R.D. O'Brien

Insensitivity of acetylcholinesterase as a factor in resistance of houseflies to the organophosphate Rabon

Abstract Acetylcholinesterase from an organophosphate-resistant strain of housefly exhibited a decrease in sensitivity to Rabon in vitro , to the extent of 206-fold for the soluble enzyme and 38-fold for the particulate. These effects upon the bimolecular reaction constant, k i , were due to a 573-fold decrease in affinity of the inhibitor for the soluble and 103-fold for the particulate enzyme, coupled with a small increase in reactivity.

Properties of Lubrol-solubilized acetylcholine receptor from Torpedo electroplax

Abstract The efficiencies of two nonionic and four anionic detergents in solubilizing acetylcholine receptors (AChR) (as detected by ACh-binding) from electric tissue of Torpedo were compared. Lubrol WX solubilized the highest concentration of active AChR/mg protein and sodium dodecyl sulfate (SDS) abolished ACh-binding; however, removal of the latter detergent resulted in a small recovery of ACh binding. The Lubrol-solubilized AChR was similar to the particulate AChR in binding ACh at two sites reversibly, with high affinities, and in showing autoinhibition at ACh concentrations higher than 1 μ m . Also, binding of ACh was abolished by boiling and was reduced by pretreatment with trypsin, pronase, and phospholipases. However, “kinetics” of ACh binding to solubilized AChR were changed: there were increases in the affinity of the high-affinity site and in the ratio of the concentrations of the high- to low-affinity site, and the Hill coefficient was reduced to 0.66. Its nicotinic nature was demonstrated by blockade of ACh binding by 13 nicotinic drugs including α-bungarotoxin and cobrotoxin. Anticholinesterases were also blockers, but 21 noncholinergic drugs had no effect. Ultrafiltration and gel chromatography were used to separate the ACh-binding macromolecules. Sepharose 6B gave partial separation of AChR from AChE. The molecular weight of the smallest functional AChR was between 50,000 and 100,000 daltons, and had a high tendency to aggregate. The majority of Lubrol-solubilized AChR had molecular weights above 300,000 daltons.

DDT: A NEW HYPOTHESIS OF ITS MODE OF ACTION.

It is suggested that DDT and perhaps other chlorinated hydrocarbon insecticides owe their activity to the formation of a charge-transfer complex with a component of the nerve axon, with consequent disturbance of function. Experimental evidence is provided for the formation of two complexes with components of cockroach nerve; the complexes have been partially purified. Their formation is accompanied by an absorption in the 245- to 270-millimicron range.

THE EFFECTS OF HYDRAZINES ON RAT BRAIN 5-HYDROXYTRYPTAMINE, NOREPINEPHRINE, AND GAMMA-AMINOBUTYRIC ACID.

Abstract The influence of hydrazine (HY), methylhydrazine (MMH), 1,1-dimethylhydrazine (UDMH), and 1,2-dimethylhydrazine (SDMH) was studied upon possible transmitter substances in rat brain, at times calculated just to precede convulsions. Mesencephalon-diencephalon, medulla, cortex, and cerebellum were separately examined. All the hydrazines increased 5-hydroxytryptamine, particularly in cortex, where a three-fold increase was found. All the hydrazines increased norepinephrine by small amounts (48% maximum), the effects being greatest in cortex and least in mesencephalon-diencephalon. MMH, UDMH, and SDMH lowered γ-aminobutyric acid (GABA) by small amounts (17% maximum) in medulla, cortex, and mesencephalon-diencephalon, but effects on cerebellum were small or absent. HY raised GABA in all parts, especially in mesencephalon-diencephalon (31%). Since the ‘nontoxic’ SDMH showed all of the above effects to the same extent as the toxic hydrazines, it is unlikely that these effects are causally involved in toxicity of these agents. An exception may be the effect of HY in raising GABA levels.

Action of organophosphates on binding of cholinergic ligands

Abstract Four organophosphates: DFP, Guthoxon, Tetram and 2-( O,S -dimethylthiophosphorylimino-)-3-ethyl-5-methyl-1,3-oxazolidine, were found (at 10 −4 M ) to block the binding of 3 H-nicotine and 3 H-decamethonium (at 10 −7 M ) to macromolecules suggested to be acetylcholine receptors (ACR) in preparations of the housefly brain and electroplaxes of Torpedo and Electrophorus . Differences in blocking abilities were attributed to variations in ACR of the different tissues, structural differences amongst the organophosphates and ligand affinities. Organophosphates showed much lower affinities for ACR than for acetylcholinesterase. Binding of 3 H-muscarone and 3 H-acetylcholine to Torpedo electroplax was measured in the presence of increasing amounts of Tetram. The organophosphate blocked the binding of both ligands to ACR at concentrations higher than 5 × 10 −4 M . At low Tetram concentrations ( −5 M ) acetylcholine binding was reduced because of its hydrolysis by some acetylcholinesterase. Whereas the binding of organophosphates to acetylcholinesterase was irreversible, their binding to ACR was completely reversible.

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Abstract Binding of [65Zn++] and [45Ca++] to the acetylcholine (ACh)-receptor, purified from the Torpedo electric organ, was studied by equilibrium dialysis. Whereas [65Zn++] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10−6M, no binding of [45Ca++] at concentrations up to 10−3M could be detected with this method. However, the binding of [acetyl-3H]choline to the receptor was blocked equally by very high Zn++ or Ca++ concentrations, and the Ki for this low affinity binding was 7 × 10−3M. The high affinity binding of [65Zn++] to the receptor was blocked best by Cd++ then Co++ and Mn++, but least by Mg++ and Ca++. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca++ that could not be removed even by extensive dialysis. When Ca++-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca++. This was equivalent to 15 moles of Ca++ for each mole of ACh bound at saturation. It is suggested that the source of this Ca++ is endogenous, and that it is tightly bound to the ACh-receptor molecule.

Antagonism by DDT of the effect of valinomycin on a synthetic membrane.

The potassium conductance which is induced by 10-6 molar valinomycin in a lecithin-decane membrane is reversed by 3 x 10-6 molar DDT. Membranes not treated with valinomycin are not affected by DDT. This blockade of potassium conductance parallels the effect of DDT on axonic conduction. Dieldrin and lindane, whose physiological actions are in some ways like those of DDT, do not affect valinomycin-induced conductance of lecithin-decane membranes.

Acetylcholinesterase isozymes from the housefly brain

Abstract 1. 1. Cholinesterase activity in insect head extracts was investigated by disc electrophoresis followed by spectrophotometric analysis. The supernatant of 100 000 × g , 1 h, revealed the presence of four isozymes in the housefly, two in the American cockroach and only one in the southern army worm. 2. 2. The four isozymes, present in the head extract of houseflies, were totally inhibited by 10 μM guthoxon or eserine but were unaffected by 10 μ M p- chloromercuribenzoate (PCMB) or tri -o- cresylphosphate . All four isozymes had higher activity with acetylthiocholine than butyrylthiocholine as substrates; indicating they are isozymes of acetylcholinesterase. 3. 3. Centrifugation at 200 000 × g for 4 h precipitated 66% of the acetylcholin-esterase originally present in the supernatant of 100 000 × g for 1 h but the four isozymes remained soluble and were estimated to have molecular weights of less than 500 000. 4. 4. Two methods of gel staining were used to detect esterase activity, the direct-coloring thiocholine method using acetylthiocholine as a substrated and the modified Gomori method using α-naphthylacetate. The first proved to be effective for detecting cholinesterase activity; but at least two of the eserine-sensitive bands found by the technique of Gomori 20 were not cholinesterase.

Effect of organophosphates in vivo upon acetylcholinesterase isozymes from housefly head and thorax

Abstract Four soluble acetylcholinesterase isozymes of head and three from the thorax of housefly (Musca domestica L.) were separated by polyacrylamide gel electrophoresis. Their inhibition in vivo was studied after poisoning with an LD50 of four organophosphates: malaoxon, paraoxon, diazinon and dichlorvos. The isozymes differed greatly in their degree of inhibition. Thoracic isozymes were found to be more sensitive to inhibition than head isozymes. In surviving flies, the recovery rates of head isozymes were much faster than thoracic isozymes. In vitro studies showed that thoracic isozymes differed 4.1- and 2.9-fold in their Km and Vmax.

Stopped-flow studies of the inhibition of acetylcholinesterase by organophosphates in the presence of substrate

Abstract The stopped-flow technique has been applied to the determination of dissociation, phosphorylation, and bimolecular reaction constants for the inhibition of acetylcholinesterase by a potent organophosphate in the presence of a chromogenic substrate. The advantages of the method for inhibition studies are compared with those of an earlier spectrophotometric technique.