DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

The role of SH-groups in the development sensitivity of ATPase in plasma membranes of plant cells to ions

The effects of the SH-groups binding agent p-chloromercurybenzoate (rho CMB) and the SH-containing compounds dithiothreitol (DTT), beta-mercaptoethanol (ME) and reduced glutathione (GSH) on activation by Mg2+ and K+ of ATPase in plasma membrane preparations from corn sprout root cells were studied. Rho CMB inhibited the ATPase activity, the degree of inhibition being directly dependent on the increase of the inhibitor concentration (from 10(-6) up to 10(-4) M); the inhibition was eliminated by the SH-containing agents (25 mM). DTT and ME added to the homogenization medium and ME added to the reaction mixture produced different effects on the ATPase activity of the membranes depending on the nature of the cations added. In the absence of the additives the ATPase activity was somewhat decreased, showing a sharp rise in the presence of Mg2+; an addition of K+ to a Mg2+-containing medium further increased the enzyme activity. GSH had no effect on the ATPase activation by the cations.     

Properties of epsilon-ATP hydrolysis by CF1-ATPase from pea chloroplasts

The ATPase activity of CF1 isolated from pea chloroplasts with epsilon-ATP, the fluorescent analog of ATP and ATP used as substrates, in the presence of Mg2+, Ca2+ and sodium sulfite (stimulator of the ATPase activity) was studied. The rate of epsilon-ATP hydrolysis in the presence of Mg2+ is nearly two times as low as that of ATP; an addition of sodium sulfite to the reaction mixture increases the reaction rate without changing the above ratio. The rate of Ca2+-dependent hydrolysis of epsilon-ATP is rather low as compared to that in the presence of Mg2+. epsilon-ADP is a competitive inhibitor of Mg2+-dependent ATPase reaction and inhibits this process in the presence of Ca2+, the inhibition being of a mixed type. Modification of CF1 by covalent binding of epsilon-ADP results in a 70-80% decrease of the Mg2+-dependent ATPase activity, the Ca2+-dependent ATPase activity is changed only insignificantly thereby. The differences in the activation of ATP and epsilon-ATP hydrolyses by Ca2+ and Mg2+ can be accounted for by the existence of two sites in the active center of CF1, which are specific for Mg2+ and Ca2+, respectively. It is concluded that the binding of epsilon-ADP occurs in the Mg2+-dependent ATPase site of the active center.     

Kinetic studies and characteristics of a modifier of Ca2+-stimulated ATPase

1. The thermostable modifier increases the Mg2+-stimulated ATPase with a parallel decrease of Ca2+-stimulated ATPase. 2. The modifier inhibited Ca2+-stimulated ATPase by diminishing the velocity without any significant effect on the apparent affinity of binding of Ca2+. 3. The inhibition of Ca2+-stimulated ATPase was independent of Ca2+ concentration and could not be reversed by increasing Ca2+ levels. 4. Monovalent cations and calmodulin increased the Mg2+-stimulated ATPase in the presence of the modifier.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Properties and function of clostridial membrane ATPase.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of (Na+,K+)-ATPase by magnesium ions and inorganic phosphate and release of these ligands in the cycles of ATP hydrolysis

The kinetic data of magnesium and inorganic phosphate inhibition of the (Na+,K+)-dependent ATP hydrolysis are consistent with a model where both ligands act independently and their release in the ATPase cycle is an ordered process where inorganic phosphate is released first. The effects of magnesium on the stimulation of the ATPase activity by Na+, K+ and ATP, and the inhibition of that activity by inorganic phosphate, are consistent with Mg2+ acting both as a 'product' and as a dead-end inhibitor. The dead-end Mg-enzyme complex would be produced with an enzyme form located downstream in the reaction sequence from the point where Mg2+ acts as a 'product' inhibitor. In the absence of K+, Mg2+ inhibition was reduced when either Na+ or ATP concentrations were increased well beyond those concentrations needed to saturate their high-affinity sites. This ATP effect suggests that the dead-end Mg-enzyme complex formation is affected by the speed of the E2-E1 conformational change. The present model is consistent with the formation of an Mg-phosphoenzyme complex insensitive to K+ which could become K+-sensitive in the presence of high Na+ concentrations. These Mg-enzyme complexes appear as intermediaries in the Na+-ATPase activity found in the absence of external Na+ and K+. These results can be interpreted on the basis of Mg2+ binding to a single site in the enzyme molecule. In addition, these experiments provide kinetic evidence indicating that the stimulation by external Na+ of the ATPase activity in the absence of K+ is due to a K+-like action of Na+ on the external K+ sites.     

Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction

1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.     

A dynein ATPase inhibitor isolated from a commercial ATP preparation.

Preparations of ATP from equine muscle contained an inhibitor of dynein Mg2+-activated ATPase. The inhibitory material was separated from the ATP by molecular sieve filtration. The several molecular species of dynein extracted from three different axonemal sources were all inhibited; myosin ATPase was not. With increasing amounts of inhibitor the inhibition did not go to completion but reached a plateau when the rate had been reduced to 1/5 the uninhibited rate. A plot of 1/[S] against 1/v at several inhibitor concentrations yielded parallel lines. There was little inhibition of dynein ATPase when Mg2+ was replaced by Ca2+. The inhibitor appeared slightly smaller in molecular size than ATP, had anionic character, and was not adsorbed to charcoal.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Influence of gramicidin S on cardiac membrane Ca2+/Mg2+ ATPase activities and contractile force development

The effects of gramicidin S (GS), an antibiotic, on the rat heart membrane ATPases and contractile activity of the right ventricle strips were investigated. GS inhibited sarcolemmal Ca2+-stimulated ATPase (IC50 = 3 microM), Ca2+/Mg2+ ATPase which is activated by millimolar Ca2+ or Mg2+ (IC50 = 3.4 microM), and sarcoplasmic reticulum Ca2+-stimulated ATPase (IC50 = 6 microM). The type of inhibition for the sarcolemmal Ca2+/Mg2+ ATPase by GS was apparently uncompetitive, while that for Ca2+-stimulated ATPases in sarcolemma or sarcoplasmic reticulum was of mixed type. Other ATPases, including mitochondrial ATPase, sarcolemmal Na+-K+ ATPase, and myofibrillar ATPase, were not inhibited by this agent. GS also decreased the rat right ventricle maximum force development (half-maximal inhibitory concentration was 2-4 microM), maximum velocity of contraction, and maximum velocity of relaxation. The resting tension was increased by GS to over 200%. The contractile actions of GS were mostly irreversible upon washing the muscle 3 times over a 10-min period. Decreased Ca2+, Mg2+, Na+, K+ concentrations in the perfusate increased the effects of GS. These findings showed that GS was a potent inhibitor of divalent cation ATPases of heart sarcolemma and sarcoplasmic reticulum and it is suggested that these membrane effects may explain the cardiodepressant action of this agent.     

Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.     

AIF4-induced inhibition of the ATPase activity, the Ca2+-transport activity and the phosphoprotein-intermediate formation of plasma-membrane and endo(sarco)plasmic-reticulum Ca2+-transport ATPases in different tissues. Evidence for a tissue-dependent functional difference.

AIF4- inhibits the (Ca2+ + Mg2+)-ATPase activity of the plasma-membrane and the sarcoplasmic-reticulum Ca2+-transport ATPase [Missiaen, Wuytack, De Smedt, Vrolix & Casteels (1988) Biochem. J. 253, 827-833]. The aim of the present work was to investigate this inhibition further. We now report that AIF4- inhibits not only the (Ca2+ + Mg2+)-ATPase activity, but also the ATP-dependent 45Ca2+ transport, and the formation of the phosphoprotein intermediate by these pumps. Mg2+ potentiated the effect of AIF4-, whereas K+ had no such effect. The plasma-membrane Ca2+-transport ATPase from erythrocytes was 20 times less sensitive to inhibition by AIF4- as compared with the Ca2+-transport ATPase from smooth muscle. The endoplasmic-reticulum Ca2+-transport ATPase from smooth muscle was inhibited to a greater extent than the sarcoplasmic-reticulum Ca2+-transport ATPase of slow and fast skeletal muscle.     

Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.     

Inhibitory probes of mitochondrial ATPase and the action of DDT

Chemical inhibitors were used as probes of mitochondrial ATPase to determine the site of action of DDT on oligomycin-sensitive mitochondrial ATPase (OS-ATPase) using whole mitochondria isolated from red coxal muscle of the American cockroach. Several plotting procedures were employed to delineate the form of inhibition. Relative potency and joint action were used to detect similar action, synergism, and antagonism between DDT and the inhibitory probes DCCD, Nbf-CI, and oligomycin. DDT demonstrated not (strictly) competitive kinetics and may be acting as an uncompetitive inhibitor. DDT and DCCD produced similar additive action. At limiting concentrations of DCCD, inhibition was reduced in the presence of DDT. Effects shown by oligomycin were not altered by DDT. DDT enhanced the effects of Nbf-CI. These interactions, together with the demonstration of not (strictly) competitive kinetics, indicate that DDT may be acting on the membrane sector as an allosteric modifier.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.