S-Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography-Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [³⁵S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using ¹⁵N-labeled GSNO ([¹⁵N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.     

Progress in fold recognition

The prediction experiment reveals that fold recognition has become a powerful tool in structural biology. We applied our fold recognition technique to 13 target sequences. In two cases, replication terminating protein and prosequence of subtilisin, the predicted structures are very similar to the experimentally determined folds. For the first time, in a public blind test, the unknown structures of proteins have been predicted ahead of experiment to an accuracy approaching molecular detail. In two other cases the approximate folds have been predicted correctly. According to the assessors there were 12 recognizable folds among the target proteins. In our postprediction analysis we find that in 7 cases our fold recognition technique is successful. In several of the remaining cases the predicted folds have interesting features in common with the experimental results. We present our procedure, discuss the results, and comment on several fundamental and technical problems encountered in fold recognition. © 1995 Wiley-Liss, Inc.     

HPLC shadowing: Artifacts in peptide characterization monitored by RIA

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with ¹²⁵I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.     

Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture

Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H₂/CO₂ or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an acetate oxidizer in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F₄₂₀ autofluorescence. The coculture completely degraded 40 mol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH₄. Studies with ¹⁴C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO₂ by the coculture and that CO₂ was concurrently reduced to CH₄. During growth, there was significant isotopic exchange between CO₂ and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO₂ and H₂ by one organism, while H₂ was subsequently used by a second organism to reduce CO₂ to CH₄. Since the H₂ partial pressure must be maintained below 10^-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936.     

The Philadelphia variant of galactokinase.

We have previously reported the existence of a polymorphism that causes black populations to have lower mean RBC galactokinase activity than comparable white populations. We have designated this allele the Philadelphia variant, GALKP, and have suggested that it is common in blacks and rare in whites. GALKP individuals have normal WBC GALK activity, in contrast to the half normal WBC GALK activities of heterozygotes for the allele (GALKG) that causes the galactokinase-deficient form of galactosemia. In one family, we have presented evidence for the existence of two sisters heterozygous for both GALKG and GALKP alleles. These individuals have 50% normal WBC GALK activity and less than 50% normal red cell activity. The latter finding indicates that the two variant GALK alleles additively affect RBC activity. The WBC results suggest that the low activity of GALK in RBC of individuals with the GALKP allele is due to its relative instability. We could obtain no evidence for such instability from studies of high reticulocyte bloods or RBC fractionation. Furthermore, we could not demonstrate that the GALK in WBC from GALKP individuals has altered electrophoretic migration.     

Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli.

Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C). Additional initial-rate measurement were performed to assess specific points. Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves. Two models, both extensions of the concerted model given by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88--118] with four protomers, could be fitted to the data within the experimental error. Model discrimination in favour of one of these models was possible by proper experimental design. In the selected model one conformational state of the enzyme forms the active complex. The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations. In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site.     

An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles.     

The chemistry of thujone: I. Synthesis of insect juvenile hormone analogs via wittig coupling

Syntheses of the C₈ and C₁₀ olefinic units cis- and trans-5-ethyl-1-iodo-hex-4-enes and cis- and trans-7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.     

Liver-cell-membrane autoantibody specific for inflammatory liver diseases.

With an immunofluorescence technique using rabbit hepatocytes isolated by a non-enzymatic method an autoantibody directed against liver-cell-membrance was identified. Sera from 361 patients with various liver diseases and 274 patients with primary non-hepatic diseases-many associated with non-organ-specific auto-antibodies-were examined. The antibody (LMA) was found in 27 out of 72 patients with hepatitis-B-surgace antigen (HBsAg)-negative chronic active hepatitis and in 17 out of 28 patients with HBsAg-negative non-alcoholic cirrhosis. Only two patients had LMA and HBsAg, and both had chronic active hepatitis. One patient with extrhepatic disease was found to have LMA, and this patient had biochemical evidence of liver disease. Hence there is a close correlation between the presence of LMA and HBsAg-negative chronic inflammatory liver diseases and its detection may help in diagnosis.     

Hydrogen fluoride saccharification of wood: lignin fluoride content, isolation of alpha-D-glucopyranosyl fluoride and posthydrolysis of reversion products

Wood chips from bigtooth aspen (Populus grandidentata Michx.) were saccharified by reaction with liquid hydrogen fluoride either anhydrous or containing up to 10% v/v water. The reaction products were separated into a solid lignin fraction and a water-soluble saccharide fraction. The fluoride content of the lignin (determined after alkaline fusion) was initially about 1 mg/g wood, but was lowered to 0.1 mg/g wood by grinding and washing. Thus little or no chemical binding of fluoride to lignin occurred during hydrogen fluoride (HF) solvolysis. Analysis of the water-soluble fraction by gel filtration on Biogel P2 columns showed a range of low-molecular-weight oligosaccharides and only 10-20% sugar monomers. Thus considerable reversion occurred during HF evacuation. Posthydrolysis conditions were optimized for these reversion products by varying temperature and acid concentration. Optimal conditions at 1 h were 140 degrees C with 100mN sulfuric acid or 225mN Hydrofluoric acid resulting in monomer yields of > 90% for 0.5% sugar solutions and > 80% for 10% sugar solutions. After reaction of pure cellulose (Filter paper) with hydrogen fluoride in the absence of water, and terminating the reaction with calcium carbonate, the reaction intermediate alpha-D-glucopyranosylfluoride was isolated with a maximal yield of 0.2 g/g paper. Upon purification via paper chromatography glucosylfluoride was identified by its specific rotation and also by gas chromatography-mass spectrometry of its tetra-O-trimethylsilyl derivative.