Co-operative binding sites for transported substrates in the multiple drug resistance transporter Mdr1.

Suramin, a known inhibitor of ATP binding enzymes with six negatively charged sulfonic acid groups, stimulated the ATPase activity of the multiple drug resistance transporter Mdr1 in low concentrations by acting as a substrate and by increasing the affinity for both verapamil and ATP. At higher concentrations suramin inhibited the ATPase activity competitively with respect to ATP and noncompetitively with respect to verapamil. This indicates an interaction of suramin with the ATP site. Verapamil itself activated the ATPase activity of Mdr1 only at moderate concentrations, but showed substrate inhibition at higher concentrations. This was also observed for progesterone, which decreased the Ki of Mdr1 for verapamil but increased the Km. Additionally, verapamil increased the Hill coefficient of Mdr1 for progesterone from 1.1 to 3.2. These results indicate the existence of multiple binding sites (at least four for progesterone) for transported substrate in Mdr1 and a complicated mode of interactions between them.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.     

Binding of nucleotides to the ATP-dependent protease La from Escherichia coli

A critical enzyme in protein breakdown in Escherichia coli is the ATP-hydrolyzing protease La, the lon gene product. In order to clarify the role of ATP in proteolysis, we studied ATP and ADP binding to this enzyme using rapid gel filtration to separate free from bound ligands. In the presence of Mg2+ or Mn2+ and 10 microM ATP, two molecules of ATP were bound to the tetrameric enzyme, while at 100 microM ATP (or higher), four ATP molecules were bound, both at 0 and 37 degrees C. Protease La thus has two high affinity sites (S0.5 less than 10(-7) M) for ATP and two lower affinity sites (S0.5 = 12-15 microM). Binding was reversible. In the absence of a divalent ion, ATP bound to only two sites. However, much lower Mg2+ concentrations (50 microM) were required for maximal ATPase binding than for maximal proteolytic and ATPase activity (2 mM). Decavanadate, which is a potent inhibitor of proteolysis, also blocked ATP binding, but orthovanadate had neither effect. Different ATP analogs bind to these sites in distinct ways. Adenyl-5'-yl imidodiphosphate binds to only one high affinity site, while adenyl-5'-yl methylene monophosphonate binds to two. Nevertheless, both non-metabolizable analogs can activate oligopeptide hydrolysis as well as ATP. Although binding of a single nucleotide can activate peptide hydrolysis, occupancy of all four sites appears necessary for maximal protein breakdown. The ATP molecules on all four sites are hydrolyzed rapidly. The Pi is released, but ADP remains on the enzyme. ADP binds to the same four sites, but this process does not require divalent ions. Protease La shows higher affinity for ADP than for ATP. Therefore, in vivo, ADP should inhibit ATP binding and protease La function.     

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)     

Rapid filtration analysis of nucleotide binding to Na,K-ATPase

Transient kinetic analysis of nucleotide binding to pig kidney Na,K-ATPase using a rapid filtration technique shows that the interaction between nucleotide and enzyme apparently follows simple first-order kinetics both for ATP in the absence of Mg(2+) and for ADP in the presence or absence of Mg(2+). Rapid filtration experiments with Na,K-ATPase membrane sheets may nevertheless suffer from a problem of accessibility for a fraction of the ATPase binding sites. Accordingly, we estimate from these data that for ATP binding in the absence of Mg(2+) and the presence of 35 mM Na(+) at pH 7.0 at 20 degrees C, the bimolecular binding rate constant k(on) is about 30 microM(-1) x s(-1) and the dissociation rate constant k(off) is about 8 s(-1). In the presence of 10 mM Mg(2+), the binding rate constant is the same as that in the absence of Mg(2+). For ADP or MgADP the binding rate constant is about 20 microM(-1) x s(-1) and the dissociation rate constant is about 12 s(-1). Results of rapid-mixing stopped-flow experiments with the fluorescent dye eosin are also consistent with a one-step mechanism of binding of eosin to the ATPase nucleotide site. The implication of these results is that nucleotide binding to Na,K-ATPase both in the absence and presence of Mg(2+) appears to be a single-step event, at least on the time scale accessible in these experiments.     

Plasma membrane adenosine triphosphatase of oat roots: activation and inhibition by mg and ATP

ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L. cv. Goodfield) roots was examined in the presence of various concentrations of MgCl(2) and ATP. A Mg(2+): ATP ratio of about 1 was required for maximal activity regardless of the concentrations used; the optimum concentration for both Mg(2+) and ATP was 9 mm. Based on the ATPase activity at different concentrations of complexed Mg.ATP and free ATP, it is concluded that Mg.ATP is the true substrate of this enzyme.Under certain experimental conditions, high concentrations of MgCl(2) and ATP inhibited the plasma membrane ATPase. On the basis of the relative amounts of free and complexed ATP and Mg(2+), it was found that the different moieties caused different amounts of inhibition. Free ATP inhibited the ATPase at concentrations in excess of 2 mm. Mg.ATP concentrations above 11 mm inhibited the enzyme. Free Mg(2+) caused only a slight inhibition of the ATPase.The Km for Mg.ATP was found to vary from 0.64 to 1.24 mm depending on the experimental conditions. This variation is thought to be due to variable amounts of Mg.ATP, which serves as an inhibitor as well as the substrate, and free ATP, which also inhibits the enzyme.     

The effect of Mg2+ on mitochondrial F0.F1 ATPase and characteristics of the nucleotide binding sites

The interaction of Mg2+ with nucleotide-washed F0.F1 ATPase from pig heart was studied. Mg2+ had no effect on nucleotide-washed F0.F1 ATPase, but it competitively inhibited the hydrolytic activity of washed F0.F1 ATPase preincubated with ADP and slightly activated the hydrolytic activity of washed F0.F1 ATPase preincubated with ATP. In the last two cases, it revealed negative cooperativity. The effect of Mg2+ on F0.F1 ATPase is therefore closely related to the characteristics of the nucleotide binding sites on mitochondrial F0.F1 ATPase.     

P-glycoprotein catalytic mechanism: studies of the ADP-vanadate inhibited state

Kinetics of inhibition of ATPase activity of pure mouse Mdr3 P-glycoprotein upon incubation with MgADP and vanadate were studied along with the trapping of [14C]ADP in presence of vanadate. The presence of verapamil strongly magnified both effects. Inhibition of ATPase was also increased by several other drugs known to bind to drug-binding sites. Inhibition by ADP-vanadate was slow and depended cooperatively on nucleotide binding. Stoichiometry of [14C]ADP trapping by vanadate was 1 mol/mol P-glycoprotein at full inhibition. Catalytic site mutants prevented [14C]ADP trapping, whereas interdomain signal communication mutants reduced it in approximate correlation with their effects upon drug stimulation of ATPase. In explanation of the results, we propose that a "closed conformation" involving dimerization and interdigitation of the two nucleotide-binding domains is necessary to allow inhibition by ADP-vanadate. The results suggest that such a conformation occurs naturally during ATP hydrolysis. It is proposed that in order for the catalytic transition state to form, the two nucleotide-binding domains dimerize to form an integrated single entity containing two bound ATP with just one of the two ATP being hydrolyzed per dimerization event.     

The mechanism of stimulation of MgATPase activity of chloroplast F1-ATPase by non-catalytic adenine-nucleotide binding. Acceleration of the ATP-dependent release of inhibitory ADP from a catalytic site.

The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

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.     

The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin

The stoichiometric actin--DNase-I complex was used to study the actin--nucleotide and actin--divalent-cation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin--DNase-I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size-exclusion chromatography. This effect is due to a loss of actin-bound nucleotide but is prevented by the presence of 0.1-0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ ('physiological conditions') the association rate constants for ATP, ADP and epsilon ATP (1,N6-ethenoadenosine 5'-triphosphate) and the dissociation rate constant for epsilon ATP were determined. Both the on and off rates were found to be reduced by a factor of about 10 when compared to uncomplexed actin. Thus the binding constant of epsilon ATP to actin is almost unaltered after complexing to DNase I (2.16 x 10(8) M-1). Titrating the increase in DNase I activity of the actin--DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation-free form of actin--DNase I complex was found to be 5 x 10(3) M-1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high-affinity metal-binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide-free and metal-ion-free actin--DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.     

Nucleotide binding sites and chemical modification of the chromaffin granule proton ATPase

The purified proton ATPase of chromaffin granules contains five different polypeptides denoted as subunits I to V in the order of decreasing molecular weights of 115,000, 72,000, 57,000, 39,000, and 17,000, respectively. The purified enzyme was reconstituted as a highly active proton pump, and the binding of N-ethylmaleimide and nucleotides to individual subunits was studied. N-Ethylmaleimide binds to subunits I, II, and IV, but inhibition of both ATPase and proton pumping activity correlated with binding to subunit II. In the presence of ADP, the saturation curve of ATP changed from hyperbolic to a sigmoid shape, suggesting that the proton ATPase is an allosteric enzyme. Upon illumination of the purified enzyme in the presence of micromolar concentrations of 8-azido-ATP, alpha-[35S]ATP, or alpha-[32P]ATP subunits I, II, and IV were labeled. However, at concentrations of alpha-[32P]ATP below 0.1 microM, subunit II was exclusively labeled in both the purified and reconstituted enzyme. This labeling was absolutely dependent on the presence of divalent cations, like Mg2+ and Mn2+, while Ca2+, Co2+, and Zn2+ had little or no effect. About 0.2 mM Mg2+ was required to saturate the reaction even in the presence of 50 nM alpha-[32P]ATP, suggesting a specific and separate Mg2+ binding site on the enzyme. Nitrate, sulfate, and thiocyanate at 100 mM or N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at 100 microM prevented the binding of the nucleotide to subunit II. The labeling of this subunit was effectively prevented by micromolar concentrations of three phosphonucleotides including those that cannot serve as substrate for the enzyme. It is concluded that a tightly bound ADP on subunit II is necessary for the activity of the enzyme.     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

Characterization of the high and low affinity components of the renal Ca2(+)-Mg2+ ATPase

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2(+)- and Mg2(+)-dependent ATP hydrolysis. With 0.2 microM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 microM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 microM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis-Menten kinetics with Km for Ca2+ of 0.08 microM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 microM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)