Robert B. Koch

Differential inhibition responses caused by DDT on oligomycin-sensitive Mg2+-ATPase activity: Nature of the requirements for DDT sensitivity☆

Abstract Earlier communications from this laboratory have shown that DDT inhibited oligomycin-sensitive Mg 2+ -ATPase (EC 3.6.1.3) but that its active component, F 1 , was not affected. In the present investigation evidence has been obtained to determine the nature of the requirements for DDT sensitivity. The results showed that DDT sensitivity was conferred to F 1 from pig heart mitochondrial preparations when it was bound to F 0 from the same preparation. The F 1 from house fly ( Musca domestica L) thorax was able to bind to F 0 from pig heart. This combination showed similar sensitivity to that of the original F 1 -F 0 combination from pig heart mitochondria. However, when F 1 from pig heart mitochondria was incorporated into F 0 depleted in oligomycin sensitivity-conferring protein (OSCP) from the same source, the resulting ATPase activity was insensitive to DDT. Addition of crude (50–200 μg) or purified (5–20 μg) OSCP in the above preparation restored DDT sensitivity. Presence of dioleyl or dipalmitoyl phosphatidyl choline or Triton X-100 in the reaction medium antagonized the DDT inhibitions. Depletion of phospholipids from submitochondrial membrane preparations (SMP) decreased ATPase activity. Addition of dioleyl or soybean phosphatidyl choline to this lipid-depleted preparation restored DDT sensitivity. Evidence presented suggests that DDT acted on F 1 in association with one or more membrane components and that OSCP and phospholipid were essential for DDT sensitivity.

Adenosine triphosphatase from cockroach coxal muscle mitochondria. Isolation, properties, and response to DDT

Abstract An insect water-soluble ATPase (IF 1 ) was isolated from mitochondria of cockroach coxal muscle. It was homogenous as assessed by polyacrylamide gel electrophoresis and electron microscopy. It was found to be composed of five subunits with molecular weights of 70,000, 68,000, 53,000, 43,000, and 34,000. IF 1 was cold labile and showed maximal activity at 47–50°C. Its ATPase activity was Mg+ dependent and was stimulated by DNP and p -hydroxy-mercuribenzoate. This activity was inhibited by sodium azide and guanidine-HCl, not inhibited by oligomycin, and only slightly inhibited at a relatively high level of DDT. The site of action of DDT is discussed in light of relative insensitivity of IF 1 ATPase to DDT compared to the high sensitivity of particulate mitochondrial ATPase activity.

Isolation, characterization, and properties of factor F1 from mitochondrial preparations of housefly (Musca domestica L.) thorax☆

Abstract A water-soluble Mg2+-dependent ATPase (coupling factor F1) was isolated from the mitochondria of housefly thorax. It comprised about 14% of the proteins from a crude preparation. The F1 preparation was nearly homogeneous as assessed by gel electrophoresis, isoelectric focusing, and electron microscopy. It was composed of five subunits with the following apparent molecular weights: α, 68,000; β, 61,000; γ, 38,000; δ, 27,000; and ϵ, 17,500. The isoelectric pH (pI) of this protein was 7.3. F1 had a pH optimum of 8.2 and a temperature optimum between 37 and 45°C. The enzyme was fairly stable at 25°C. Nearly complete loss of activity was noticed at 0°C, while at 0 or 25°C, glycerol (20%) partially stabilized the enzyme activity against such inactivation. The Km value of the enzyme with respect to ATP was 0.4 mM. The activity was stimulated by low concentrations of 2,4-dinitrophenol. The enzyme was inhibited by azide, p-hydroxymercuribenzoate, and guanidine hydrochloride. Oligomycin and the pesticides pyrethrin, cyhexatin, and DDT have no effect on the enzyme activity. However, all of these chemicals inhibited intact Mg2+- ATPase. The results are discussed in the light of differential responses of soluble and intact ATPase to these pesticides.

Partial purification and characterization of a Ca2+-stimulated lipoxygenase from soybean seeds

A highly purified Ca2+-stimulated lipoxygenase was isolated from the Hill variety of soybean seeds. Separation of Ca2+-stimulated lipoxygenase from lipoxygenase active in the absence of Ca2+ (lipoxygenase-1) was readily obtained using a DEAE-cellulose column. Sample size applied to the ion exchange column was found to be critical. Both enzymes were bound to the column, although some highly active Ca2+-stimulated lipoxygenase eluted with buffer in the presence of bound lipoxygenase-1. Ca2+-stimulated lipoxygenase bound to DAAE-cellulose required the use of a NaCl gradient for elution. Ca2+-stimulated lipoxygenase showed an apparent isoelectric point at pH 5.90 and optimum activity at pH 7.5 and at 1.1 mM calcium. Lipoxygenase-1 was inhibited over 95% in the presence of 60 μM methyl mercuric chloride, while Ca2+-stimulated lipoxygenase showed a maximum of only 20% inhibition under the same conditions.

Effects of purified components of jellyfish toxin (Stomolophus melleagris) on adenosine triphosphatase activities

Abstract Toxin from the jellyfish Stomolophus meleagris was found to stimulate both Na + -K + and mitochondrial Mg 2+ ATPase activities at low toxin concentrations and to inhibit these ATPase activities at higher (> 0·2 mg/ml) concentrations. Using discontinuous membrane partition chromatography the toxin was fractionated into six fractions. Of these six fractions, one strongly activated when five inhibited mitochondrial Mg 2+ ATPase. One fraction strongly activated, a second mildly activated, while the other four inhibited Na + -K + ATPase about 50 per cent. Only two fractions affected oligomycin-insensitive ATPase, and both of these exhibited a mild stimulation. It is concluded that the ability of this toxin to alter membrane permeability to Na + is due, at least in part, to components in the toxin which act directly on ATPase.

Inhibition of dog brain synaptosomal Na+-K+ ATPase and K+-stimulated phosphatase activities by long chain n-alkyl-amine and -piperidine, and N″-alkylnicotinamide derivatives

Abstract The effects of piperidine, primary amine, and nicotinamide aliphatic derivatives on dog brain snyaptosomal Na + -K + ATPase were investigated. These derivatives inhibited the enzyme activity in a manner dependent on alkyl chain length. Kinetic studies revealed that inhibition of Na + -K + ATPase activity by long chain alkyl derivatives (C 12 -C 18 ) was biphasic and non-competitive with respect to the inhibitor and substrate (ATP) concentrations respectively. These long alkyl derivatives caused changes in the Hill coefficient that suggest the occurrence of a possible conformational change in the enzyme molecule. Dual-inhibitor experiments showed that both saturated and unsaturated pentadecylpiperidine (C 15 -pip) derivatives inhibited Na + -K + ATPase by the same mechanism. Oleylamine (C 18:1 -NH 2 ), and N -dodecylnicotinamide (C 12 -NA + Cl − ) derivatives apparently inhibited the enzyme activity by a mechanism different from that of piperidine derivatives. Low concentrations of C 15:1 -pip, C 18:1 -NH 2 , and C 12 -NA + Cl − inhibited dog brain Na + -K + ATPase activity only, but at higher inhibitor concentrations K + -stimulated phosphatase activity was inhibited as much as Na + -K + ATPase. It is concluded that long chain n -alkyl derivatives of piperidines, amines, and nicotinamide have a cationic detergent-like action on Na + -K + ATPase, possibly by the disruption of protein-phospholipid interactions. The mechanism by which these compounds inhibit the overall Na + -K + ATPase may involve two different inhibitory sites: one, a high affinity inhibitory site, binding to which inhibits the Na + -stimulated phosphorylation reaction, and the other, a low affinity inhibitory site, binding to which inhibits the K + -stimulated dephosphorylation reaction. These possible mechanisms may provide an explanation for the observed biphasic inhibition kinetics.

ATPase and dehydrogenase activities from house flies susceptible and resistant to organochlorine insecticides

Abstract Comparisons were made on ATPase, glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-P-GDH) activities in strains of the house fly, Musca domestica L., susceptible and resistant to organochlorine insecticides. Oligomycin sensitive (OS) Mg 2+ -ATPase was more sensitive to inhibition by DDT and aldrin than Na + K + -ATPase. OS-Mg 2+ -ATPase was present at similar levels in susceptible and resistant strains of house flies and was equally sensitive to inhibition by the pesticides. Levels of Na + K + -ATPase were 40% lower in the two resistant strains of house flies than in the susceptible strain. In contrast to OS-Mg 2+ -ATPase in the resistant strains, the Na + K + -ATPase was about 10 times less sensitive to inhibition by DDT and aldrin than in a susceptible strain. However, in vivo , DDT was a strong inhibitor of OS-Mg 2+ -ATPase and a very weak inhibitor of Na + K + -ATPase. Activities of G-6-PDH and 6-P-GDH were 1.2- to 4.6-fold higher in the resistant strain compared to the susceptible strains. Increased dehydrogenase activities were noted in susceptible flies after in vivo exposure to DDT (100 ppm). Variations in levels of activity and in sensitivity to inhibition of the enzymes tested indicate that toxicity and resistance to organochlorine insecticides may be related to different enzymes associated with ATP production and utilization.

Properties of an antibody to Kelevan isolated by affinity chromatography: Antibody reactivation of ATPase activities inhibited by pesticides☆

Abstract Antibody molecules were produced by injection of BSA-Kelevan into chickens and rabbits. Pure antibody was obtained by a single pass of blood serum through an affinity column. The affinity gel was prepared by covalently binding BGG-Kelevan to activated Sepharose 4B-CN. Purity of the antibody was determined by ultracentrifugation and gel electrophoresis. Properties of the antibody included: sedimentation coefficient = 6.2, pI = 7.0, calculated MW = 150,000, and precipitin band formation using the microouchterlony test. The antibodies in free or immobilized form were able to prevent or reverse Kepone inhibition of ATPase activity from a variety of tissues from different sources. About 70 μg (approx 0.4 μM) of purified antibody was sufficient to restore the activity of mitochondrial (oligomycin-sensitive) Mg2+ ATPase activity which had been inhibited (in vitro) by 1 μM Kepone. The antibody was effective in preventing enzyme inhibition by other organochlorine pesticides with widely differing molecular structures. However, nonchlorinated inhibitors of mitochondrial oligomycin-sensitive Mg2+ ATPase activity were much less affected by the antibody. The available evidence suggests that the antibody binding site for the hapten may be specific for secondary or induced bonding forces due to the carbon-chlorine bonds rather than for a specific molecular structure.

In vitro response of atpase activities in tissue subcellular particle preparations to a series of mono-unsaturated C18 fatty acids

The positions of the double bond and the cis/trans configurations of six mono-unsaturated C18 fatty acids (FA) showed selectivity for inhibition and stimulation of ATPase activities of tissue homogenate fractions. The 13,000 g and 100,000 g sediments (fractions B and C respectively) of tissues homogenates were used as sources of Na+-K+ ATPase and of oligomycin-sensitive (OS) and -insensitive (OIS) Mg2+ ATPase activities. Tissue source included bovine brain and rat brain, kidney, heart and liver. Cis mono-unsaturated C18 FA caused an apparent uncoupling of mitochondrial coupling factor F1 (Mg2+ ATPase). This was indicated by the loss of oligomycin and 1,1,1-trichloro-2,2 bis (p-chlorophenyl) ethane (DDT) sensitivity in the presence of 50 microM FA (12-C18:1). Thus, a precipitous decrease in OS-Mg2+ AtPase activity of mitochondria was accompanied by an equally steep increase in OIS-Mg2+ ATPase activity. This was especially apparent in the heart tissue preparation. The uncoupling action of the FA was not observed with the trans mono-unsaturated C18 FA, Na+-K+ ATPase activity from a bovine brain nerve ending particle (B) fraction was also sensitive to 12-C18:1 FA. However, inhibitory response of Na+-K+ ATPase activity were different for OS-Mg2+ ATPase activity. The latter (OS) was not sensitive to the trans 12-C18:1, while the former (Na+-K+) was sensitive to both cis and trans forms of 12-C18:1 and inhibition appeared to be dependent on the position of the double bond.

Antibody reactivation of OS-ATPase from house fly (Musca domestica L) inhibited by organochlorine pesticides

Abstract Antibody reactivation of ATPase activity inhibited by organochlorine pesticides was investigated using preparations from the thorax of house flies (Musca domestica L). Purified anti-Kelevan antibody from chicken serum was used for reactivation of OS-ATPase activity inhibited by in vivo or in vitro exposure to Kepone, DDT, and aldrin. Results indicate that the antibody was capable of removing the pesticides from the inhibitory site(s) on the enzyme, whether inhibition occurred in vivo or in vitro.