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PubMed1981

Effect of histamine on cyclic AMP levels in the submandibular gland.

Histamine and 4-methylhistamine increased on cyclic AMP level in chopped guinea-pig submandibular glands. 4-Methylhistamine was more active than histamine. 2-(2-Pyridyl)ethylamine was less active than histamine or almost inactive. Metiamide markedly antagonized the histamine-induced cyclic AMP increase but mepyramine had no inhibitory effect on this increase. These results suggest that there are H2-receptors which mediate the cyclic AMP response to histamine in the guinea-pig submandibular gland.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Pharmacological characterization of histamine receptors in the mouse seminal vesicle: sensitivity to H1- and H2-receptor agonists and antagonists.

Neither histamine nor the more specific H1- or H2-receptor agonists (0.2-220 micrometers) produced a contraction of the mouse seminal vesicle. However, all these agonists decreased resting tone and caused a dose-related inhibition of the electrically evoked twitch responses in these concentrations. The slopes of the percentage response versus log molar concentration curves for all agonists did not differ significantly from that of histamine. The rank order and relative potency of the compounds tested were: histamine (100%) greater than dimaprit (65%) greater than 4-methylhistamine (36%) greater than 2-methylhistamine (4.5%) greater than 2-(2-thiazolyl) ethylamine (1.7%). Inhibition of the twitch response by histamine was antagonized by cimetidine (3.5-140 micrometers) but not by mepyramine (0.1-1.0 micrometer). The slopes of Schild plots for cimetidine against histamine and dimaprit did not differ from unity, and the calculated pA2 values of cimetidine were 5.70 and 5.55, respectively. Histamine (2.2 and 6.5 micrometers) inhibited the contraction elicited by a submaximal concentration of exogenous noradrenaline (1 micrometer); this inhibition could also be selectively antagonized by cimetidine. These results demonstrate that histamine and selective H1- and H2-receptor agonists have an inhibitory action on the mouse seminal vesicle, and that this action is mediated via a post-junctional H2-receptor. The calculated pA2 values of cimetidine against histamine and dimaprit also suggest that the receptor in the mouse seminal vesicle of the conventional H1-type. The results further indicate that an H1-receptor is absent in the mouse seminal vesicle.

Animals↗
PubMed1994

Release of acetylcholine in the ventral striatum is influenced by histamine receptors.

To investigate whether histamine receptor ligands influence the in vivo-release of acetylcholine in the ventral striatum, this brain region was superfused with histamine receptor agonists or antagonists through a push-pull cannula and drug effects on the release of acetylcholine were investigated. Histamine, the H1 receptor agonist 2-thiazolyl-ethylamine and the H3 receptor antagonist thioperamide enhanced acetylcholine release, while the H3 receptor agonist (R)-alpha-methylhistamine was ineffective. The results indicate that H1 receptors and H3 receptors modulate acetylcholine release. The thioperamide-induced increase of acetylcholine release might be exerted via H3-receptors located on cholinergic terminals. Alternatively, thioperamide might enhance acetylcholine release by increasing endogenous histamine release via H3 autoreceptors. It is concluded that, via stimulation of striatal H1- and H3 receptors, histaminergic neurons are involved in the regulation of cholinergic neuronal activity in the ventral striatum.

Acetylcholine↗
PubMed1980

Dimaprit inhibition of zymosan-stimulated beta-glucuronidase release from human granulocytes.

Histamine inhibits the in vitro release of granulocytic lysosomal beta-glucuronidase when incubated with complement activated zymosan particles and this is an H2-receptor response. The highly specific histamine H2-receptor agonist, dimaprit, also inhibits this secretory enzyme release but is less potent (-log molar ED50 6.71 with histamine vs. -log ED50 5.97 with dimaprit, p less than 0.05). No change in beta-glucuronidase release was found with the H1-agonst, 2-(2 pyridyl)-ethylamine. The antagonist activity of metiamide was similar with the two agonists (KB = 2.9 x 10(-8) M with histamine and KB = 3.6 x 10(-8) M with dimaprit). Diphenhydramine did not change the granulocyte response to either histamine or dimaprit.

Adult↗
PubMed1988

Indirect evidence for a role of phosphatidylinositol turnover in the cardiac response to H1-receptor stimulation.

The influence of lithium on the positive inotropic effect of the H1-agonist 2-pyridyl-ethylamine (PEA) and of the H2-receptor agonist 4-methylhistamine was studied in isolated guinea-pig ventricular strips electrically stimulated at 1 Hz. Lithium (1-10 mM) was devoid of any effect on cardiac contraction; the positive inotropic effect of 4-methylhistamine was unaffected in the presence of 10 mM lithium. On the other hand, lithium (1-10 mM) dose-dependently shifted the dose-inotropic effect curve for PEA to the right; an antagonistic effect, qualitatively similar to that of lithium, was induced by the myoinositol antagonist 2-2'-anhydro-2-C-hydroxymethyl-myoinositol, at a concentration of 100 microM. Moreover the antagonistic effect of the higher lithium concentration (10 mM) was almost completely prevented in preparations superfused with 10 mM myoinositol. Since it is known that lithium is able to reduce the cellular availability of myoinositol by an interference with the phosphatidylinositol (PI) cycle, these results suggest that the H1-receptor-mediated increase in contractility may be linked to an increased turnover of PI, while the H2-receptor-mediated one is not.

Animals↗
PubMed2003

Synthesis and antimicrobial activities of some new nitroimidazole derivatives.

In this study, some new nitroimidazole derivatives were obtained from 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylamine dihydrochloride (4) and 1-(2-bromoethyl)-2-methyl-5-nitroimidazole (5), which were prepared using metronidazole. Compound 4 was reacted with arylisothiocyanates (6) to obtain 1-[2-(2-methyl-5-nitroimidazol-1-yl)ethyl]-3-arylthioureas (7) and the latter with alpha-bromoacetophenones (8) to give 3-[2-(2-methyl-5-nitroimidazol-1-yl)ethyl]-2-arylimino-4-aryl-4-thiazolines (9). Also 1-[2-(2-methyl-5-nitroimidazol-1-yl)ethyl]-2-phenyl-4-arylideneimidazolin-5-ones (11) were prepared by reaction of 4 with 2-phenyl-4-arylidene-5-oxazolones (10). The reaction of the other starting material 5 with 5-arylidenethiazolidin-2,4-dione (12) gave 3-[2-(2-methyl-5-nitroimidazol-1-yl)ethyl]-5-arylidenethiazolidin-2,4-dione (13) derivatives. Structural elucidation of the compounds was performed by IR, 1H-NMR and MASS spectroscopic data and elemental analysis results. Antimicrobial activities of the compounds were examined and moderate activity was obtained.

Anti-Bacterial Agents↗
PubMed1998

Hypersusceptibility to DMCM-induced seizures during diazepam withdrawal in mice: evidence for upregulation of NMDA receptors.

The present study investigated the role of NMDA (N-methyl-D-aspartate) receptors in the hypersusceptibility to seizures induced by the benzodiazepine inverse agonist DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) during diazepam withdrawal in mice, using behavioral and biochemical approaches. The seizure threshold of DMCM was markedly decreased during diazepam withdrawal, reflecting withdrawal hyperexcitability in response to physical dependence. The decrease in the seizure threshold of DMCM in diazepam-withdrawn mice was inhibited by the non-competitive NMDA receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate; 50 microg/kg, s.c.) and ifenprodil (20 mg/kg, i.p.). The effective doses of these compounds were lower than those required to prevent DMCM-induced seizures in chronically vehicle-treated mice. Since MK-801 and ifenprodil do not only bind to NMDA receptors but also to sigma receptors, the present study also investigated the effects of sigma receptor ligands. The decrease in the seizure threshold of DMCM in diazepam-withdrawn mice was not modified by the sigma receptor agonist, (+)-pentazocine (5 mg/kg, s.c.), or the sigma receptor antagonist, NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride; 5 mg/kg, i.p.). Furthermore, the latency to the expression of wild running induced by intracerebroventricular administration of NMDA (60 ng/mouse) was also significantly lower in diazepam-withdrawn mice than in vehicle-treated control mice. On the other hand, there was no difference in the spermidine concentration between vehicle-treated control and diazepam-withdrawn mice. In a receptor binding experiment, the Bmax value for [3H]-MK-801 binding was significantly increased in cerebrocortical tissues from diazepam-withdrawn mice, while the Kd value did not change in either group. However, the acute addition of a high concentration of diazepam (10 and 100 microM) in vitro did not alter [3H]-MK-801 binding in cerebrocortical membrane preparations. The behavioral experiments suggest that NMDA receptor antagonists may suppress benzodiazepine withdrawal responses, while the biochemical study reveals upregulation of the NMDA receptor, which may play an important role in the hypersusceptibility to DMCM-induced seizure in diazepam-withdrawn mice.

Animals↗
PubMed1998

Ciproxifan and chemically related compounds are highly potent and selective histamine H3-receptor antagonists.

We determined the affinities of five newly synthesized histamine H3-receptor antagonists in an H3-receptor binding assay and their potencies in a functional H3-receptor model. Furthermore, we determined their potencies in a histamine H2- and H1-receptor model. The compounds differ from histamine in that the ethylamine side chain is replaced by an aryl-substituted propyloxy chain and they differ from one another by varying substituents of the aryl rest. Iodoproxyfan, a highly potent and selective antagonist at H3 receptors, is structurally related to these five compounds. The specific binding of [3H]-Nalpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the five compounds at pKi values ranging from 8.24 to 9.27. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]noradrenaline was antagonized by all compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.78 to 9.39. The five compounds under study possess negligible potencies at histamine H2 and H1 receptors studied in the guinea-pig right atrium and ileum, respectively (pD'2 or pKp values < or = 5.2). The present paper shows that the five compounds under study possess high affinities and potencies at histamine H3 receptors, four out of the five compounds in this respect being equipotent with iodoproxyfan. Like iodoproxyfan, the five compounds show an at least 1000-fold selectivity for H3 as compared to H2 and H1 receptors.

Animals↗
PubMed2001

Effects of a novel, selective, sigma1-ligand, MS-377, on phencyclidine-induced behaviour.

Phencyclidine (PCP)-induced head-weaving is inhibited by a novel selective sigma1-ligand, (R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), but not by dopamine D2 antagonists. In the present study, we examined the effects of two potent and selective sigma1-ligands, MS-377 and N,N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl) ethylamine (NE-100), on PCP-induced rearing behaviour, hyperlocomotion and ataxia in comparison with the currently available antipsychotic agents with affinity for D2 receptors, haloperidol, sultopride and risperidone. Male Wistar rats or ddY mice were administered MS-377, NE-100, haloperidol, sultopride or risperidone, and PCP was administered 60 min later (in the case of NE-100 10 min later). Rearing behaviour, hyperlocomotion and ataxia were examined 10 min after PCP administration. MS-377, haloperidol, sultopride and risperidone dose-dependently inhibited PCP-induced rearing and hyperlocomotion, but did not antagonize PCP-induced ataxia. In contrast, the other selective sigma1-ligand, NE-100, did not affect any of the PCP-induced behaviour patterns in this study. These results suggest that there are at least two types of ligands for sigma1-receptors and that some sigma1-ligands, including MS-377, have more comprehensive effects against PCP-induced abnormal behaviour than other sigma1-ligands or D2 antagonists.

Animals↗
PubMed2002

Inhibition by sigma receptor ligand, MS-377, of N-methyl- D-aspartate-induced currents in dopamine neurons of the rat ventral tegmental area.

RATIONALE: MS-377 [( R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl) piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate] is a novel anti-psychotic drug candidate with high affinity for sigma receptors but devoid of binding affinity for PCP binding site of NMDA receptor/ion channel complex. OBJECTIVES: The effects of MS-377 on NMDA receptor and/or its ion channel complex were examined to elucidate the antipsychotic properties of MS-377. METHODS: We examined the effect of MS-377 on NMDA ( N-methyl- D-aspartate)-induced current in acutely dissociated dopamine neurons of rat ventral tegmental area (VTA) using patch clamp whole cell recording. RESULTS: MS-377 applied in a bath inhibited the peak current evoked by NMDA applied via the U-tube method for 2 s in a concentration-dependent manner. Other sigma receptor ligands, BD-1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), NE-100 ( N, N-dipropyl-2-[4-methoxy-3-(2-phenylenoxy)-phenyl]-ethylamine monohydrochloride) and haloperidol also inhibited NMDA-induced current in a concentration-dependent manner. Interestingly, concomitant application of MS-377 with BD-1063, NE-100 or haloperidol at concentrations that had no effects on NMDA-induced current, potentiated the MS-377-induced inhibition. CONCLUSIONS: The results suggest that MS-377, as well as other sigma receptor ligands, indirectly acts on the sigma receptor to inhibit glutaminergic transmission mediated by NMDA receptor/ion channel complex in VTA dopamine neurons, thereby inhibiting dopamine release in target VTA areas.

Animals↗
PubMed2003

Rapid fluorimetric assay for primary amine groups in water samples.

Bond Elut C(18) solid-phase extraction cartridges were used for pre-concentration followed by derivatization with o-phthaldialdehyde-N-acetylcysteine (OPA-NAC) of primary amines in water. Optimal conditions were: conditioning the cartridges with borate buffer pH 10.4, retention of the primary amines, addition of the OPA-NAC(3.7 mmol L(-1)) 1:1 molar ratio and borate buffer pH 8, elution of the isoindol with MeOH-borate buffer (9:1) pH 10.2 and fluorescence measurement. The equations of the calibration graphs for methylamine, ethylamine, propylamine, butylamine, pentylamine, and beta-phenylethylamine at lambda(excitation)=330 nm and lambda(emission)=440 nm, in the optimal conditions are presented. The solid-phase extraction procedure improved ten times the detection limits of the solution derivatization. Those values are in the 0.01-0.06 mg L(-1) interval in function of the amine. Also, it is possible to estimate the total primary aliphatic amine concentration in water, expressed as molar concentration of -NH(2) group or -NH(2)-N mg L(-1). On the basis of these studies, the method was applied for the determination of primary amino groups in tap, ground, factory and source water samples.

Journal Article↗
PubMed2004

Low-level atrazine exposure decreases cell proliferation in human fibroblasts.

There is continuing concern that release of certain chemicals into the environment leads to human exposure to toxins, particularly through contaminated ground and surface waters. The triazine herbicide atrazine (2-chloro-4-ethylamine-6-isopropylamino-S-triazine) is routinely found as a contaminant in many surface and groundwaters. Little is known of the long-term effects of atrazine exposure on human cells. The objective of this research was to examine whether low-level exposure of atrazine to normal human fibroblast cells in culture had measurable deleterious effects. We show that low-level, short-term exposure of atrazine to normal fibroblasts results in decreased cell proliferation. In two separate assays to measure cell proliferation, either a 1.5-fold decrease in the cell number occurred (saturation density analysis) or a 2-fold decrease in cell proliferation was found (MTT analysis). Experiments to measure apoptosis in cells exposed to low levels of atrazine did not show DNA fragmentation or increased caspase activity. Thus, this analysis shows that short-term exposure to environmentally relevant, low levels of atrazine can be assessed by a decrease in cellular proliferation of normal human cells.

Apoptosis↗
PubMed1996

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells.

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.

Alkalies↗
PubMed2004

Trichosporon wieringae sp.nov., an anamorphic basidiomycetous yeast from soil, and assimilation of some phenolic compounds, polysaccharides and other non-conventional carbon sources by saprophytic Trichosporon species.

A morphological and physiological description of an anamorphic basidiomycetous yeast species isolated from soil, named Trichosporon wieringae, is presented. The phylogenetic position within the genus, based on nuclear base sequencing of the D1/D2 region of the large subunit of rDNA and of the ITS region reveals close relationship to the Porosum clade of the genus Trichosporon Behrend, T. gamsii being the closest relative. The ability of non-pathogenic Trichosporon species to assimilate uric acid, ethylamine, l-4-hydroxyproline, some aromatic compounds and polysaccharides as sole sources of carbon and energy was studied. An identification key to these species, based on growth tests on these non-conventional carbon sources, is proposed. The type strain of T. wieringae is CBS 8903 (JCM 12201).

Carbon↗
PubMed2005

Affinity labeling mu opioid receptors with novel radioligands.

1. A series of novel opiate ligands based upon 6alpha-naloxamine have been examined in opioid receptor binding assays. 2. Coupling an ethylamine spacer alone to 6-alpha-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa1 opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives. 3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [125I]NalAmiBen and the azido derivative [125I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [125I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [125I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [125I]NalAziBen binding showed a band at approximately 70-80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [125I]NalAmiBen.

Affinity Labels↗
PubMed2006

Expression optimization and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Escherichia coli novablue.

A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His(6)-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 degrees C and 9, respectively. The apparent K (m) and V (max) values for gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor in the transpeptidation reaction were 37.9 microM and 53.7 x 10(-3) mM min(-1), respectively. The synthesis of L -theanine was performed in a reaction mixture containing 10 mM L -Gln, 40 mM ethylamine, and 1.04 U His(6)-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.

Chromatography, Gel↗
PubMed2001

Determination of stereochemical configuration of the glycerol moieties in glycoglycerolipids by chiral phase high-performance liquid chromatography.

This study reports a simple and sensitive method for determining the absolute configuration of the glycerol moieties in glycoglycerolipids. The method is based on chiral phase high-performance liquid chromatography (HPLC) separations of enantiomeric di- and monoacylglycerols released from glycosyldi- and monoacylglycerols, respectively, by periodate oxidation followed by hydrazinolysis. The released di- and monoacylglycerols were chromatographed as their 3,5-dinitrophenylurethane (3,5-DNPU) and bis(3,5-DNPU) derivatives, respectively. The derivatives were separated on two chiral phases of opposite configuration, (R)- and (S)-1-(1-naphthyl)ethylamine polymers for diacylglycerols and N-(R)- -(1-naphthyl)ethylaminocarbonyl-(S)-valine and N-(S)-1 -(1-naphthyl)ethylamino-carbonyl-(R)-valine for monoacylglycerols. Clear enantiomer separations, which permit the assignment of the glycerol configuration, were achieved for sn-1,2(2,3)-diacyl- and sn-1(3)-monoacylglycerols generated from linseed oil triacylglycerols by partial Grignard degradation on all the chiral stationary phases employed. Using the method, we have determined the glycerol configuration in the glycosyldiacylglycerols (monogalactosyl-, digalactosyl-, and sulfquinovosyldiacylglycerols) and glycosylmonoacylglycerols (monogalactosyl-, digalactosyl-, and sulfoquinovosylmonoacylglycerols) isolated from spinach leaves and the coralline red alga Corallina pilulifera. The results clearly showed that the glycerol moieties in all the glycoglycerolipids examined have S-configuration (sn-1,2-diacyl- and sn-1-monoacylglycerols). The new method demonstrates that chiral phase HPLC provides unambiguous information on the configuration of the glycerol backbone in natural glycosyldi- and monoacylglycerols, and that the two-step liberation of the free acylglycerols does not compromise glycerol chirality.

Chromatography, High Pressure Liquid↗
PubMed2004

Asymmetric in vitro synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D.

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30 degrees C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30-40% 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol (R,S configuration) and 60-70% 1,2-diacyl-sn-glycero-3-phospho-3'-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3' position of the glycerol molecule.

Bacterial Proteins↗