[Comparative study of the interaction of acetylcholinesterases of human erythrocytes and the heads of houseflies with phosphorylated alkylchloroformoximes]. / Sravnitel'noe izuchenie vzaimodeistviia atsetilkholinésteraz éritrotsitov cheloveka i golov komnatnykh mukh s fosforilirovannymi alkilkhlorformoksimami.

Studies have been made on the interaction of four types of phosphorylated alkylchloroformoximes, i.e. analogues of an insecticide-acaricide valexon, with acetylcholinesterases from human erythrocytes and from the heads of the housefly Musca domestica. Antiacetylcholinesterase activity of the drugs depended both on the structure of the organophosphorus compounds, and the origin of the enzyme, indicating the existence of differences in the active surface of these acetylcholinesterases. Incorporation of one or two chloride atoms into alkylchloroformoxime group of the cleaved part of the organophosphorus compounds increased anticholinesterase activity with respect to both enzymes. Diethyl derivatives of these drugs exhibited higher specificity with respect to housefly enzyme as compared to human acetylcholinesterase.

Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.

Multiple defects in the activity of adenylate cyclase from the Drosophila memory mutant rutabaga.

Adenylate cyclase in homogenates of Drosophila melanogaster is heterogeneous with respect to its affinity toward MgATP and its subcellular distribution. Km values for MgATP range, under similar assay conditions, from approximately 10(-5) M to approximately 10(-3) M, depending on the body region and on the subcellular localization of the enzyme. The majority of the enzyme in whole-body preparations is particulate, but various body regions differ in the relative proportion of the soluble enzyme. The memory mutant rutabaga lacks up to 35% of the total particulate activity. Even ligands that stimulate directly the catalytic subunit are incapable of bringing the activity of the mutant's enzyme to normal levels. The defect is differentially pronounced in various body parts and is associated with an altered responsiveness of the enzyme to Mg2+, to Ca2+, and to forskolin. It is suggested that rutabaga is lesioned in a subpopulation, or a functional state, of adenylate cyclase, which may play a role in memory formation.

The synthesis of acetylcholine by plants.

Choline acetyltransferase was demonstrated in nettles (Urtica dioica), peas (Pisum sativum), spinach (Spinacia oleracea), sunflower (Helianthus annuus) and blue--green algae by using a Sepharose--CoASH affinity column. The column effected a 1500-fold purification of the enzyme from nettle homogenates and was required for demonstrating activity in the other higher plants. Demonstration of the enzyme in blue-green algae suggests that acetylcholine was a biochemical necessity in the earliest photosynthetic organisms.

Acetylcholinesterase from the house-fly head. Molecular properties of soluble forms.

1. Polyacrylamide gel electrophoresis in Tris/glycine buffer (pH 8.3) revealed five forms of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the 100 000 X g, 1-h supernatants of aqueous fly-head extracts from the DDT/S strain. Five other housefly strains (CSMA, Bayer 21/199, Cradson/P, Malathion/R and DDT/R)were shown qualitatively to have the same soluble forms of the enzyme. 2. Plots of the electrophoretic mobility versus polyacrylamide concentration indicated that the multiple forms constituted a size isomer family. From the retardation coefficients derived from these plots, molecular weight estimates were obtained; these suggested that the smallest active component was a form of approx. 80 000 daltons. The higher aggregates, however, did not appear as simple oligomers of this component. 3. Density gradient sedimentation supported the electrophoretic findings. The smallest active component, with a sedimentation coefficient of 5.3 S, was confirmed as a molecular species of acetylcholinesterase that has not previously been obtained from house-flies; higher aggregates gave sedimentation coefficients of 7.4, 7.8. 8.1, and 11.8 S. 4. Gel-filtration on calibrated Sephadex G-150 columns provided further evidence that the smallest active component was a form of about 80 000 daltons. 5. Autolysis converted much of the particulate enzyme and all of the soluble forms into a species of approx. 160 000 daltons indistinguishable from the native 7.4-S form. Both the autolysed enzyme and the native 7.4-S form were susceptible to cleavage by disulphide reducing agents, and released catalytically active subunits that corresponded to the 5.3-S form of 80 000 daltons. The data were compatible with a monomer-dimer relationship between the 5.3-S and 7.4-S forms. 6. The possibility is suggested that a form of molecular weight approx. 80 000 constitutes the "fundamental unit" of insect cholinesterase.

Acetylcholinesterase of the house-fly head. Affinity purification and subunit composition.

1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.

Penetration and comparative metabolism of leptophos in susceptible and resistant houseflies.

The metabolism and rate of penetration of leptophos (O-methyl O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate) was determined in a susceptible strain and a strain of housefies which was 50-fold resistant to leptophos. Penetration of leptophos into resistant flies was substantially slower than into susceptible flies but large differences in metabolism, both quantitatively and qualitatively, were not observed. No difference was observed in the sensitivity of flyhead and thorax acetylcholinesterase to leptophos-oxon in vitro, and tolerance to leptophos by the resistant strain is explained in terms of decreased rates or penetration and minor differences in metabolism.

Nicotinamide-adenine dinucleotide phosphate enzymes in the mosquito during growth and aging.

1. Optimum conditions were established for determining the activities of the NADP(+)-linked enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, in mosquito tissues. 2. The activity of each dehydrogenase was determined in samples of mosquitoes of different ages throughout the life-span. The specific-activity curves attained maximal values in the pupal or early adult period. From these maxima an 81% decrease in glucose 6-phosphate-dehydrogenase and 67% decrease in 6-phosphogluconate-dehydrogenase activities occurred after the tenth day of adult life; a 77% decrease in isocitrate-dehydrogenase activity occurred before the fifth day. 3. The activity differences were found in different body regions as well as in whole organisms. 4. Starvation of the larva or adult did not result in decreases in enzyme activity. 5. These findings support the hypothesis that the activities of enzymes that form NADPH are related to the biosynthetic activity, for the enzyme activities increased during the period of cellular growth and decreased during the aging period.