Modeling the stimulation by glutathione of the steady state kinetics of an adenosine triphosphate binding cassette transporter

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the “nonessential activator model” where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis.     

Pre-steady state kinetic studies on H(+)-ATPase from Candida albicans.

The mechanism of ATP hydrolysis by plasma membrane H(+)-ATPase from Candida albicans has been investigated by following the kinetics of H(+) liberation/absorption and the UV difference spectrum in a stopped flow spectrophotometer. A distinct pre-steady state phase of ATP hydrolysis could be defined. While the rapid mixing of P(i) and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H(+) per molecule of ATPase. Rapid mixing of ATP with ATPase releases about 2 H(+) per molecule of ATPase, of which around 1.3 H(+) are reabsorbed. The magnitudes of both H(+) release and absorption were found to be independent of ATP concentration. The rate of H(+) release (k(f)) shows ATP dependence while the rate of H(+) absorption is independent of ATP concentration. The rate of H(+) liberation with ADP, on a concentration basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP. The stoichiometry of ATP binding to PM-ATPase was found to be unity from UV-difference spectrum studies. The k(f) values for H(+) release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a 1:1 ratio of ATP:ATPase. The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrolysis.     

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F₁F_o-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K_0.5=0.27 mM) and low (K_0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg²⁺ saturating condition (high affinity site, K_0.5=0.10 mM, and low affinity site, K_0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.     

Purification and characterization of dinitrophenylglutathione ATPase of human erythrocytes and its expression in other tissues

S-(2,4-dinitrophenyl)glutathione (Dnp-SG) ATPase of human erythrocytes has been purified to apparent homogeneity by affinity chromatography. In reduced denaturing gels, the subunit Mr value of Dnp-SG ATPase was found to be 38,000. Dinitrophenyl glutathione (Dnp-SG) stimulated the hydrolysis of ATP by the purified enzyme whereas oxidized glutathione (GSSG) did not, indicating that Dnp-SG and GSSG are transported from the erythrocytes by different transporters. Results of Western blot analysis using the antibodies against Dnp-SG ATPase subunits indicated that the enzyme was expressed in human liver, lung, placenta and pancreas.     

Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes.

Dinitrophenyl S-glutathione (Dnp-SG) ATPase has been purified from human muscle to apparent homogeneity using Dnp-SG affinity chromatography and immunoaffinity chromatography using antibodies raised against human erythrocyte Dnp-SG ATPase. The enzyme purified from human muscle showed a subunit M(r) value of about 38 kDa in denaturing gels. The M(r) value of the native enzyme as determined by Sephadex G-200 gel filtration was found to be about 80 kDa, which indicates that it is a dimer. The N-terminus of the enzyme was blocked. Its immunological and kinetic properties were similar to Dnp-SG ATPase of human erythrocytes. Besides catalyzing the ATP hydrolysis in the presence of Dnp-SG, the muscle enzyme also catalyzed ATP hydrolysis in the presence of various leukotrienes, namely LTC4.LTD4, LTE4, and N-acetyl LTE4. The specific activity of the enzyme toward LTC4 was relatively higher than other GSH-xenobiotic conjugates. The muscle enzyme exhibits a low Km value for all leukotrienes as compared to Dnp-SG, indicating high affinity of the enzyme for leukotrienes as activators. The enzyme also catalyzed ATP hydrolysis in the presence of GSH conjugates of endogenously generated fatty acid epoxides. Our results might suggest that Dnp-SG ATPase is involved in the transport of GSH conjugates, leukotrienes, and other organic anions in muscle, erythrocytes, liver, and probably other tissues.     

Steady state kinetics at high enzyme concentration. The myosin MgATPase

The rate of ATP hydrolysis by myosin at high concentrations with an ATP-regenerating system increases linearly with increasing added ATP up to a sharp break at the equivalence point of 1 ATP/myosin active site. Theoretical modeling indicates that the data require a KM on the order of the 30 nM value predicted by the rapid kinetic work (Lymn, R. W., and Taylor, E. W. (1970) Biochemistry 7, 2975-2983). Changes in the experimental conditions are found to change the slope of the initial increase in ATPase rate, but not to change the equivalence point. Proteolytic subfragments of myosin do not exhibit a linear initial increase in rate indicating that they are not homogeneous. Purified myosin is also found to show a small additional increase in ATPase rate at much higher ATP levels with a corresponding increase in flux through a pathway with a low extent of oxygen exchange. This high Km component with low oxygen exchange is distinct from the contaminating ATPase reported previously (Sleep, J. A., Hackney, D. D., and Boyer, P. D. (1980) J. Biol. Chem. 255, 4094-4099) which is shown here to be the CaATPase of the sarcoplasmic reticulum.     

Stoichiometric photolabeling of two distinct low and high affinity nucleotide sites in sarcoplasmic reticulum ATPase

Highly purified 3'-arylazido-ATP (aATP) was obtained by high performance liquid chromatography. In the dark, this photoactivatable ATP analog was a competitive inhibitor of ATP hydrolysis catalyzed by purified sarcoplasmic reticulum (SR) ATPase with a Ki of 10 microM. The analog itself was hydrolyzed by the enzyme in the dark. A biphasic curve of velocity of hydrolysis of the analog versus aATP concentration was obtained, indicating the presence of high and low affinity sites with K0.5 of approximately 10 microM and 300 microM, respectively. Upon irradiation with visible light, a biphasic curve was obtained for the level of covalent photolabeling of the enzyme versus [beta-32P]aATP concentrations. Levels of 6.5-9 nmol of analog/mg of protein and 20-22 nmol of analog/mg of protein were obtained when labeling with 20-30 or with 400 microM aATP, respectively, showing the existence of 1 mol of high affinity sites/mol of ATPase and 1-1.5 mol of low affinity sites/mol of enzyme. The rate of light-dependent incorporation of [beta-32P]aATP was decreased by the presence of ATP, Pi, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene-ATP, or Ca2+ in the illumination media. Photolabeling of the high affinity sites had little effect on the velocity of ATP hydrolysis but significantly inhibited the splitting of additional aATP added in the dark. Photolabeling the low affinity sites caused irreversible inhibition of the ATPase activity. The inhibition was prevented by having ATP in the illumination medium, which protected it from labeling. Gel filtration chromatography in the presence of detergent showed that radioactive photolabel was incorporated in the SR ATPase protein. The results indicate that aATP is a useful tool for stoichiometrically labeling and probing the nucleotide binding domains of the SR ATPase.     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase

Incubation of [gamma-32P]ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F1) results in binding of substrate primarily in a single, very high affinity (KA = 10(12) M-1) catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10(6)-fold, that is, to Vmax rates (Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101-12105). For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. Recently, Bullough et al. (Bullough, D.A., Verburg, J.G., Yoshida, M., and Allison, W.A. (1987) J. Biol. Chem. 262, 11675-11683) reported that when 5 to 20 microM concentrations of nonradioactive ATP were added as a cold chase to an enzyme-substrate complex consisting of F1 and ATP bound to the high affinity catalytic site, hydrolysis of the chase was commensurate with the turnover rate of the enzyme, whereas the hydrolysis of bound ATP was considerably slower. These authors suggested that the high affinity catalytic site on F1 is not a normal catalytic site. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F1 is a normal catalytic site.     

Increased substrate selectivity during transition from Ca2+-activated to K+,EDTA-activated nucleoside triphosphatase activity of heavy meromyosin

A comparison of kinetic parameters (Km(app) and V) of hydrolysis by heavy meromyosin of natural (ATP and ITP) and modified nucleoside triphosphates showed that in the K+, EDTA-ATPase conformation the enzyme exhibited a higher selectivity towards the structure of the substrate nucleoside moiety than in the case of the Ca2+-stimulated nucleoside triphosphatase activity. In the presence of Ca2+, all the N1- and N6-substituted analogs of ATP as well as ITP, etheno-ATP and the dialdehyde derivative of ATP were hydrolyzed at a high rate irrespective of their markedly decreased affinity for heavy meromyosin. In the presence of K+, EDTA the ATPase activity showed a tendency for a total decrease of the analog affinity for nucleoside triphosphates, i.e., the impossibility of tight binding of the substrate phosphate residues to the protein in the absence of bivalent cations, which was concomitant with an increase in the hydrolysis rate. However, it was found that only in N1-substituted analogs any appreciable changes in the substrate properties were absent. All the other nucleoside triphosphates tested (N6-carboxy-methoxy-ATP, N6-(N'-acetylaminoethoxy)-ATP, etheno-ATP, ITP and the dialdehyde derivative of ATP having a rupture in the ribose ring) lost their ability to be hydrolyzed by heavy meromyosin. The experimental results as well as the literature data are suggestive of differences in the spatial structure of the active center in two different myosin conformations associated with a high catalytic activity, i.e., K+, EDTA-ATPase and Ca2+-ATPase.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

Epsilon subunit of Escherichia coli F1-ATPase: effects on affinity for aurovertin and inhibition of product release in unisite ATP hydrolysis

The epsilon subunit of Escherichia coli F1-ATPase is a tightly bound but dissociable partial inhibitor of ATPase activity. The effects of epsilon on the enzyme were investigated by comparing the ATPase activity and aurovertin binding properties of the epsilon-depleted F1-ATPase and the epsilon-replete complex. Kinetic data of multisite ATP hydrolysis were analyzed to give the best fit for one, two, or three kinetic components. Each form of F1-ATPase contained a high-affinity component, with a Km near 20 microM and a velocity of approximately 1 unit/mg. Each also exhibited a component with a Km in the range of 0.2 mM. The velocity of this component was 25 units/mg for epsilon-depleted ATPase but only 4 units/mg for epsilon-replete enzyme. The epsilon-depleted enzyme also contained a very low affinity component not present in the epsilon-replete enzyme. In unisite hydrolysis studies, epsilon had no effect on the equilibrium between substrate ATP and product ADP.P1 at the active site but reduced the rate of product release 15-fold. These results suggest that epsilon subunit slows a conformational change that is required to reduce the affinity at the active site, allowing dissociation of product. It is suggested that inhibition of multisite hydrolysis by epsilon is also due to a reduced rate of product release. epsilon-depleted F1-ATPase showed little of no modulation of aurovertin fluorescence by added ADP and ATP. Aurovertin fluorescence titrations in buffer containing ethylenediaminetetraacetic acid (EDTA) revealed that epsilon-depleted enzyme had high affinity for aurovertin (Kd less than 0.1 microM) regardless of the presence of nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

Kinetics of ATP synthesis in pea cotyledon submitochondrial particles

A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K_m values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K_m for ADP was involved in ATP synthesis. α,β-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K_m of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K_m of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K_m of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Cooperative effects of Ca2+ and Sr2+ on sarcoplasmic reticulum adenosine triphosphatase

The intrinsic fluorescence of purified Ca-ATPase from skeletal sarcoplasmic reticulum was measured in the presence of various concentrations of Ca2+, Sr2+, and Ba2+. Ca2+ and Sr2+ induce positive cooperative fluorescence enhancement, whereas Ba2+ does not change the fluorescence of ATPase. ATP does not seem to modify the kinetic parameters of Ca2+ and Sr2+ binding to ATPase. Nevertheless, p-nitrophenylphosphate hydrolysis, activated by Ca2+ or Sr2+ at various pHs, changes the affinity and the cooperative behavior for both cations and two components appear in the Hill plots. For Ca2+, nH of 1.6 to 3.5 were obtained, and 1.06 to 1.83 for Sr2+; nH changes of the second component seem to be pH dependent. Differences in the ratio between rates of Ca2+ transport and substrate hydrolysis by sarcoplasmic reticulum were found, i.e., two for ATP and one for p-nitrophenylphosphate. For Sr2+ this ratio was one for either ATP or p-nitrophenylphosphate.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Effect of polyamines on actomyosine-ATP-ase activity in smooth muscles of the uterus

The ability of the aliphatic polyamines to inhibit [figure: see text] the ATPase activity of smooth muscle actomyosine satisfies the succession: spermine > spermidine > putrescine that is correlated with magnitude of positive charge at physiological value of pH. The most effective inhibitor of the ATP hydrolysis process is the spermine, which highest inhibitory action is manifested at 10(-3) M concentration, in lesser concentration (10(-5) M) activates the actomyosine ATPase. While defining the kinetic parameters of the ATP hydrolysis reaction catalyzed by uterus myometrium the correlation between inhibiting the ATPase activity of myometrium contractile complex under introduction into the incubation medium of 10(-3) M spermine and decreasing the affinity of actomyosine for ATP was made; the activating effect of spermine on ATPase activity of actomyosine complex in the presence of 10(-5) M spermine correlated with the increase of actomyosine affinity for Mg2+.     

Nucleotide specificity of canine cardiac sarcoplasmic reticulum. Differential alteration of enzyme properties by detergent treatment

We previously demonstrated that, in contrast to the hydrolysis of ATP, the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium. Based on a variety of qualitative and quantitative considerations (cf. Tate, C. A., Bick, R. J., Chu, A., Van Winkle, W. B., and Entman, M. L. (1985) J. Biol. Chem. 260, 9618-9623), we suggested that the hydrolysis of ATP and GTP appears to be effected by the same enzyme. In the present paper, we examined the sensitivity of both enzymatic activities to low concentrations of detergent. With nonsolubilizing concentrations of the nonionic detergent, octaethylene glycol monododecyl ether, the hydrolysis of GTP was rendered partially calcium-sensitive resulting from a slightly increased total (Ca2+ + Mg2+)-GTPase activity and a markedly inhibited calcium-independent (Mg2+-dependent) GTPase activity. Calcium-dependent ATPase activity was increased with octaethylene glycol monododecyl ether, mimicking the effect of the ionophore, A23187. Calcium-dependent ATPase activity and detergent-induced calcium-dependent GTPase activity were similar in (a) calcium sensitivity, (b) sensitivity to mersalyl, and (c) pressure inactivation through dilution and centrifugation, all of which differed from the untreated calcium-independent GTPase activity. Calcium-dependent ATPase activity differed from calcium-dependent GTPase activity with (a) a higher nucleotide affinity, (b) a lower vanadate sensitivity, and (c) a calcium sensitivity for phosphoenzyme formation. Thus, the detergent-induced perturbation of the GTPase resulted in an enzyme with many characteristics qualitatively and quantitatively similar to the calcium ATPase.     

Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.