Mammalian fatty acid synthase activity from crude tissue lysates tracing (13)C-labeled substrates using gas chromatography-mass spectrometry

Fatty acid synthase (FASN or FAS, EC 2.3.1.85) is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl- and malonyl-coenzyme A (CoA) esters. This article describes a new method that directly quantifies uniformly labeled (13)C(16)-labeled palmitate ([(13)C(16)]palmitate) by tracing [(13)C(2)]acetyl-CoA and [(13)C(3)]malonyl-CoA using an in vitro FASN assay. This method used gas chromatography-mass spectrometry (GC-MS) to detect [(13)C(16)]palmitate carboxylate anions (m/z 271) of pentafluorobenzyl (PFB) derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [(13)C(16)]palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates ensured equal FASN amounts, preserved steady-state kinetics, and enabled calculation of FASN-specific activity. FASN activity determined by [(13)C(16)]palmitate synthesis was consistent with values obtained from β-nicotinamide adenine dinucleotide 2'-phosphate (NADPH) oxidation assays. Analysis of FASN activity from tissue extracts was not hampered by contaminating enzymes or preexisting fatty acids. Crude mammary gland and liver lysates had significantly different activities at 82 and 65nmolmin(-1)mg(-1), respectively, suggesting that tissue-specific activity levels differ in a manner unrelated to FASN amount. GC-MS quantification of [(13)C(16)]palmitate synthesis permits sensitive evaluation of FASN activity from tissues of varied physiological states and of purified FASN activity in the presence of modifying proteins, enzymes, or drugs.     

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

Effects of metabolic substrates and inhibitors on weak organic anion transport in rat renal proximal tubules

Stimulatory effects of intermediates of the tricarboxylic acid cycle on renal uptake of a weak organic anion, fluorescein, were studied with the aid of the method of contact microfluorimetry of individual convoluted proximal tubules ascending to the surface of the rat renal cortex slices. The study was undertaken for verifying the hypothesis that energization of renal excretion of anionic exenobiotics is mediated through their transport across the basolateral membrane in exchange for cytoplasmic alpha-ketoglutarate serving as a counter-anion. Effects of inhibitors of the tricarboxylic acid cycle such as fluoroacetate, malonate and 5-methoxyindole-2-carboxylate on the fluorescein uptake and renal gluconeogenesis in the presence of the metabolic substrates were investigated in order to outline metabolic pathways that could be responsible for elevation of the cytoplasmic alpha-ketoglutarate. Obtained data evidence that the stimulatory effects of the tricarboxylic acid cycle intermediates on the transport process under study depend on the metabolic state of the mitochondria and involve an activation of certain reactions but not the cycle as a whole. It has been suggested that an elevation of the cytoplasmic alpha-ketoglutarate resulting from this activation can be conditioned by export of isocitrate from the mitochondria with its subsequent transformation into alpha-ketoglutarate in the cytoplasm in the isocitrate dehydrogenase reaction.     

Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with ¹³C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO₂ via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that α-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.     

13C-NMR study of glycerol metabolism in rabbit renal cells of proximal convoluted tubules

Perchloric acid extracts of rabbit renal proximal convoluted tubular cells (PCT) incubated with [2-13C]glycerol and [1,3-13C]glycerol were investigated by 13C-NMR spectroscopy. These 13C-NMR spectra enabled us to determine cell metabolic pathways of glycerol in PCT cells. The main percentage of 13C-label, arising from 13C-enriched glycerol, was found in glucose, lactate, glutamine and glutamate. So far it can be concluded that glycerol is a suitable substrate for PCT cells and is involved in gluconeogenesis and glycolysis as well in the Krebs cycle intermediates. Label exchange and label enrichment in 13C-labelled glucose, arising from [2-13C]glycerol and [1,3-13C]glycerol, is explained by label scrambling through the pentose shunt and a label exchange in the triose phosphate pool. From relative enrichments it is estimated that the ratio of the pyruvate kinase flux to the gluconeogenetic flux is 0.97:1 and that the ratio of pyruvate carboxylase activity relative to pyruvate dehydrogenase activity is 2.0:1. Our results show that 13C-NMR spectroscopy, using 13C-labelled substrates, is a powerful tool for the examination of renal metabolism.     

Sulfhydryl groups are essential for organic cation exchange in rabbit renal basolateral membrane vesicles

The effect of N-ethylmaleimide (NEM), an irreversible sulfhydryl modifying reagent, on the transport of organic cations in the renal basolateral membrane was examined. The studies were conducted examining the exchange of [3H]tetraethylammonium (TEA) for unlabeled TEA in basolateral membrane vesicles isolated from the outer cortex of rabbit kidneys. NEM inactivated TEA transport in a dose-dependent fashion with an IC50 value of 260 microM. The rate of TEA transport inactivation followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 4.0. The data imply that inactivation involves the binding of at least four molecules of NEM per active transport unit. This is most consistent with the presence of four sulfhydryl groups at this site. The substrate TEA displayed a dose-dependent enhancement of NEM inactivation, with 50% enhancement occurring at 365 microM TEA. Another organic cation, N1-methylnicotinamide, known to share a common transport mechanism with the TEA/TEA exchanger is also capable of increasing the reactivity of sulfhydryl groups to NEM. These results demonstrate that there are essential sulfhydryl groups for organic cation transport in the basolateral membrane. In addition, the capability of organic cations to alter the susceptibility to sulfhydryl modification suggests that these groups may have a dynamic role in the transport process.     

Three-dimensional 1H-TOCSY-relayed ct-[13C,1H]-HMQC for aromatic spin system identification in uniformly 13C-labeled proteins

Summary Three-dimensional ¹H-TOCSY-relayed ct-[¹³C,¹H]-HMQC is a novel experiment for aromatic spin system identification in uniformly ¹³C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect ¹H and ¹³C chemical shift evolution periods and for the generation of ¹H-¹H antiphase magnetization to accelerate the ¹H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of ¹³C-¹H zero-quantum and double-quantum coherences is to first order insensitive to ¹³C-¹H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.     

Differentiation between unlabeled and very-low-level fully 15N,13C-labeled nucleotides for resonance assignments in nucleic acids

Based on different characteristics between unlabeled and fully ¹⁵N,¹³C-labeled nucleotides, we develop a method for unambiguous resonance assignments in nucleic acids following site-specific fully ¹⁵N,¹³C isotope incorporation at very low levels¹. The J-couplings between heteronuclei provide for distinction between the NMR signals of the fully labeled nucleotides and those of the natural abundance nucleotides. The method is demonstrated for DNA oligonucleotides², in the dimeric G-quadruplex [d(GGGTTCAGG)]₂and in the 22-nucleotide human telomeric fragment d[AG₃(TTAG₃)₃]. We expect this approach to be useful for selective monitoring of important functional domains and of their interactions in large nucleic acids.     

Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides

Summary New 2D and 3D ¹H-¹³C-¹⁵N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in ¹³C-and ¹⁵N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective ¹³C-refocusing and ¹⁵N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ¹H-¹⁵N and 3D¹H-¹³C-¹⁵N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.     

Production of the (14)C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce (14)C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-(14)C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4?mg C L(-1). Concentrations of 12.6?g (14)C-labeled glycerol L(-1) (1?%?v/v) resulted in the production of 17.1?mg (14)C-Cry1Ab L(-1) cultivation medium. (14)C mass balances showed that approx. 50?% of the label was lost by respiration and 20?% remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05?% of the total (14)C originated from Cry1Ab. In the presence of 2.04?MBq (14)C-labeled carbon sources, a specific activity of up to 268?Bq?mg(-1) (14)C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63?MBq and an extended cultivation period of 144?h. This study demonstrates that (14)C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple (14)C-labeled carbon source. It also provides a general strategy to produce (14)C-labeled proteins useful for soil metabolic studies.     

Kininogen substrates for trypsin and cathepsin D in human,rabbit and rat plasmas

Studies have compared “total”, HMW kininogen and leukokininogen levels in human, rabbit and rat plasmas using trypsin, glass powder and cathepsin D as kininogenases or activators of kininogenases. Rat plasma was found to have about 10 fold more leukokininogen than the other plasmas assayed. When trypsin was used to estimate total kininogen, rat plasma liberated maximal amounts of kinin only in the presence of high concentrations of trypsin (1 mg/ml incubation mixture). In addition, it was found that trypsin in these concentrations liberated from rat plasma both bradykinin and a previously unidentified kinin which we have termed “T-kinin”. The results overall indicate that in the case of rat and rabbit plasma, currently used methods for estimations of total kininogen may not be accurate. T-kinin may represent a leukokininogen or a hitherto undescribed kininogen.     

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r2 = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.     

A study on the digestion of human muscle biopsies for trace metal analysis using an organic tissue solubilizer.

A commercially available tissue solubilizer, based on tetraalkyl-ammonium hydroxide in toluene, has been compared with conventional digestion producers in order to determine the minimum amount of tissue needed for the determination of trace elements such as Al, Cd, Cu, and Mn in human biopsy material using flameless atomic absorption spectrophotometry. The solubilizer can be recommended for digestion of biopsy samples in order to determine Cu, Cd, and Mn, but not Al.     

Application of 13C(omega 1)-half-filtered [1H,1H]-NOESY for studies of a complex formed between DNA and a 13C-labeled minor-groove-binding drug

The complex formed between the anticancer drug 4-[p-[p-(4-quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d-(GCATTAATGC)2 was investigated using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(omega 1)-half-filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [1H,1H]-NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [1H,1H]-NOESY provides an improved experimental basis for structural studies of drug/DNA complexes.     

Differential Effects of a Substituted Pyridazinone, BASF 13-338, on Pathways of Monogalactosyldiacylglycerol Synthesis in Arabidopsis

We have examined the effects of the substituted pyridazinone herbicide, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338, Sandoz 9785), on the desaturation of linoleic acid (18:2) on different molecular species of monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) in leaf tissue of Arabidopsis thaliana (L.) Heynh. Specific changes in lipid composition allowed identification of different substrates for desaturation of 18:2 to linolenic acid (18:3). 18:2/16:2 MGDG was desaturated in the chloroplast to form 18:3/16:3 MGDG. Levels of 18:3/16:3 MGDG were reduced by treatment with BASF 13-338, suggesting that both the formation of 18:3 at the sn-1 position, and the formation of 16:3 at the sn-2 position of 18:2/16:2 MGDG were inhibited by this compound. Kinetic studies using exogenously incorporated [¹⁴C] 18:1 indicated that 18:2/18:3 MGDG originated from an 18:2/18:3 diglyceride precursor derived from PC. The formation of 18:3 at the sn-1 position of 18:2/18:3 MGDG was also inhibited by BASF 13-338. In contrast the desaturation of 18:2 proposed to occur at the sn-2 position of PC outside the chloroplast, was not affected.     

Trypsin-catalyzed kinetically controlled synthesis of a precursor dipeptide of thymopentin in organic solvents, using a free amino acid as nucleophile

Trypsin-catalyzed, kinetically controlled synthesis of a precursor, dipeptide of thymopentin (TP-5), Bz-Arg-Lys-OH (or-OEt) in organic solvents was studied. Bz-Arg-OEt was used as the acyl donor and Lys-OH and Lys-OEt were used as the nucleophiles. Ethanol was selected as the organic solvent from ethanol, methanol, acetonitrile, and ethyl acetate tested under the experimental conditions. As expected, Lys-OEt is not a suitable nucleophile in trypsin-catalyzed reaction, due to its competition with the protective Arg-OEt as acyl donor for the active site of trypsin, while Lys-OH does not have this problem. The optimal reaction condition for the synthesis of Bz-Arg-Lys-OH was set up as 20% Tris-HCl buffer, pH 8.0, 35 degrees C for 6 h with the yield of 52.5%, or for 18-24 h with the yield of about 60%.