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Enzyme-catalysed degradation of the nerve agents tabun, sarin, ethyl sarin and soman by three variants of an organophosphate-degrading enzyme was studied at low concentrations of nerve agent. The concentration of nerve agent at a given time was determined by its ability to inhibit the enzyme acetylcholinesterase. Experiments were conducted in 96-well microtitre plates. Values of the ratio of k(cat) (turnover number) to K(m) (Michaelis-Menten constant) were calculated. For tabun, this value (for the most effective OpdA variant) exceeded any value published to date for other enzymes. The value was within an order of magnitude for the highest value reported for sarin, but there appears to be no published value for ethyl sarin for comparison. The OpdA enzymes were relatively inefficient in degrading soman.

Recent advances in knowledge of the properties of antimicrobial peptides (AMPs) are reviewed. AMPs are typically small, positively charged, amphipathic peptides that interact electrostatically and non-stereospecifically with the bacterial cell membrane, resulting in its permeabilization and cell death. Classes of AMPs, their mechanisms of action, hemolytic activity, and cytotoxicity towards host cells are discussed. A particular focus is AMPs with potential for use in defense against biological warfare agents. Some AMPs cytotoxic to Bacillus anthracis have been described. Synthesis of these peptides in multivalent form leads to a synergistic increase in antibacterial activity. Strategies to enhance the potency, stability, and selectivity of AMPs are discussed.

The cubic lyotropic mesophase composed of the ganglioside G(M1) and the synthetic surfactant phytantriol has been employed as a scaffold to prepare a polyvalent inhibitor of cholera toxin (CT). Surfactant mixtures containing up to 20% (w/w) G(M1)/phytantriol afforded a hydrated insoluble gel-like material, which retained an inverse cubic phase (Q) structure in excess water and at 22 degrees C, as confirmed using small-angle X-ray scattering. The G(M1)-functionalized mesophases bind up to 98.8% of CT from solution containing 100 ng/mL of CT with a dissociation constant (K(d)) of 67-73 nM. The estimated IC50 values for the mesophase systems were 0.1-0.27 microM. The inhibitory effect of the mesophases may be enhanced through the high internal surface area of the inverse cubic phase in addition to the optimal self-orientation of G(M1) ligand to match the distribution of binding sites on the toxin surface. As a result, polyvalent inhibitor materials manufactured using these mesophase structures do not require precise control of ligand distribution, which offers advantages with respect to complexity of synthesis and formulation when compared to more rigid and conformationally restricted materials. This approach provides a route to a unique class of polyvalent inhibitors, which should be broadly applicable to a range of bacterial and plant toxins.

Lyotropic mesophases of the inverse hexagonal or cubic type are nanostructured materials that result from the self-assembly of amphiphilic surfactant molecules in water. The extremely large area of the surfactant-water interface inherent within these structures makes them attractive media for sorbent or encapsulant systems. Here, we report on the development of a new class of polyvalent materials that are based on the incorporation of bioactive ligands within lyotropic mesophases. In particular, we have studied the potential for these materials to behave as polyvalent antitoxins by incorporating synthetic galactose amphiphiles, which mimic the natural cell surface ligand for the protein toxin ricin. The study demonstrates that cubic morphology lyotropic mesophases containing galactose amphiphiles exhibit high specificity ricin uptake, with favorably high dissociation constants and high capacities. We suggest that lyotropic mesophase polyvalent ligands are thus promising materials for the incorporation of a broad range of cell surface recognition moieties and hence may have wide applicability as materials capable of partaking in biological recognition processes.

A selection of galactose and lactose analogues was evaluated for their potency in inhibiting the binding of ricin to immobilised asialofetuin, which is a model of the cell-surface receptor for ricin. The aim was to identify compounds that could be used as antagonists of ricin toxicity in vivo, and as more selective, and therefore safer, antitoxins. Although one of these analogues had been identified by molecular modelling in a previous study as a potentially potent inhibitor, it and the other carbohydrates studied were less effective than galactose and lactose themselves (I(50) = 1.39 and 0.74 mM, respectively). In an attempt to increase the potency of carbohydrate-based inhibitors, galactose was coupled to the surface of dendrimers. No synergistic interactions were observed from this multivalent approach. Encouraging results, however, were obtained with a self-assembled lyotropic mesophase gel containing novel synthetic galactose-based surfactants, which was able to sequester ricin from aqueous solution in a 2-phase system., (Copyright 2006 John Wiley & Sons, Ltd.)

Ganglioside GM1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to GM1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (GM1-ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for GM1 (methanol or phosphate-buffered saline), the pH of aqueous solvents (7.4-10.2) and the temperature (4-37 degrees C). High and near-maximal rates of absorbance change in the assay were found for lower concentrations of GM1 (100 ng ml(-1)) and for shorter incubation times (a few hours) than reported in the literature. A method was devised to provide a semi-quantitative estimate of the amount of GM1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized GM1 required > or =1.5 h for maximal assay results. The failure of free GM1 in solution to displace cholera toxin once bound to immobilized GM1 indicated that binding to immobilized GM1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin-GM1 complex., (Copyright 2005 John Wiley & Sons, Ltd.)

Optimum conditions for the binding of ricin to the glycoprotein asialofetuin immobilized on microtitre plates were investigated for the purpose of evaluating inhibitors of ricin B-chain lectin activity. Such inhibitors are of potential value in the use of immunotoxins based on ricin. This assay was first reported in 1986, but has not been characterized fully. Maximum binding of asialofetuin to the plate was observed at a concentration of ca. 4 microg ml(-1). Binding increased with time of incubation (1-24 h), pH (7.4-9.9) and temperature (2-37 degrees C). The pH effects were more marked at lower temperatures. Saturable binding of ricin to immobilized asialofetuin was observed, and at least 80% of maximum binding was observed by 10 min of incubation time. The binding was found to be very tight, such that an appreciable proportion of ricin added to the wells was bound at low concentrations, and binding was only partially reversible by addition of free galactose. Consequently, only estimates of the ricin-asialofetuin and ricin-galactose dissociation constants could be determined: 1.9 nM and 83 microM, respectively. Binding of ricin A- and B-chains was found to be 47% (at a 200-fold higher concentration) and 26% (at a twofold higher concentration) of that of the whole ricin molecule, respectively. The assay permits qualitative comparison of inhibitors of ricin B-chain lectin activity.

Microcystins are a family of more than 50 structurally similar hepatotoxins produced by species of freshwater cyanobacteria, primarily Microcystis aeruginosa. They are monocyclic heptapeptides, characterised by some invariant amino acids, including one of unusual structure which is essential for expression of toxicity. Microcystins are chemically stable, but suffer biodegradation in reservoir waters. The most common member of the family, microcystin-LR (L and R identifying the 2 variable amino acids, in this case leucine and arginine respectively) has an LD50 in mice and rats of 36-122 microg/kg by various routes, including aerosol inhalation. Although human illnesses attributed to microcystins include gastroenteritis and allergic/irritation reactions, the primary target of the toxin is the liver, where disruption of the cytoskeleton, consequent on inhibition of protein phosphatases 1 and 2A, causes massive hepatic haemorrhage. Microcystins are tight-binding inhibitors of these protein phosphatases, with inhibition constants in the nanomolar range or lower. Uptake of microcystins into the liver occurs via a carrier-mediated transport system, and several inhibitors of uptake can antagonise the toxic effects of microcystins. The most effective of these is the antibiotic rifampin (a drug approved for clinical use), which protects mice and rats against microcystin-induced lethality when given prophylactically and, in some cases, therapeutically.

The therapeutic efficacy of tacrine, atropine and glycopyrrolate alone or in combination with the oxime HI-6 against soman was evaluated in anaesthetized rats. Arterial blood pressure, heart rate, respiratory frequency and body temperature were monitored in vivo. Blood cholinesterases were determined after each drug or soman challenge. At the lowest concentration tested (2.5 mg kg-1), tacrine was effective in improving the survivability of the rat by a factor of 2.6 (protection ratio), whereas the protection by atropine or glycopyrrolate was either insignificant or only marginally effective (protection ratio ranged from 1.0 to 1.9). In combination with HI-6, atropine increased the ratio to 4.6. In contrast, tacrine with HI-6 failed to improve the efficacy of the regimen, while glycopyrrolate plus HI-6 showed only slight improvement. The four physiological parameters monitored were relatively constant during the time course of the experiment in both the control and those with drug therapy. The more noticeable changes occurred toward the end of the experiment when sufficient amount of soman was injected to cause lethality. Death of the animal was usually preceded by a surge of arterial blood pressure and heart rate and a decrease in respiratory frequency. These physiological parameters rapidly deteriorated to zero just before the animal died. Blood and plasma cholinesterases were significantly inhibited after the animal received a relatively small dose of soman (20 micrograms kg-1) and were almost completely inactivated after the lethal dose of soman was administered. However, these changes of enzyme activity did not correspond well with the survivability of the rat. The inclusion of HI-6 with the three antimuscarinics appeared to be capable of protecting some cholinesterases against soman.

The effects of the oximes 2-pyridine aldoxime methiodide (PAM), HI-6, HS-6, toxogonin and TMB-4 on the rate of carbamylation of membrane-bound bovine erythrocyte acetylcholinesterase were studied. The second-order rate constant of carbamylation (ki) and the first-order rate constant of decarbamylation (k3) were calculated from the proportion of free acetylcholinesterase at equilibrium and the rate of approach to equilibrium. Twenty insecticidal carbamates plus physostigmine and pyridostigmine were studied. The oximes increased ki for several carbamates, with HI-6 causing an increase in the most number of cases (12) and PAM the least (3). HI-6 was also a potent accelerator of decarbamylation (increase in k3) in all cases, whereas PAM caused a significant decrease in k3 in 15 cases and a nonsignificant decrease in the other 7. Toxogonin and TMB-4 increased k3 or had no significant effect. The results were generally consistent with a proposal in the literature that there is a correlation between increased ki and increased toxicity of the carbamate in the presence of an oxime.

A review was conducted of papers describing the use of N-methyl-2-pyridinealdoxime (PAM), toxogonin or HI-6 as antidotes to the nerve agents tabun, sarin, soman and VX. The review included use of the oxime alone, oxime plus atropine and oxime plus atropine plus diazepam, given therapeutically, i.e. after nerve agent, in all cases. Experiments with any of these compounds given prophylactically were not considered. The review also included protocols of pyridostigmine prophylaxis and oxime-atropine therapy (with or without diazepam). It was difficult to draw conclusions as to the best oxime to use, because of lack of data in many cases. The identity of the oxime did not appear to be important when pyridostigmine prophylaxis was combined with atropine-oxime-diazepam therapy; in these cases, very good protection was observed in guinea pigs against all four nerve agents. The choice of oxime based on the data presently available may well depend on factors other than protection against lethality, such as cost and availability of the oxime and human toxicity of the oxime. This last factor was also reviewed, and the results showed that toxogonin is likely to cause more side-effects than PAM or HI-6. The efficacy of the oximes against the emerging threat agent GF was also reviewed.

Membrane-bound bovine erythrocyte acetylcholinesterase was inhibited with physostigmine or carbaryl, and the rate constants of carbamylation and decarbamylation were determined from the proportion of inhibited acetylcholinesterase in the steady state, and the rate of approach to the steady state. The oximes 2-PAM, HI-6, HS-6, TMB-4 and toxogonin, at 0.1 mM, all decreased the rate of carbamylation by physostigmine, but increased the rate of carbamylation by carbaryl. TMB-4 and toxogonin were the most effective oximes in potentiating carbamylation by carbaryl, with an enhancement of the second-order rate constant of 54- and 17-fold respectively. The greatest reduction in the rate constant for carbamylation by physostigmine (3.7-fold) was caused by HI-6. HS-6 and HI-6 increased the rate of decarbamylation, while 2-PAM reduced the rate of decarbamylation if physostigmine was the carbamate. 2-PAM and HI-6 were also studied with soluble bovine erythrocyte acetylcholinesterase, and similar results were obtained. The results extend those in a recent report by other authors who studied the half-life of carbamylation for acetylcholinesterase and butyrylcholinesterase in an attempt to understand the mechanism by which oximes increase the toxicity of carbaryl in vivo. These authors proposed binding of the oximes to an allosteric site on the enzyme. While not discounting this possibility, the present results, taken with other reports in the literature, suggest that binding of the oximes to the anionic subsite of the active site of the enzyme is also feasible. The present results also offer an explanation for another recent report, in which anomalous results were presented for decarbamylation of physostigmine-inhibited and carbaryl-inhibited erythrocyte acetylcholinesterase in the presence of 2-PAM or HI-6.

The experiments reported herein were designed to identify the best assay of diacetylmonoxime (DAM) in biological fluids, with particular emphasis on detection limits. Initially, four variations of the assay of urea with excess DAM were compared. The best method, published in 1977, was one that includes thiosemicarbazide, 4-aminoantipyrine and ceric ammonium sulphate in the acid reagent; it is fast, gives a reasonably stable chromophore and displays good linearity. However, the reaction was two or more times less sensitive when applied to the assay of DAM, with urea in excess, by interchanging the amounts of urea and DAM. Further, the calibration graphs did not pass through the origin, and one of the methods gave a mixture of two chromophores. None approached the sensitivity of a DAM-urea reaction specifically designed to assay biacetyl (formed from DAM by acid hydrolysis) and published in 1968. This method, using antipyrine and arsenicosulphuric acid, is also fast, with good linearity and a stable chromophore, but is sensitive to interference by plasma and urine. An alternative photometric assay that does not involve urea was equally sensitive. It had the advantage of less interference by plasma and urine but was more time-consuming. Both of these photometric methods had a limit of detection of ca. 0.2 microgram DAM, similar to that of a high-performance liquid chromatography (HPLC) assay. Sample clean-up is necessary before application of the HPLC assay.

1. Crude and purified synaptosomes were prepared from the cerebral cortex of the rat or the guinea pig and used to study the uptake and release of [3H]GABA and [3H]glutamate. 2. Baclofen at 10(-5) M inhibited stimulated release of [3H]GABA from crude rat and guinea pig synaptosomes, but not from purified rat synaptosomes. 3. 1-2 mM tabun decreased the uptake of [3H]GABA and increased the uptake of [3H]glutamate by purified guinea pig synaptosomes. 4. Soman and tabun at 10(-6) M and 10(-5) M inhibited basal release of [3H]GABA and [3H]glutamate from crude guinea pig synaptosomes. Tabun at 10(-5) M decreased stimulated release of [3H]GABA while soman had no effect. 5. The results do not sustain the possibility that nerve agents cause convulsions by affecting the uptake or release of GABA or glutamate. However indirect evidence was obtained that soman and tabun inhibit catabolism of GABA and glutamate.

1. Homogenates of guinea pig right atrium (M2 receptors) were treated with N-ethylmaleimide, after which the ability of carbachol to inhibit binding of [3H]quinuclidinyl benzilate was studied. 2. At 37 degrees C, 10(-4) M and 10(-3) M, but not 10(-5) M, N-ethylmaleimide increased the affinity of carbachol for the receptor. At 2 degrees C, 10(-5) M and 10(-4) M, but not 10(-3) M, N-ethylmaleimide decreased carbachol affinity. At 2 degrees C, after the homogenate had been incubated at 37 degrees C without N-ethylmaleimide, 10(-5) M N-ethylmaleimide decreased carbachol affinity, as at 2 degrees C without preincubation, but 10(-3) M N-ethylmaleimide increased carbachol affinity, as at 37 degrees C. 10(-4) M N-ethylmaleimide was without effect. 3. The results are discussed with respect to a previously proposed model in which N-ethylmaleimide interacts with two sites, causing an increase or decrease in agonist affinity respectively.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine) inhibited binding of [3H]flunitrazepam to benzodiazepine receptors of guinea pig hippocampus with an inhibition constant of 46 microM at 2 degrees C and 37 degrees C. gamma-Aminobutyric acid (GABA) decreased the affinity of tacrine for the receptor, suggesting that tacrine may act as an inverse agonist. A Hill coefficient less than 1 was observed under all conditions. Allosteric interactions may explain this behaviour, since 100 microM tacrine increased the rate of dissociation of [3H]flunitrazepam from the receptor. Tacrine inhibited the binding of 11 nM [3H]GABA to GABA receptors of guinea pig cerebral cortex with I50 = 188 microM. Bicuculline methiodide was 4 times as potent (I50 = 49 microM). The interaction of tacrine with GABA or benzodiazepine binding sites is unlikely to be of clinical significance.

Inhibition of bovine erythrocyte acetylcholinesterase (AChE) by 1,2,3,4-tetrahydro-9-acridinamine (tacrine) was independent of time of incubation and was partially reversed by dilution and by increased substrate concentration. It was fully reversed by dialysis. Similar results were obtained with AChE from other sources. The results are consistent with some reports in the literature, but not with others; none of these reports examined all four criteria of reversibility. The results do not explain the prolonged inhibition of AChE in vivo or the ability of tacrine to protect animals against the lethal effects of organophosphate anticholinesterases.

1. Inhibition of the binding of [3H]quinuclidinyl benzilate to homogenates of guinea pig right atrium (M2 receptors) by varying concentrations of carbachol was studied. 2. Pretreatment of membranes with 5 x 10(-5) M N-ethylmaleimide at 2 degrees C shifted the carbachol inhibition curve to the right, indicating decreased affinity of the receptor for carbachol. However pretreatment at 37 degrees C moved the curve to the left. 3. The ability of guanyl-5'-yl imidodiphosphate to reduce agonist affinity was largely eliminated by treatment with N-ethylmaleimide at both temperatures. 4. Conflicting reports in the literature and the present results can be explained by invoking a model in which N-ethylmaleimide has a high affinity for a heat-labile site and a lower affinity for a heat-insensitive site. Reaction with the first site decreases agonist affinity, but at 37 degrees C this site is largely inactivated and reaction with the second site, which leads to increased agonist affinity, predominates.

The phosphatidylinositol phosphodiesterase of rat brain shows little activity under conditions likely to pertain in vivo (neutral pH and micromolar Ca(2+) concentrations). A short incubation of a brain supernatant with trypsin, or a longer pre-incubation of the supernatant alone, produce new forms of the enzyme, which are active under such conditions. A possible role of receptor-linked proteinases in initiating phosphatidylinositol catabolism is discussed.

1. A rat brain supernatant and microsomal fraction contained a phospholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7-8.5) as well as at pH 5-5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH.

Gallamine and tubocurarine increased the rate of decarbamylation of carbamylated Triton-solubilized rabbit brain acetylcholinesterase and interacted synergistically with 3,3-dimethyl-1-butanol in the acceleration of decarbamylation at low ionic strength. Gallamine and tubocurarine also accelerated decarbamylation at a physiological ionic strength, but in this case the interaction with 3,3-dimethyl-1-butanol was not synergistic. Tubocurarine decreased the rate of ageing of isopropylmethyl-phosphonyl-acetylcholinesterase at low ionic strength, but gallamine and tubocurarine had no effect on ageing at a physiological ionic strength, nor did gallamine at low ionic strength. However reductions in the rate of ageing were observed for gallamine/3,3-dimethyl-1-butanol and tubocurarine/3,3-dimethyl-1-butanol mixtures at low ionic strength. Gallamine increased the maximum velocity of hydrolysis of acetylthiocholine at low ionic strength, but not at physiological ionic strength. Gallamine increased the rate of carbamylation of acetylcholinesterase by physostigmine and neostigmine at low ionic strength; however the data were not consistent with a simple model of complexing inhibition by these carbamates. Overall, the kinetic properties of Triton-solubilized rabbit brain acetylcholinesterase were less sensitive to modification by proposed allosteric effectors than bovine erythrocyte acetylcholinesterase, the allosteric properties of which have been reported previously, and millimolar concentrations of gallamine and tubocurarine were required to produce observable effects at physiological ionic strength. Further experiments are required to determine whether acetylcholinesterase is similarly insensitive to allosteric regulation in vivo.

1. Injection of [Me-14C]choline into sheep indicated that the small amount of phosphatidylcholine present in abomasal digesta was largely (69%) of non-dietary or ruminal origin. 2. Long-term feeding of [Me-3H]choline to sheep produced insignificant labelling of plasma phosphatidylcholine, indicating that more than 99% of the choline body pool was of non-dietary origin. 3. In contrast, when rats were fed with [Me-3H]choline for similar periods, 18-54% of the tissue phosphatidylcholine was derived from dietary choline. 4. The loss of [14C]choline and 32P from the plasma phosphatidylcholine after a single injection of these isotopes indicated a markedly slower turnover of choline in the sheep compared with the rat. This observation, coupled with a lack of liver glycerophosphocholine diesterase, provides an explanation for the insensitivity of the sheep to an almost complete microbial destruction of dietary choline before alimentary-tract absorption.

The local anaesthetic procaine showed the properties of an allosteric effector of bovine erythrocyte acetylcholinesterase at low ionic strength; it antagonised inhibition of substrate hydrolysis caused by decamethonium, decreased the rate of ageing of isopropylmethylphosphonyl-acetylcholinesterase, increased the rate of decarbamylation of dimethylcarbamyl-acetylcholinesterase, and interacted synergistically with the nucleophilic alcohol 3,3-dimethyl-1-butanol in the acceleration of decarbamylation. These allosteric effects almost completely disappeared as the ionic strength was increased to a physiological level, and they could not be demonstrated at the physiological ionic strength with membrane-bound human erythrocyte acetylcholinesterase. There was no evidence of significant cooperativity in the binding of procaine to the enzyme, nor in the binding of the substrate acetylthiocholine in the presence of procaine, contrary to reports in the literature for other sources of acetylcholinesterase. Procaine was not hydrolysed by acetylcholinesterase (EC 3.1.1.7) although it is a substrate for serum cholinesterase (EC 3.1.1.8). The possibility that the results at low ionic strength can be explained on the basis of procaine binding to the active site of acetylcholinesterase (at low concentrations) and also to a peripheral allosteric site (at higher concentrations) is discussed. The results confirm the complexity of the kinetics of acetylcholinesterase, and extend the range of compounds with the ability to modify rates of decarbamylation and ageing.

In April 1986, the endangered Perdido Key beach mouse (Peromyscus polionotus trissyllepsis) existed only as a small population of less than 30 animals on the western end of Perdido Key at Gulf State Park, Alabama This population was vulnerable to extinction from a variety of causes. Fifteen pairs of mice from Alabama were moved approximately 20 km on the same island to Gulf Islands National Seashore Florida between November 1986 and April 1988. The Alabama population was surveyed by live-trapping before each removal and showed a large increase during this study. Eleven pairs of mice were released into enclosures to stimulate burrowing and reduce dispersal at the release site. The last four pairs w e released unrestricted into the dune habitat Trapping in July 1988 revealed that virtually all available dune habitat (11,000 linear m; approximately 160 ha) had been occupied by the mice. Fifty-five individuals were captured including four of the released mice. Exchanges between the populations are recommended to prevent loss of genetic diversity. Future research should investigate demographics, dispersal pattern, and the application of DNA fingerprinting techniques to determine rates of gene flow in the population. The Perdido Key beach mouse provides an excellent model for studying the effects of a population bottleneck on genetic diversity and testing the predictions of population viability analysis.

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.

Effects of guanyl nucleotides on the affinity of cholinergic agonists at the [3H]quinuclidinylbenzilate binding site have been compared with their effects on the intact left atrium. The binding of carbachol was described by a two-site model; guanyl nucleotides had little effect on the affinity at either site but increased substantially the proportion of low affinity sites. The negative inotropic effect of acetylcholine (ACh) was unaffected by exogenous guanyl nucleotides or variation in external Mg++ or Ca++. ACh (2 microM) shortened the action potential, reduced spike amplitude and usually increased the maximum diastolic potential. GTP and guanyl-5'-yl-imidodiphosphate in the presence of ACh increased action potential amplitude and duration and increased further the diastolic potential. Low external Mg++ had similar effects to guanyl-5'-yl-imidodiphosphate but their effects were not additive. It is suggested that these compounds delay the activation of a transient outward K+ current by ACh.

1. The anaerobic rumen protozoon Entodinium caudatum was incubated either intact or with various radioactive precursors of phospholipids after ultrasonication. 2. Pulse-chase experiments showed a rapid turnover of phosphatidylinositol and much slower turnovers of phosphatidylethanolamine and phosphatidylcholine. 3. E. caudatum imbibed choline very rapidly; this was immediately and exclusively converted into phosphatidylcholine which was shown by radioautography after 10 min to be distributed throughout the cell membranes. 4. Phosphatidylcholine was synthesized through a phosphorylcholine-CDP-choline pathway, the methylation or base-exchange pathways not being present. 5. Under suitable conditions [Me-14C]choline can be substantially (50-60%) converted into CDP-choline by sonicated E. caudatum and this provides an excellent method of preparing this biosynthetic intermediary. 6. [2-14C]Ethanolamine was taken up much less readily than choline. The former was incorporated into phosphatidylethanolamine by the CDP-ethanolamine pathway. 7. Doubly labelled [32P]phosphatidyl[2-3H]ethanolamine was converted into ceramide phosphorylethanolamine and N-(1-carboxyethyl)phosphatidyl-ethanolamine, without change in the isotopic ratio. Ceramide phosphoryl [2-14C]-ethanolamine was converted into phsophatidylethanolamine. 8. Palmitic acid, oleic acid and linoleic acid were taken by E. caudatum cells and incorporated into phospholipids. By contrast, although stearic acid was taken up it was hardly incorporated into phospholipids.

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidycholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A2 of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphinogomyelin which is not hydrolysed by the former enzyme.

By administering (14)C-labelled grass to a sheep it has been shown that the ruminal bio-hydrogenation of the esterified linolenic acid of the galactolipids in chloroplast membranes only occurs after a rapid lipolysis. A slow accumulation of [(14)C]phytanic acid is presumed to arise by oxidation of phytol residues removed from chlorophylls.

1. Phosphatidylinositol-phosphodiesterase (EC 3.1.4.10) was strongly inhibited when ovophosphatidylcholine and saturated phosphatidylcholines with acyl chain lengths of more than eight carbon atoms were mixed with its substrate. In contrast, dihexanoylglycerophosphocholine produces a marked activation attended by a breakdown of the bilayer structure of the substrate. 2. C12, C14, and C16 lysophosphatidylcholines gave a progressive inhibition of the enzyme with increasing chain length; C10 lysophosphatidylcholine activated the reaction. 3. The enzyme was strongly inhibited by blood plasma and serum and by blood lipoproteins. 4. Phosphatidic acid, and certain lysophosphatidic acids (oleoyl and palmitoylglycerophosphates) activated the hydrolysis at a physiological pH, whereas decanoylglycerophosphate had little effect. 5. Phosphatidic acid at a concentration of 1% molar total lipid P produced an enhancement of the hydrolysis of the phosphatidylinositol substrate contained in a lipid environment which approximated to the inner (cytoplasmic) lamella of the plasma membrane bilayer of the liver sinusoidal cell surface. 6. The possible role of phospholipid molecules adjacent to phosphatidylinositol in controlling the hydrolysis of the latter in the proximity of a cell surface receptor is discussed. It is suggested that phosphatidic acid formed by the action of diacylglycerol kinase could amplify any increased hydrolysis of phosphatidylinositol which results from stimulation of the cell by an agonist.

Many phospholipases of the A or C types show a very limited ability to hydrolyse phospholipids such as phosphatidylcholine or phosphatidylinositol which adopt a bilayer form when hydrated. The addition of unsaturated 1,2 or 1,3 diacylglycerols to such substances produces a marked stimulation of the attack. In contrast, the hydrolysis of phosphatidylethanolamine, which normally exists in the hexagonal II structure, is not stimulated by diacylglycerol. It is suggested that the liberation of diacylglycerol which often occurs during cell stimulation, may play a part in activating the associated cascade of phospholipid reactions.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.

The major pathway by which liver lysosomal enzymes degrade phosphatidylinositol is through an EDTA-insensitive formation of phosphorylinositol. This is in distinct contrast with the Ca2+-dependent production of phosphorylinositol from phosphatidylinositol, which is located in the cytosol. Lysosomal enzymes can also totally deacylate phosphatidylinositol, producing glycerophosphorylinositol.

The kinetics of the inhibition of eel and rabbit brain acetylcholinesterase by a number of organophosphates and carbamates has been studied. Five organophosphates (tabun, sarin, soman, GF, and VX) and two carbamates (neostigmine and physostigmine) were used. Small but significant differences have been found between the two enzyme preparations in both the dissociation constant and the rate constants for the irreversible step. The values of k2 ranged from 0.56 to 1.08 sec-1 for the eel enzyme and 0.19 to 0.73 sec-1 for the rabbit enzyme. Kd varied from 0.3 to 24.5 microM for the eel and 0.3 to 9.3 microM for the rabbit enzyme. The similarity between the enzymes is remarkable considering the differences in the species of origin.

The antiviral drug amantadine has anticholinergic effects in the guinea-pig atrium at concentrations greater than 1 X 10(-4)M. It is a competitive inhibitor of [3H]-quinuclidinyl benzilate binding to the muscarinic receptor, but antagonizes the negative inotropic effect of acetylcholine in a non-competitive manner. It increases the duration of the atrial action potential and also increases the force of atrial contraction. These effects are evident at approximately 10 times lower concentrations than the antimuscarinic effects. The increase in contractility can be reversed by propranolol (5 X 10(-7)M) but the increase in action potential duration is potentiated by propranolol. Shortening of the action potential duration by acetylcholine was reversed by amantadine, but at approximately ten times lower levels than were needed to reduce the negative inotropic effect. Interactions between beta adrenoceptor binding of [3H]-dihydroalprenolol and amantadine could not be demonstrated. Similarly, binding of [3H]-nitrendipine to the calcium channel is not influenced. It is suggested that amantadine may exert its positive inotropic effect by interaction with the potassium channel, causing a delay in outward current.

Rat brain contains a soluble, high molecular weight phospholipase A1 of alkaline pH optimum which shows a preference for phosphatidylethanolamine as substrate. There is evidence that the same enzyme exists in liver and kidney. At low osmotic concentrations of buffer the enzyme is markedly stimulated by CaCl2. However, MgCl2 and MnCl2 are equally as effective although at concentrations above 2 mM the activation falls away with MnCl2. The phospholipase A1 is stimulated by divalent metal ion chelators (EDTA, EGTA, CDTA) and by sodium phosphate and sodium sulphate. The activity is inhibited by hexanol, benzyl alcohol, diethylether and detergents. Although the activity can be inhibited by saturated and unsaturated fatty acids, no evidence could be obtained that the activators function by counteracting the inhibitory action of fatty acids liberated at the interface of the substrate and incubation medium. It is suggested that to achieve good enzymic hydrolysis a certain type of organised hydrated phosphatidylethanolamine structure is required in which the negative zeta potential has been reduced by metallic cations in the incubation medium.

1. The secretion from sheep pancreas and a supernatant fraction prepared from the gland contained an EDTA-insensitive acid phospholipase A1 which readily deacylated phosphatidylinositol (pH optimum, 5.3), 1-acylglycerophosphoinositol and phosphatidic acid, but had limited action of phosphatidylcholine and phosphatidylethanolamine even with deoxycholate present. The enzyme was not a triacylglycerol lipase. 2. The action of the phospholipase A1 on phosphatidylinositol was inhibited effectively by Ca2+ and Mg2+, probably by interaction of these ions with the substrate. 3. In the presence of calcium the decomposition of phosphatidylinositol and lysophosphatidylinositol by the supernatant fraction was overwhelmingly by phosphodiesterase action (EC 3.1.4.10), producing inositol monophosphate and its cyclic derivative. Its pH optimum was about 6.0 but with considerable activity extending to pH 8.5. 4. The phosphodiesterase was not secreted in the pancreatic juice.

The complex lipids of the naturally-occurring general-fatty-acid-auxotroph Butyrivibrio S2 [Hazlewood & Dawson (1979) J. Gen. Microbiol. 112, 15-27] grown with palmitic acid as sole fatty-acid supplement have been investigated and some have been isolated in a state of purity and analysed. The majority are phospholipids (84%) and many contain galactose. They typically possess few esterified long-chain fatty-acid residues (C16:0), but are rich in esterified butyric acid and C16-alkenyl groups. Most of the phosphorus-containing lipids, including the two major lipids of the organism, contain esterified diabolic acid, a long-chain vicinal dimethyl-substituted dicarboxylic acid [Klein, Hazlewood, Kemp & Dawson (1979) Biochem. J. 183, 691-700] in definite stoichiometric relationship to phosphorus. No phosphatidylglycerol was present, but its monobutyroyl ester was detected as a minor component. Galactofuranosyldiacylglycerol (plasmalogen) and its monobutyroyl ester, cetyl alcohol and diacylglycerol were also identified.

The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.

(1) An analysis has been conducted of the lipids present in three obligately anaerobic bacteria isolated from the ovine rumen belonging to the genus Butyrivibrio. Two of these organisms are rich in phospholipase (A1 + A2) activity, and appear to be different strains of the species fibrisolvens. (2) The only N-containing lipids comprise N-acyl-phosphatidylethanolamine occurring as a minor component in all organisms and a new lipid, diglyceride galactosylphosphorylethanolamine in one of these. (3) All three organisms contained the n-butyryl ester of phosphatidyl-glycerol and in one this represented the major phospholipid present. Valeryl, iso-valeryl, propionyl and myristoyl esters of phosphatidylglycerol were also detected. (4) Two organisms contained glycerylphosphorylgalactosyldiglyceride and one of these also contained a large proportion of a less polar galactophospholipid which is probably a diacyl derivative of the former lipid. (5) All three organisms contained monogalactofuranosyl diglyceride and from one a n-butyryl ester of this galactolipid was isolated. (6) In all of the lipids examined the "diglyceride' moiety consisted almost entirely of plasmalogenic diglyceride (alk-1-enyl, acyl, glycerol).

A naturally occurring fatty acid-requiring Butyrivibrio sp. (strain S2), isolated from the ovine rumen, deacylates plant galactolipids, phospholipids and sulpholipids to obtain sufficient fatty acid for growth. Growth in vitro was promoted by adding to the growth medium a single straight-chain saturated fatty acid (C13 to C18) or vaccenic acid. Palmitoleic and oleic acids also supported growth but gave lengthy lag phases probably due to their toxicity. Linolenic and linoleic acids supported good growth but they were completely hydrogenated to trans-11-octadecenoic acid which was incorporated into the bacterial complex lipids. No chain elongation, chain shortening or desaturation of the added fatty acids occurred and all were substantially incorporated into bacterial lipids of the plasmalogen type, partially as a new type of hydrophobic grouping derived from two molecules of fatty acid. The absence of fatty acid unsaturation poses the question of the maintenance of membrane fluidity within this bacterium.