Detection of D-erythro and L-threo sphingosine bases in preparative sphingosylphosphorylcholine and its N-acylated derivatives and some evidence of their different chemical configurations

Sphingosylphosphorylcholine prepared from native sphingomyelin by the Kaller procedure was found to comprise about 70% of the L-threo (2S, 3S) isomer and 30% of the D-erythro (2S, 3R) isomer. This analytical result was obtained by gas-liquid chromatography (GLC) of trimethylsilyl derivatives of N-acetylsphingosines which were prepared by enzymatic hydrolysis of synthetic N-acetylsphingosylphosphorylcholines with Clostridium perfringens phospholipase C. Some other evidence of the different chemical configuration between the erythro and threo isomers of synthetic N-acylated sphingosylphosphorylcholines was also provided by thin layer chromatography (TLC), optical rotatory dispersion (ORD), and fast atom bombardment (FAB) mass spectrometry.     

D- AND L-STEREOISOMERS OF ALLYLGLYCINE: CONVULSIVE ACTION AND INHIBITION OF BRAIN L-GLUTAMATE DECARBOXYLASE

DL-Allylglycine was resolved into the L- and D-stereoisomers using hog kidney acylase. Both isomers were active as convulsants after administration to mice. The dose of D-allylglycine required to induce convulsions was greater than that of the L-isomer. Studies on the concentration of the two isomers in brain suggest that the lower effectiveness of D-allylglycine is partially due to its slower penetration into the brain through the blood-brain barrier. Both isomers of allylglycine inhibited brain glutamate decarboxylase in vitro to approximately the same extent, however, in vivo L-allylglycine inhibited the enzyme more strongly than the D isomer. Concentrations of allylglycine which caused a significant inhibition of L-glutamate decarboxylase in vivo were ineffective in inhibiting the enzyme in vitro. Oxidation products derived from L- or D-allylglycine by the action of either L- or D-amino acid oxidase caused an almost complete inhibition of the enzyme in vitro. It is suggested that a common intermediate derived from the two isomers (possibly 2-keto-4-pentenoic acid) is responsible for the in vivo inhibition of L-glutamate decarboxylase and possibly also for the induction of convulsions.     

Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichis coli

Abstract Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli . The best substrates were, in decreasing order of specific activity, D- erythro -4-methylglutamic acid, D- erythro - methylglutamic acid, DL-homocysteic acid, (±)- trans -1-amino-3-carboxy-cyclopentanecarboxylic acid and (±)- trans -1-amino-3-carboxy-cyclohexanecarboxylic acid. Among the different stereoisomers, only the D- erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates. Apart from the D- erythro -3 and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine.     

Stereochemistry of reactions catalyzed by glutamate decarboxylase.

When the decarboxylation of L-glutamic acid by the glutamate decarboxylase from Escherichia coli is carried out in D2O, the product gamma-aminobutyric acid contains a single deuterium atom. The stereochemistry of this material was established by conversion to levorotatory methyl 4-phthalimido [4(-2)H] butyrate. The dextrorotatory isomer of the latter compound was synthesized from S-[2(-2)H] glycine by a series of reactions not affecting the stereochemistry at the chiral center. Thus, the decarboxylation of glutamic acid occurs with retention of configuration. Decarboxylation of L-alpha-methylglutamic acid by this enzyme produced levorotatory gamma-aminovaleric acid and thus also occurs with retention of configuration.     

DL-threo-4-fluoroglutamic acid. A chain-terminating inhibitor of folylpolyglutamate synthesis

The effects of 4-fluoroglutamate on the reaction catalyzed by partially purified rat liver folylpolyglutamate synthetase have been investigated. DL-threo-4-Fluoroglutamate was an effective, concentration-dependent inhibitor of polyglutamylation of both tetrahydrofolate and methotrexate, while the erythro isomer was weakly inhibitory. 4-Fluoroglutamate acted as an alternate substrate; the DL-threo isomer was incorporated only slightly less effectively than L-glutamate, while the erythro isomer was poorly incorporated. The resulting product, a pteroylglutamyl-gamma-(4-fluoro)glutamate, was a very poor substrate for further glutamylation. Thus, when tetrahydrofolate and 4-fluoroglutamate were substrates, the sole Zn/HCl cleavage product co-chromatographed on high performance liquid chromatography with chemically synthesized p-aminobenzoylglutamyl-gamma-(4-fluoro)glutamate. When [3H]methotrexate (4-NH2-10-CH3PteGlu) and 4-fluoroglutamate were the substrates, one product was obtained which co-chromatographed on high performance liquid chromatography with chemically synthesized 4-NH2-CH3PteGlu-gamma-(4-fluoro)glutamate. Further evidence that the product from [3H]methotrexate was a dipeptide came from gamma-glutamyl hydrolase digestion experiments and quantitative amino acid analysis. The appearance of trace amounts of a product having properties consistent with the addition of a second 4-fluoroglutamate occurred only under forcing conditions. The chemically and enzymatically synthesized fluoroglutamate-containing products were at least 15 times poorer than the analogous diglutamyl compound as substrates for rat liver folylpolyglutamate synthetase. These results are consistent with inhibition of polyglutamate synthesis by 4-fluoroglutamate through a "leaky" chain termination mechanism.     

Quantitative effects of unsaturated fatty acids in microbial mutants. VII. Influence of the acetylenic bond location on the effectiveness of acyl chains.

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (greater than 40 degrees C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis-double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2-5 and 13-17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli. The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli. In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except delta2 and delta3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8-11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae.     

Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli.

Glutamate decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the irreversible alpha-decarboxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5). The contribution of the active-site lysine residue (Lys276) to the catalytic mechanism of E. coli glutamate decarboxylase has been determined. Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable. The purified mutants contain very little (K276A) or no (K276H) cofactor at all. However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly. The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form. Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400-fold less than that of the reaction between glutamate and pyridoxal 5'-phosphate in solution. Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism. Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.     

Influence of stereoisomers of 4-fluoroglutamate on rat brain glutamate decarboxylase

Inhibition of rat brain glutamate decarboxylase (GAD, EC 4.1.1.15) by individual stereoisomers of 4-fluoroglutamate (4-F-Glu) and 2-fluoro-4-aminobutyrate (2-F-GABA) was studied. All stereoisomers of 4-F-Glu inhibited decarboxylation of L-glutamate catalysed by the enzyme preparation. At 1 x 10(-2) M concentration, the most potent inhibitor of GAD was D-erythro-4-F-Glu with about 70% inhibition in the presence of 1.23 x 10(-2)M L-glutamate. The inhibition by all stereoisomers was of the competitive type. Ki values ranged from 2 x 10(-3)M for the D-erythro isomer to 1.1 x 10(-2)M for the D-threo and L-erythro isomers. The influence of all stereoisomers was reversible as shown by dialysis except for a small amount in the case of the D-erythro isomer. The inhibition was independent of external pyridoxal-5'-phosphate added. No inhibition of rat brain GAD was found with 2-fluoro-4-aminobutyrate stereoisomers.     

Mechanism of the stereospectific irreversible inhibition of bacterial glutamic acid decarboxylase by (R)-(--)-4-aminohex-5-ynoic acid, an analogue of 4-aminobutyric acid.

4-Aminohex-5-ynoic acid inhibits bacterial glutamic acid decarboxylase in a time-dependent irreversible manner. The inhibition is stereospecific and requires the abstraction of the propargylic hydrogen from 4(R)-(--)-4-aminohex-5-ynoic acid. This leads to the generation of a reactive alkylating agent in the active site which can react with a nucleophilic residue. At complete inhibition, there is incorporation of one molecule of inhibitor per pyridoxal binding site. If the decarboxylation of glutamate occurs with retention of configuration, the irreversible inhibition of this enzyme by the 4-(R) isomer can be rationalized on the basis of reversibility of the protonation step in the normal catalytic mechanism.     

Studies on amino acid decarboxylases in Escherichia coli

The isolation of highly active preparations of glutamate decarboxylase, arginine decarboxylase and histidine decarboxylase from Escherichia coli is described. These preparations showed strict specificity, and were in each case resolved into apo- and co-enzyme as shown by the significant restoration of activity that took place on addition of pyridoxal 5-phosphate.     

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues.     

Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium.

An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

THE GROWTH OF BOTRYTIS CINEREA PERS., FUSARIUM CAERULEUM (LIB.) SACC., AND PHOMA FOVEATA FOISTER IN THE PRESENCE OF TETRACHLORONITROBENZENE ISOMERS

The linear growth of Botrytis cinerea, Fusarium caeruleum and Phoma foveata in culture was reduced in the presence of vapour from any of the three isomers of tetrachloronitrobenzene. The isomers were fungistatic but not fungicidal.
Differences in activity were observed amongst the isomers. The 2:3:4:6 isomer was the most active against all three test fungi. 2:3:5:6-TCNB (tecnazene) was more active than 2:3:4:5-TCNB against Botrytis cinerea , but less active against Fusarium caeruleum and Phoma foveata . Two strains of Fusarium caeruleum resistant to the 2:3:5:6 isomer were not resistant to the other two isomers, although they were more resistant than their 2:3:5:6-TCNB sensitive parent strains.
Sporulation and sclerotial production by Botrytis cinerea were completely suppressed by 2:3:5:6-TCNB and 2:3:4:6-TCNB but not by the 2:3:4:5: isomer.     

Stereospecific elimination of deuterium as a method for determining the stereochemistry of a number of metabolites of the tetrahydrocannabinols

The stereospecific elimination of the 3-deuterium atom from metabolites of [2H]-analogues of delta 1-tetrahydrocannabinol (delta 1-THC), delta 6-THC and delta 7-THC has been investigated as a possible method for determining the stereochemistry of metabolites substituted with hydroxy or acid groups in the terpene ring. Elimination of HCOOTMS was found to involve the 3-hydrogen of the axial but not the equatorial isomer of hexahydrocannabinol-7-oic acid, a metabolite of all three cannabinoids. Similar stereospecific eliminations were observed during the loss of TMSOH from the TMS derivatives of 5 alpha-hydroxy-delta 6-THC, 6 alpha-hydroxy-delta 1-THC and 1 alpha, 2 beta-dihydroxy-delta 1-THC. Loss of TMSOH from 1 alpha, 7- and 1 beta, 7-dihydroxy-HHC involved the 3-hydrogen in both cases but the isomers could be distinguished as their alkane-boronate derivatives; only the derivative of 1 alpha, 7-dihydroxy-HHC lost the boronate ring with stereospecific removal of the 3-hydrogen. The stereochemistry of the four isomers of 1,6-dihydroxy-HHC could not be determined in this way as the [M-TMSOH]+ ions from all four compounds had lost the 3-hydrogen, presumably as the result of 1,6-bond cleavage.     

Uropygiols: confirmation of structure by proton magnetic resonance

Lipids were extracted from excised uropygial glands of domestic chickens and the wax diesters were isolated by preparative thin-layer chromatography (TLC). The diesters were hydrolyzed and the liberated diols were resolved by boric acid TLC into two fractions. These were investigated by proton magnetic resonance at 360 MHz of the free diols and of their acetonide derivatives. The results showed that the cis and trans acetonides, formed from the erythro and threo isomers of the diols, respectively, could be distinguished by the degree of magnetic nonequivalence of the two acetonide methyl groups in each molecule. On the presumption that the cis isomer should show the greater nonequivalence of the methyl groups, this configuration was assigned to the acetonides of these diols which had the lesser TLC mobility on boric acid/silica gel. This agrees with the assignment of configuration made by earlier workers on the basis of the relative TLC mobility of the diol isomers on boric acid/silica gel, but was contrary to a previous assignment based on gas-liquid chromatographic (GLC) retention times. We conclude that the erythro isomers of the diols are characterized by lower mobility on boric acid TLC, as well as on silica gel TLC, and form acetonides that have longer retention times on GLC, and greater nonequivalence of the acetonide methyl groups in the NMR spectrum, than do the acetonides of the threo isomers.     

Stereospecificity in enzymic and non-enzymic oxidation of beta-O-4 lignin model compounds

The degradation of the erythro and threo isomers of the non-phenolic lignin model compound 2-(2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)-1,3-propanediol was examined. Enzymic and non-enzymic oxidation of the diastereomers was performed with Trametes versicolor lignin peroxidase and cerium(IV) ammonium nitrate, respectively. Mixtures of approximately equal amounts of the diastereomers were partially degraded and subsequently analyzed with TLC and 1H-NMR. Analysis of reaction mixtures from enzymic as well as non-enzymic oxidation, revealed a preferential degradation of the threo form. Preliminary analyses of enzymic reaction mixtures of either the erythro or threo isomer suggest they yield in part different products. The observations made would have implications for the understanding of how enzymes attack lignins. They should also be taken into consideration in experiments where model compounds are being used to mimic native lignin.     

Pseudomonas cepacia 2,2-dialkylglycine decarboxylase. Sequence and expression in Escherichia coli of structural and repressor genes

A 3969-base pair PstI-PstI fragment of Pseudomonas cepacia DNA containing the gene for the pyridoxal 5'-phosphate dependent 2,2-dialkylglycine decarboxylase (pyruvate) (EC 4.1.1.64) was cloned in Escherichia coli. The insert was sequenced by the dideoxy method using nested deletions from both ends, revealing a central 1302-base pair region that codes for the decarboxylase subunit. The recombinant enzyme was expressed in E. coli, purified to homogeneity, and sequenced at the amino terminus. Also, a cofactor-labeled active site peptide was sequenced. The carboxyl terminus of the deduced amino acid sequence is homologous with the carboxyl terminus of mammalian ornithine aminotransferase; the active site sequence is similar to the active site sequences of several other aminotransferases. No homologies with known decarboxylase sequences could be found. Expression of the decarboxylase gene is negatively controlled by a 687-nucleotide sequence upstream of and diverging from the structural gene. Expression is induced by S-isovaline, 2-methylalanine, and D-2-aminobutanoic acid, but not by glycine, D- or L-alanine, L-2-aminobutanoic acid, R-isovaline, or other alkyl amino acids.     

Investigation of the preferred Mg(II)-adenine-nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

In the presence of Mg2+ the ecto-(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolizes the PS diastereoisomer of adenosine 5'-[alpha-thio]diphosphate, (PS)-ADP [alpha S], and the ecto-(nucleoside triphosphatase) selectively catabolizes the PS isomer of adenosine 5'-[beta-thio]triphosphate, (PR)-ATP[beta S], but exhibits no selectivity towards ATP[alpha S] isomers. In the presence of Cd2+ selectivity to ADP[alpha S] and to ATP[beta S] isomers is reversed; in the presence of Co2+, selectivity is lost. We conclude that each enzyme preferentially recognises the lambda (screw-sense) bidentate Mg(II)-nucleotide complex at its active site.