the N-terminal segment of PLD57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD57; this peptide had no PLD activity by itself. Increasing the pH of PLD42/20 to 8.9, followed by chromatography of PLD42/20 on a HiTrap Q column at pH 8.5 separated the 42- and 20-kDa proteins. The 42-kDa complex had about the same specific activity with or without the 20-kDa fragment. The lack of PA activation for the 42-kDa protein and for PLD42/20 indicates that an intact C-terminal region of PLD57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD57.