Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Some kinetic and molecular properties of yeast phosphofructokinase

Yeast PFK had a sedimentation coefficient of 16.7 S both in the absence and in the presence of ATP, and did not dissociate even at very low protein concentrations. Sodium dodecyl-sulphate caused dissociation of the protein to sub-units of 3.2 S.The effects of pH on substrate affinities are described. In the presence of UTP, acting as non-inhibiting phosphate donor, the behaviour of the enzyme towards F-6-P was co-operative, with a Hill coefficient of 2.2.     

Anion regulation of active transport of protons across the membrane of the synaptic vesicles of the brain

The dependence of active transport of H+ on the presence of anions in synaptic vesicle membranes from rat brain was studied. The H+ transport was measured by monitoring the acidification of the vesicles with a permeant weak base-acridine orange. The fluorescence changes in the latter were proportional to the magnitude of artificially imposed pH gradients (delta pH). The ATP-dependent generation of delta pH was completely dependent on the presence of a permeant anion, was maximal at 150 mM Cl- and was inhibited, when the medium osmolarity was further increased by sucrose or KCl. At 150 mM only Br-, similar to Cl-, behaved as permeant anions, whereas I- was effective only at low (5-20 mM) concentrations. The anions--SCN-, ClO4-, HSO3- and I-(10-20 mM) as well as 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonate (K0.5 = 14 microM) blocked the ATP-dependent generation of delta pH observed in the presence of Cl-, while other anions tested (F-, phosphate, bicarbonate, some organic anions) were virtually without effect and did not support the H+ transport. The dependence of the rate and extent of H+ accumulation on Cl- concentration was sigmoidal with a Hill coefficient of 2.8 and a Km value of 85-90 mM. The effects of anions point to the presence in the membrane of synaptic vesicles of an anion (chloride) channel whose conductance can regulate the H+ transport by switching it from an electrogenic to an electroneutral (coupled entry of H+ and Cl-) mode of operation.     

BIOCHEMICAL ASPECTS OF INSECTICIDE RESISTANCE IN COTTON BOLLWORM FROM HANDAN OF HEBEI PROVINCE*

Abstract The cotton bollworms, Helicoverpa armigera Hübner, collected from Handan of Hebei Province, have evolved high resistance to pyrethroid, organophosphate and carbamate insecticides, The sensitivity of acetylcholinesterase (AChEs) to paraoxon and methomyl varied with the development stages of the cotton bollworm. After the treatments with LD₅ and LD₅₀ of parathion and methomyl to the cotton bollworms, the affinity of AChE to acetylthiocholine (ATCh) and acetyl-β-bmethyl-thio choline (MeTCh) increased significantly except the treatment of parathion using LD₅₀ dosage while the sensitivity of AChEs to paraoxon significantly decreased. The sensitivity of AChEs to methomyl strongly increased in the treatment of parathion using LD₅₀ dosages while strongly decreased in other treatments. The affinity of carboxylesterase to β-naphthyl acetate (β-NA) was higher in groups of treatment with insecticides than in group of control. The glutathione-s-transferase (GST) activity significantly decreased in the induced groups using LD₅ dosages, while increased in the selection groups using LD₅₀ dosages. The effects of parathion and methomyl on the phosphatases of cotton bollworm were related to the dosages of application and the time after treatment and the effect on the alkaline phosphatase was stronger than on acid phosphatase.     

Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.     

Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure–function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 2,500-fold from the conditioned medium to near homogeneity. While K_M's of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, V_max's were substantially different. The IC₅₀'s for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59 × 10⁴ nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.     

Cooperative effects in the binding of pyridoxal 5'-phosphate to mitochondrial apo-aspartate aminotransferase

Titrations of mitochondrial apo-aspartate aminotransferase with pyridoxal 5'-phosphate in the presence of AMP, contrary to what has been observed in the case of the cytosolic isoenzyme [(1983) FEBS Lett. 153, 98-102], show sigmoidal isotherms, with Hill coefficients ranging from nH = 1.4, in the absence of AMP, to nH = 1.8, in the presence of 5.9 mM AMP. The experimental data were successfully fitted by the Monod-Wyman- Changeaux model. The best fit, in the absence of AMP, was obtained with L = 30, KR = 4.72 X 10(-7) M and KT = 1.18 X 10(-5) M. Binding curves in the presence of AMP fit the model by keeping KR as a constant. This implies that AMP could bind to the apoenzyme only in the T state. In contrast, binding curves in the presence of phosphate ion (Pi) showed a less pronounced cooperativity, the Hill coefficient dropping to nH = 1.0 in the presence of 0.1 mM Pi. The above results suggest a regulatory role of AMP and Pi in the reconstitution of aspartate aminotransferase.     

Binding of Ca2+ to glycoprotein from bovine heart mitochondria

It is shown that glycoprotein from bovine heart mitochondria which forms Ca2+-selective conductance channels in a bilayer lipid membrane possesses Ca2+-binding activity. Ca2+-binding sites of two kinds were revealed in the glycoprotein molecule: high affinity sites with Kd = 2.8 X 10(-6) M and low affinity sites with Kd 1.1 X 10(-5) M. Ca2+-binding by the high affinity sites occurs co-operatively. The Hill coefficient is about 2.     

Nitrobenzylthioinosine binding cooperativity in chromaffin tissue membranes.

The nucleoside transporter present in chromaffin tissue membranes has been studied by [3H]nitrobenzylthioinosine (NBTI) binding. This ligand presents a high affinity, with a Kd value of 2.1 +/- 0.2 nM and a Bmax of 1.7 +/- 0.2 pmol/mg protein. From the Scatchard and the semilogarithmic graphical representations a positive cooperativity was deduced, with a Hill coefficient of 1.7 +/- 0.4. In displacement studies of NBTI by the non labelled compound, the Hill coefficient was also higher than 1 (1.44 +/- 0.11) in the presence of ATP. This nucleotide seems necessary to maintain the number of high affinity binding sites.     

Cooperative interaction of pyruvate dehydrogenase from pigeon breast muscle]

Cooperative interaction of pyruvate with the pyruvate dehydrogenase (PD) complex from pigeon breast muscle was shown. The sigmoidal dependence of the reaction rate on pyruvate concentration was observed for the PD complex. The Hill coefficient is equal to 1,5; no inhibition by the substrate (up to 2.2.10(-3) M) was found. The kinetic behaviour of the isolated pyruvate dehydrogenase component (PDH) analyzed under similar conditions, is more complex; this may be probably due to the presence of oligomeric forms with different molecular weights and specific activities. The competitive inhibitor of the PD complex--an amide of pyruvic acid (PA) (Ki=6.3-10(-6) M) activates the enzyme at low concentrations (less than 2,10(-6) M). When PA is present, the dependence of the reaction rate on pyruvate concentration gives a usual hyperbolic curve, v of [S]o. It is concluded that pyruvate may have a regulatory effect on the activity of muscle PD complex.     

Sensitive detection of organophosphorus pesticides using a needle type amperometric acetylcholinesterase-based bioelectrode. Thiocholine electrochemistry and immobilised enzyme inhibition

An acetylcholinesterase (AChE) based amperometric bioelectrode for a selective detection of low concentrations of organophosphorus pesticides has been developed. The amperometric needle type bioelectrode consists of a bare cavity in a PTFE isolated Pt-Ir wire, where the AChE was entrapped into a photopolymerised polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ). Cyclic voltammetry, performed at Pt and AChE/Pt disk electrodes, confirmed the irreversible, monoelectronic thiocholine oxidation process and showed that a working potential of +0.410 V vs. Ag/AgCl, KCl(sat) was suitable for a selective and sensitive amperometric detection of thiocholine. The acetylthiocholine detection under enzyme kinetic control was found in the range of 0.01-0.3 U cm(-2) of immobilised AChE. The detection limit, calculated for an inhibition ratio of 10%, was found to reach 5 microM for dipterex and 0.4 microM for paraoxon. A kinetic analysis of the AChE-pesticide interaction process using Hanes-Woolf or Lineweaver-Burk linearisations and secondary plots allowed identification of the immobilised enzyme inhibition process as a mixed one (non/uncompetitive) for both dipterex and paraoxon. The deviation from classical Michaelis Menten kinetics induced from the studied pesticides was evaluated using Hill plots.     

Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.     

A comparison of the inhibition of deacetylase in primary cultures of rat and human hepatocytes effecting metabolism and DNA-binding of 2-acetylaminofluorene

The metabolism of 2-acetylaminofluorene (AAF) in primary cultures of rat and human hepatocytes was investigated to determine if the activation of this well-studied chemical carcinogen proceeds via similar routes of metabolism between species. The total level of AAF metabolite(s) bound to hepatocellular DNA was determined in the presence of deacetylase inhibitors, diethyl(p-nitrophenyl) phosphate (paraoxon) or bis(p-nitrophenyl) phosphate (BPNPP). These compounds are known to inhibit deacetylase and to decrease the mutagenicity of AAF. Experiments with rat and human hepatocytes demonstrated inhibition in the deacetylation of AAF (5×10⁻⁴ M) with paraoxon or BPNPP. The BPNPP (5×10⁻⁴ M inhibited 99% of the AF formation in the human hepatocytes and 88% inhibition in the rat hepatocytes. Paraoxon at 10⁻⁴ M demonstrated a 98% inhibition of deacetylation with humans and a 92% inhibition with rats. The rat hepatocytes also showed a 53% decrease in DNA binding in the presence of paraoxon. In contrast with human hepatocytes, while paraoxon decreased the AF metabolite by > 97%, there was no change in total DNA binding.     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently

The kinetics of active Ca2+ transport in inside-out red cell membrane vesicles and the Ca2+-ATPase activity of the purified Ca2+ pump were studied and the effects of calmodulin, acidic phospholipids, and controlled trypsinization were compared. In the presence of calmodulin the maximal rate and the apparent affinity of the pump for Ca2+ were greatly increased in both preparations. The lowest value of Km(Ca) was between 0.5 and 0.7 microM depending on the concentration of calmodulin and on the enzyme preparation. Positive cooperativity for Ca2+ activation with a Hill coefficient of 1.6-1.7 was observed in all cases. When acidic phospholipids (phosphatidylinositol 4-phosphate was routinely used) were added to the inside-out vesicles or to the purified enzyme, maximal transport rates equal to those obtained with calmodulin were measured but the Km(Ca) decreased to 0.25 microM and the positive cooperativity disappeared (the Hill coefficient approached 1). Highly active, calmodulin-independent proteolytic fragments of molecular mass of 81 and 76 kDa were produced with controlled trypsinization. When the trypsin treatment was directed to obtain primarily the 81-kDa fragment, the preparation showed characteristics similar to those of the intact Ca2+ pump in the presence of calmodulin; that is, the same Vmax was obtained, the Km(Ca2+) was 0.5-0.6 microM, and the Hill coefficient was about 1.6. Addition of phosphatidylinositol 4-phosphate or allowing further proteolysis to produce the 76-kDa fragment, shifted the Km(Ca) to 0.25 and reduced the Hill coefficient to 1, without changes in the maximal rate. Based on these results it is suggested that the maximal velocity and the Ca2+ affinity on the erythrocyte Ca2+ pump may be regulated independently and that independent polypeptide regions of the enzyme are involved in the regulations.     

Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.     

Phenobarbital modulates the (Na+, K+)-stimulated ATPase and Ca2+-stimulated ATPase activities by increasing the bilayer fluidity of dog brain synaptosomal plasma membranes

The binding of [14C]phenobarbital into synaptosomal plasma membranes of dog brain follows a sigmoid path. The "best fit" curve of this binding is the one described by the Hill equation (r2 less than 0.93 and Hill coefficient, n = 1.32). (Na+, K+)-stimulated ATPase and Ca2+-stimulated ATPase activities are modulated by phenobarbital. Arrhenius plots of (Na+, K+, Mg2+)-dependent ATPase revealed that phenobarbital (2 mM) lowered the transition temperature and altered the Arrhenius activation energies of this enzyme. The allosteric inhibition by F- of the (Na+, K+)-stimulated ATPase was studied in control and phenobarbital-treated membranes. The lowering of the transition temperature and changes in Arrhenius activation energy about the transition temperature in combination with changes observed in the allosteric properties of the (Na+, K+)-stimulated ATPase by F-, produced by phenobarbital, would be expected if it is assumed that phenobarbital "fluidizes" synaptosomal plasma membranes.     

Kinetic properties of sodium transport pathways in the river lamprey Lampetra fluviatilis erythrocytes

To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 to 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9-10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6-1.0 microM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration of Na+: for the semimaximal stimulation of H+ outcome amounted to 19 mM. The obtained results indicate the presence in lamprey erythrocytes of only one binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+ binding from the extracellular side of the Na+/H+ exchanger. Its efflux from cells in the Na+ -free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equation with n 1.5. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm the concept of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.