Carlos E. Domenech

Pesticide action and membrane fluidity. Allosteric behavior of rat erythrocyte membrane-bound acetylcholinesterase in the presence of organophosphorous compounds.

It is well documented that the organophosphorous pesticides or their metabolites are powerful inhibitors of acetylcholinesterase with consequent disruption of nervous activity [l-2] . Variations in the interaction energies as low as 700 cal/mol would be enough to trigger significant changes in the Hill coefficient from membrane-bound enzymes (n-values) [3]. It was indicated that the cooperative behavior of several membrane-bound enzymes correlated with the membrane fluidity and that these relationships raised the possibility of evaluating changes in membrane fluidity [4] . This novel approach was illustrated in the case of cholesterol [5], cortisol, progesterone [6], insulin [6-71 and thyroid hormones and thyrotropin interplay [8]. In this work we offer biochemical evidence regarding a new molecular basis in the action of organophosphate agents on the membrane fluidity through studies

Induction of acid phosphatase and cholinesterase activities in Ps. aeruginosa and their in-vitro control by choline, acetylcholine and betaine

SummaryCholine, acetylcholine and betaine used as a sole carbon source, effectuate in Ps. aeruginosa an acid phosphatase activity in addition to a cholinesterase activity. Induction of both enzyme activities was repressed by succinate or glucose. Cyclic AMP failed to relieve the repression produced by these compounds. Substrates not related to choline and used as a sole source of carbon, were inefficient to produce induction of both enzymes.The in-vitro action of choline, acetylcholine and betaine on Ps. aeruginosa acid phosphatase and cholinesterase has also been studied. To perform these studies periplasmic extracts obtained by EDTA-lysozyme treatment of the cells grown on choline or betaine as sole source of carbon, were used. Acid phosphatase activity was competitively inhibited by betaine, whereas the inhibition produced by choline and acetylcholine showed competitive and noncompetitive components. Cholinesterase activity was noncompetitively inhibited by betaine. At low acetylthiocholine concentration choline was an inhibitor of cholinesterase, whereas at high substrate concentration choline raised the hydrolysis rate of acetylthiocholine.These findings allow the conclusion that acid phosphatase and cholinesterase are specifically induced by choline and its metabolites derivatives. Kinetic results led us to postulate that acid phosphatase and cholinesterase contain a similar allosteric site. This site would either be of an anionic nature or show affinity to a methyl group or display both characteristics.

Phospholipids inTrypanosoma cruzi: Phosphoinositide composition and turnover

The polyphosphoinositides fromTrypanosoma cruzi were isolated by preparative thin-layer chromatography (TLC) and identified. When myo-[3H]inositol was present in the culture medium for five days, analyses showed the presence of phosphatidylinositol (PI), lysophosphatidylinositol (lysoPI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). Short-term incubation with32Pi led to higher percentages of incorporation into phosphatidylethanolamine (PE), lysophosphatidylethanolamine (lysoPE) and PI compared to the other glycerophospholipids. The phosphoinositides (PI, PIP and PIP2) contained a larger proportion of unsaturated than saturated fatty acids. High proportions of 18∶2 were found in the three phosphoinositides analyzed, whereas the major saturated fatty acid was 18∶0. Watersoluble inositol phosphates (IP, IP2 and IP3) were also identified.

Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site

SummaryDifferent compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.

Distribution of lactate dehydrogenase isozymes in subcellular fractions of rat tissues

Abstract Subcellular fractions from adult rat testis and kidney have been investigated for their lactate dehydrogenase isozymes content. A fraction from testis homogenates, showing predominance of mitochondria with dense matrix and dilated cristae, was revealed to contain most of the additional isozymes (“X”) specific of testis and sperm. These molecular forms showed a high degree of compartmentation. A small proportion of total lactate dehydrogenase activity in kidney is associated with the microsomal fraction. That portion is entirely represented by isozyme 5, a molecular species usually not detected in whole kidney homogenates. Patterns of elution of the bound enzyme with different salt concentrations and detergents indicated that the forces involved in that association must be complex and that they possibly can operate in vivo .

Choline transport in Pseudomonas aeruginosa

SummaryCholine used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km=3 µM) and another of low affinity (Km=400 µM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na+ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.

Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite

SummaryIn this work the action of the following compounds upon Ps. aeruginosa acid phosphatase has been studied: 1) alkylammonium compounds; 2) aminoalcohols and aminoacids with different substituents (−H, −CH3OH and −CH3) attached to the nitrogen atom; 3) alcohols analogous to some compounds of the above series, but without the amino group.It was found that the enzyme inhibition was more effective with N-trimethylated compounds than with the triethylated ones. The degree of inhibition depended on the number of methyl groups bound to the nitrogen atom. Taking into account the choline and betaine series the hydroxyl derivatives showed more affinity for the enzyme than the carboxylated ones. In each series the Ki values increased with the decrease of methyl groups bound to the nitrogen atom. The presence of a positively charged nitrogen atom in the molecule of the effector was essential. These results enable us to confirm that in the molecule of Ps. aeruginosa acid phosphatase there exists an anionic site with one subsite with affinity for methyl groups.

Pesticide action: Different response of erythrocyte membrane acetylcholinesterase to inhibition by organophosphorus compounds under varied dietary conditions

Abstract The inhibitory effect of malathion in conjunction with maloxon or other derivatives on the activity of rat erythrocyte membrane-bound acetylcholinesterase has been investigated after feeding diets known to affect the lipid composition of the membrane. The inhibition of AchE by preincubation of membrane preparations with this organophosphorus compound, possibly with contaminants, differed with the diet: the curve was sigmoidal after supplementation with corn oil and hyperbolic after supplementation with lard. In the latter case the kinetics of the inhibition became sigmoidal by assaying in the presence of cortisol or isoproterenol or by previous solubilization of the enzyme. These results indicate that some organophosphorus compounds can differentiate conformational states of acetylcholinesterase present in the erythrocyte membrane.

Acetylcholinesterase from rat red cells and cholinesterase ofPseudomonas aeruginosa: Different types of inhibition by atropine

SummaryThe inhibition by atropine of cholinesterase from Pseudomonas aeruginosa has been studied in parallel with the membrane bound acetylcholinesterase from rat red cells.Acetylcholinesterase of rat red cells, like other animal cholinesterases, was competitively inhibited while the cholinesterase fromPseudomonas aeruginosa was partially non competitively inhibited by atropine.These results clearly indicated a different behavior of cholinesterase fromPseudomonas aeruginosa in comparison with the enzyme ofPseudomonas fluorescens and other animal cholinesterases.

Molecular forms of l-α-hydroxy acid oxidase from rat kidney

Abstract l -α-Hydroxy acid oxidase ( l -amino acid:oxygen oxidoreductase, EC 1.4.3.2) has been purified from “heavy” and “light” mitochondrial subfractions and from the soluble fraction of rat kidney homogenates. Studies by gel electrophoresis, column chromatography and stability to heating indicated that the enzyme from “heavy” mitochondria differs from that of “light” mitochondria and cytosol. They are two distinct molecular forms with different net charge but similar molecular size. Effect of substrate and product upon these forms has been studied with a variety of α-hydroxy acids and α-keto acids. α-Hydroxy valerate and -isocaproate produced inhibition of enzymatic activity at concentrations higher than 50 mM. Certain α-keto acids act as non-competitive inhibitors of the enzymes, α-keto isocaproate being the most active. Catalytic behavior and pH optima for the different molecular forms were very similar.