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

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 ?, 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 K_m 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.     

DDT effects on certain ATP related systems in the peripheral nervous system of the lobster Homarus americanus

Effects of DDT (1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane) on various ATP utilizing enzymes in the lobster peripheral nerve were studied. On the basis of inhibition by ouabain and DDT, four classes of ATPase enzymes were recognized. They are: (1) ATPase activity that is sensitive to both ouabain and DDT inhibition, or Type A, (2) ATPase activity that is sensitive to DDT inhibition only, or Type B, (3) ATPase activity that is sensitive to ouabain only, and (4) ATPase activity that is not sensitive to either ouabain or DDT. The Type A ATPase is considered to be a part of the total (Na⁺K⁺) ATPase enzyme associated with the electrogenic pump. The Type B ATPase consisted of an uncharacterized Na⁺, K⁺, and Mg²⁺ stimulated ATPase and includes also a small portion of Mg²⁺ stimulated ATPase. Ca²⁺ stimulated ATPase activity was also detected but was not significantly affected by DDT. Proteins with actomyosin-like properties were also recognized to be present, though this superprecipitation process was only slightly affected by DDT.Other systems studied include the transfer of (γ-³²P) ATP to endogenous proteins and added histone in the presence and absence of c-AMP. DDT generally stimulated the process of ³²P incorporation, while it inhibited a portion of the specific c-AMP dependent protein kinase activity.It was concluded from these studies that DDT has a potential to inhibit or otherwise interfere with a variety of enzymatic reactions that utilize ATP as a substrate. Of these systems, the Type B ATPase bore overall resemblance to the possible target for DDT.     

Two different types of inhibitory effects of pyrethroids on nerve Ca- and Ca + Mg-ATPase activity in the squid, Loligo pealei

The biochemical process by which various pyrethroid insecticides affect membrane-bound ATPase activities of the squid nervous system was examined. Of the five ATP-hydrolyzing systems tested, only Ca²⁺-stimulated ATPase activities are seen to be clearly affected by pyrethroids. It was found that the “natural type” pyrethroids (e.g., pyrethrin and allethrin) primarily inhibit Ca-ATPase activity whereas the “highly modified type” pyrethroids (e.g., cypermethrin and decamethrin) mainly inhibit Ca + Mg-ATPase. permethrin, which is considered to possess structural similarities to both the natural type and the highly modified type pyrethroids, was found to have an intermediate property in terms of its inhibitory potency to both Ca- and Ca + Mg-ATPase activities. The level of inhibition of Ca²⁺-stimulated ATPase activities was generally high in the retinal axons and optic lobe synaptosomes but lowest in the axoplasmic preparations.     

DDT and pyrethroids: Receptor binding and mode of action in the house fly

The mode of action of DDT and pyrethroids was investigated in the house fly, Musca domestica L, using drug:receptor binding techniques. Both in vivo and in vitro binding studies demonstrated the existence of membrane receptors which bind specifically to [¹⁴C]DDT and [¹⁴C]cis-permethrin. The receptors show properties to be expected of a critical target site of these insecticides. These include negative temperature correlation with binding, relatively nonsensitivity to DDE, and sensitivity to Ca²⁺. The receptor sites are readily saturated at 45–90 nM [¹⁴C]DDT and have an apparent disassociation constant (K_d) of 12.2 nM. The maximum number of binding sites was estimated to be 17 pmol DDT/mg membrane protein (0.34 pmol/house fly head). Competition studies showed DDT, cis-permethrin, and cypermethrin bind to the same receptor but not at precisely the same site. The addition of Ca²⁺ to the incubation buffer significantly inhibited the binding of both [¹⁴C]DDT and [¹⁴C]cis-permethrin, suggesting the receptor binding is Ca²⁺ sensitive and may have a role in ion conductance.     

Influence of calmodulin and ATP on DDT inhibition of Na/Ca exchange in the axolemma-rich nerve preparation from the American lobster

The effects of DDT on the Na/Ca exchange system were studied by using an axolemma-rich nerve membrane preparation from walking legs of Homerus americanus, the American lobster. The Na/Ca exchange system was measured by using two criteria: Na⁺ stimulated ⁴⁵Ca²⁺ uptake by the membrane vesicles and Na⁺Ca²⁺ protein kinase-phosphatase (formally called Ca-ATPase) as measured by the production of ³²P-labeled inorganic phosphate from [γ-³²P]ATP in the presence of Na⁺ and Ca²⁺. Activities of both systems were stimulated by the addition of exogenous calmodulin. DDT's inhibitory actions on both systems were reduced when an excess of calmodulin was added. Also addition of large amounts of ATP (or γ-thio-ATP) to the former system had an effect to reduce levels of DDT inhibition. DDT appears to act on the protein kinase proper as well as calmodulin itself in this system in a reversible manner.     

Interaction of insecticides with the Ca2+-pump activity of sarcoplasmic reticulum

The organophosphorus insecticides, parathion and azinphos (10^?5-10^?4M), significantly stimulate the Ca²⁺-pump activity of sarcoplasmic reticulum, while malathion has a limited effect. The rates of Ca²⁺ translocation and ATP hydrolysis are both stimulated and, apparently, the $\text{Ca}{}^{\mathrm{2+}}\text{ATP}$ ratio is improved. Parathion and azinphos maximally increase this ratio by 26 and 14%, respectively. The organochlorine compounds, DDT and aldrin, also stimulate the Ca²⁺ pump, and lindane has a reduced effect. These effects are smaller than those observed for parathion and azinphos. The order of effectiveness is similar to the toxicity of the compounds to mammals and can be described as follows: parathion > azinphos > DDT ≈ aldrin > malathion ≈ lindane.     

A comparison of DDT and its biodegradable analogues tested on ATPase enzymes in cockroach

Analogues of DDT (ethoxymethyl and methoxymethio derivatives) compared with DDT for their inhibitory action on the ATPase system from tissues of the cockroach, Periplaneta americana show similar, but less inhibitory effects. The mitochondrial (oligomycin-sensitive) Mg²⁺ ATPase activity from coxal muscle preparations was more sensitive to DDT than the two analogues; whereas, the muscle and nerve cord homogenates showed about equal sensitivity to the biodegradable analogues. The mitochondrial Mg²⁺ ATPase from nerve cord preparation was more sensitive to the three compounds than the Na⁺K⁺ ATPase activity. The significance of these results in relation to recent reports on the effect of DDT on Na⁺K⁺ ATPase is discussed.     

Quantitative structure-activity studies of substituted benzyl chrysanthemates: 9. Calcium uptake inhibition in crayfish nerve cord and lobster axon homogenates in vitro by sunthetic pyrethroids

Twenty-five synthetic pyrethroids and related chemicals were assessed for their effects on the uptake of Ca²⁺ (as ⁴⁵Ca²⁺) by crude homogenates prepared from crayfish (Procambarus clarkii) nerve cords and 20 were assessed on the uptake by homogenates prepared from lobster (Panulirus japonicus) axons. A parabolic relationship was demonstrated between inhibition of Ca²⁺ uptake for this series of chemicals and lipophilicity (log P) in both species when tested at 5 μM. Optimal log P for maximal inhibition was located at about 6.6 and 84(±6)% inhibition was obtained with resmethrin. Compounds of higher or lower log P were either weaker inhibitors, not inhibitors or occasionally resulted in slightly increased levels of Ca²⁺ uptake. No direct correlations between the potential for the pyrethroids to inhibit Ca²⁺ uptake and the potential for these agents to increase the frequency of spontaneous discharges in crayfish nerve cords, to induce repetitive firing in American cockroaches, or the lethality to cockroaches or to any other neurophysiological or toxicity parameter could be established. It was concluded that although some synthetic pyrethroids are moderately potent inhibitors of Ca²⁺ uptake into nerve cord and axonic preparations (i.e., I₅₀ for trans-resmethrin equals 1 μM) this inhibition alone does not relate to the neurophysiological changes in isolated nerve preparations or to the toxicity of these agents in insects.     

DDT and sodium transport in the eel electroplaque

DDT inhibits the ATPase activity of the intact eel electroplaque. At a concentration of 10^?5M, DDT inhibited 46% of the total ATPase activity, and 10^?4M DDT inhibited 62% of the total ATPase activity and 62% of the ouabain-sensitive ATPase activity. The latter concentration of DDT reduced the rate of Na efflux from intact electroplaques and slowed the rate of recovery of the membrane potential following a large depolarization produced by carbamylcholine application. Repetitive direct stimulation of the innervated membrane at 10 Hz during the application of 10^?4M DDT produced a significant irreversible depolarization. Ouabain, 10^?4M, produced similar effects. The possible role of the inhibition of active NaK transport in producing the symptoms of DDT poisoning is discussed.     

Promotion of norepinephrine release and inhibition of calcium uptake by pyrethroids in rat brain synaptosomes

A series of 25 pyrethroids were assessed for their effects on Na⁺-dependent norepinephrine release and on Ca²⁺ uptake in vitro using a crude rat brain synaptosomal preparation. The most effective pyrethroids required a concentration of 3–10 μM to promote norepinephrine release. Plotting release data versus lipophilicity (as log P) for each compound resulted in a parabolic curve with log P_opt being 5.4 for maximal release. The release promoted by most of the compounds assessed at 30 μM could not be or was only partially reversed by either tetrodotoxin or substituting choline for Na⁺ conditions which readily reversed the release promoting effects of veratridine. Thus, many pyrethroids, particularly those without the α-cyano group, did not display their expected effects on the Na⁺ channel in rat brain. When assessed at 5 μM, pyrethroids inhibited, had no effect, or caused increases in the amount of Ca²⁺ incorporated in the presence of ATP. The effectiveness of the various pyrethroids to inhibit Ca²⁺ uptake again displayed a parabolic relationship with log P_opt being 6.4. It was concluded that the variations in pyrethroid effects on norepinephrine release and Ca²⁺ uptake are not solely related to their particular chemical structures, but to lipophilicity. The effects of many pyrethroids on Ca²⁺ metabolism, particularly displacement of bound Ca²⁺, better explain the transmitter release promoting properties in vitro rather than a direct effect on the Na⁺ channel. No direct relationship between known toxicity to mammals and Ca²⁺ inhibition by pyrethroids was established.     

The sensitivity of mitochondrial Mg2+ ATPase to DDT and analogs at different temperatures

Oligomycin-sensitive (O-S) Mg²⁺ ATPase from American cockroach muscle was more sensitive to DDT, TDE, methoxychlor, and DDE at cool temperatures than at warm temperature, thus showing a negative temperature effect. In contrast, inhibition by acaricides dicofol, chlorfenethol, and Plictran shows a positive temperature effect. Oxidative phosphorylation in a mitochondrial preparation from cockroach coxal muscle was reduced by DDT, but the reduction was greater at a higher temperature (32°C) than at a cooler temperature (22°C). In addition, Na⁺K⁺ ATPase from cockroach nerve cord showed a positive temperature effect with DDT. The inhibition by DDT was much less on Na⁺K⁺ ATPase than on O-S Mg²⁺ ATPase. The negative temperature effect by DDT and analogs on O-S Mg²⁺ ATPase parallels toxicity effects on insects and fish as reported by numerous researchers. The results provide further evidence for this energy-regulating enzyme being a critical component in the biological action of DDT.     

Effect of isopropyl-3-chlorocarbanilate and isopropyl-3-chlorohydroxycarbanilate analogs upon oxidative phosphorylation in plant mitochondria

The effects of the herbicide, isopropyl-3-chlorocarbanilate, and its hydroxylated metabolites, isopropyl-5-chloro-2-hydroxycarbanilate and isopropyl-3-chloro-4-hydroxycarbanilate, upon NADH oxidation, P_i uptake or release, and ATP formation were studied in corn mitochondria. The results indicated that 0.1 mM isopropyl-3-chlorocarbanilate and the 2-hydroxy-metabolite inhibited NADH oxidation by 30% whereas only the 2-hydroxy-metabolite inhibited NADH-linked ATP formation (85–100%). Dinitrophenol and the 2-hydroxy-metabolite exerted similar effects upon respiration, phosphorylation, and ATPase activity. The 4-hydroxy-metabolite (0.1 mM) exerted no effect upon respiration, phosphorylation, or ATPase activity. The β-O-glucoside conjugates of the hydroxymetabolities of isopropyl-3-chlorocarbanilate did not inhibit NADH-linked respiration or phosphorylation at 0.1 mM concentrations. Comparative studies with corn, cucumber, and soybean mitochondria indicated that the parent herbicide and its metabolites affected respiration and phosphorylation activities in a similar manner.     

The in vivo sensitivity of ATPases to DDT,DDE, and physical immobilization in American cockroaches

American cockroaches injected with sublethal doses of DDT (0.75 μg/roach) at 5-day intervals showed a 40% reduction in oligomycin-sensitive Mg²⁺ATPase from muscle homogenates, and a 23% reduction of Na⁺-K⁺ATPase from nerve cords. Thus, the maximum effect measured occurred with the same enzyme and tissue as determined from in vitro studies. The metabolite, DDE, used at 15 μg per roach, gave no significant change in activity of the ATPase system following injection. In contrast, high single doses of DDT (7.5 μg/roach) and 100 μg DDE and dicofol per roach caused over 30% increase in oligomycin-sensitive Mg²⁺ATPase of muscle and a 10–15% increase in Na⁺-K⁺ATPase of nerve cords measured 24 and 48 hr later. While a similar response was observed for Mg²⁺ATPase activities in cockroaches that were immobilized, the increase in enzyme activities were much greater than that caused by the pesticides.     

Studies on the mechanism of estrogenic actions of o,p′DDT: Interactions with the estrogen receptor

The ability of o,p′DDT to bind to the 8S moiety in the uterine cytosol or to interfere with the binding of ³H-estradiol-17β (³H-E₂) to that binding component was investigated utilizing a 10–30% sucrose gradient sedimentation analysis. Attempts to demonstrate the binding of radiolabeled o,p′DDT to the 8S receptor in the mouse and rabbit were not successful, presumably due to the relatively low specific activity of the radiolabeled o,p′DDT, however, binding to the “nonspecific” 4S site(s) was detected. On the other hand, the addition of nonlabeled o,p′DDT inhibited the binding of ³H-E₂ to the 8S receptor. Thus, o,p′DDT (2 μM) suppressed by 58% the binding of ³H-E₂ (2 nM) in the 8S region in ovariectomized adult mice. Similarly, in immature rats three concentrations of o,p′DDT (16, 32, and 96 μM) inhibited by 39.5, 52.9, and 59.7% respectively, the binding of ³H-E₂ (2.8 nM). Similar results were obtained with uterine preparations from mature rats. However, the suppression of binding of ³H-E₂ in the 8S region resulted in an increased binding in the 3–4S region.A Scatchard plot analysis of the binding of ³H-E₂ in the presence of o,p′DDT revealed the same number of binding sites as in the absence of o,p′DDT, indicating that o,p′DDT did not “destroy” the binding capacity. Also, this analysis revealed that o,p′DDT merely caused a decrease in the ratio of the bound to free E₂, indicating that o,p′DDT binds to the receptor and thus interferes with E₂ binding.In addition, our observations that the administration of o,p′DDT to immature female rats causes a marked increase in the levels of the uterine nuclear binding sites (nuclear estogren receptor) is a further indication that o,p′DDT acts as a typical estrogenic compound. However, whether o,p′DDT has antiestrogenic activity as well has not been established.     

Action of deltamethrin on the calcium/calmodulin-dependent protein kinase from the rat brain

The action of deltamethrin on the calcium/calmodulin-dependent protein kinase (CaM-Kinase II) and phosphatase system in the rat brain synapse was studied under various experimental conditions to optimize these enzyme activities and to facilitate the studies of the mechanism of interaction of this pesticide with several components of this enzyme system. To obtain a clear-cut inhibition of this enzyme by deltamethrin the following conditions must be met: (a) the enzyme system should be purified by precipitation with ammonium sulfate (450 g litre^?1) prior to the addition of deltamethrin, (b) both Ca²⁺ and calmodulin (CaM) should be added to the incubated media before the addition of [y-³²P]ATP, (c) deltamethrin should be incubated at least 10 min (but less than 30 min) with the enzyme system before [y-³²P]ATP addition, (d) the incubation temperature should be above 20°C (optimum 30°C), (e) [y-³²P]ATP concentration should be in the order of 10^? M (concentration adjusted using cold ATP), and (f) the incubation time with [y^?P]ATP for incorporation of ³²P into the protein should be in the neighborhood of 60 s. Under these conditions, the inhibitory potency of various active and inactive isomers or analogs of pyrethroids and DDT was tested. The order of the inhibitory power of these active forms of pesticides was 1 R-deltamethrin > (S)(RS) fenvalerate ≥ p,p′-DDT. Other compounds were not active at the concentration tested, indicating the differential sensitivity of this enzyme and the existence of a correlation of inhibitory power to insecticidal activity.     

S-Ethyl dipropylthiocarbamate, N,N-Diallyl-2-chloroacetamide,and 2-chloroallyl diethyldithiocarbamate influence on sulfhydryl-dependent sucrose accumulation

l-[U-¹⁴C]sucrose accumulation by phloem sieve tube members (PSTM) of wheat (Triticum aestivum L. ‘Holley’) and sorghum (Sorghum bicolor L. ‘G522 DR’) was inhibited by the nonpermeant sulfhydryl inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), and this inhibition was reversed by the permeant sulfhydryl protectants dithiothreitol (DTT) and dithioerythritol (DTE). S-Ethyl dipropylthiocarbamate (EPTC) (≤0.1 mM) did not inhibit [¹⁴C]sucrose accumulation by wheat or sorghum PSTM. N-N-Diallyl-2-chloroacetamide (CDAA) (1 mM) inhibited [¹⁴C]sucrose accumulation by sorghum but not by wheat PSTM. The inhibition of [¹⁴C]sucrose accumulation in sorghum PSTM by the membrane permeant CDAA was reversed by DTT. Sorghum growth was inhibited by <1 μM CDAA. Membrane permeant 2-chloroallyl diethyldithiocarbamate (CDEC) (0.1 mM) inhibited [¹⁴C]sucrose accumulation by PSTM of sorghum but not wheat. The inhibition of sucrose accumulation in sorghum PSTM by 0.1 mM CDEC was reversed by DDT.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

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

An insect water-soluble ATPase (IF₁) 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. IF₁ 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 IF₁ ATPase to DDT compared to the high sensitivity of particulate mitochondrial ATPase activity.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

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

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.