Steady-state ATPase Research Articles

How Potassium Affects the Activity of the Molecular Chaperone Hsc70: I. POTASSIUM IS REQUIRED FOR OPTIMAL ATPase ACTIVITY

Several functions of the 70-kilodalton heat shock cognate protein (Hsc70), such as peptide binding/release and clathrin uncoating, have been shown to require potassium ions. We have examined the effect of monovalent ions on the ATPase activity of Hsc70. The steady-state ATPase activities of Hsc70 and its amino-terminal 44-kDa ATPase fragment are minimal in the absence of K+ and reach a maximum at approximately 0.1 M [K+]. Activation of the ATPase turnover correlates with the ionic radii of monovalent ions; those that are at least 0.3 A smaller (Na+ and Li+) or larger (Cs+) than K+ show negligible activation, whereas ions with radii differing only approximately 0.1 A from that of K+ (NH4+ and Rb+) activate to approximately half the turnover rate observed with K+. Single turnover experiments with Hsc70 demonstrate that ATP hydrolysis is 5-fold slower with Na+ than with K+. The equilibrium binding of ADP or ATP to Hsc70 is unperturbed when K+ is replaced with Na+. These results are consistent with a role for monovalent ions as specific cofactors in the enzymatic hydrolysis of ATP.

Catalytic cooperativity of beef heart mitochondrial F 1-ATPase revealed by using 2′,3′- O-(2,4,6-trinitrophenyl)-ATP as a substrate; an indication of mutually activating catalytic sites

The interaction of 2′,3′- O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bovine mitochondrial F 1-ATPase (MF 1) was examined under substoichiometric and stoichiometric conditions to investigate the relationship between the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis. The hydrolysis of bound TNP-ATP under substoichiometric condition proceeded slowly, with a first order rate constant of 0.014 s −1. However, hydrolysis was greatly accelerated by addition of excess ATP. The hydrolyzed product, TNP-ADP, did not dissociate from the enzyme even after the addition of excess ATP. These properties were the same for both native and nucleotide depleted enzyme. The difference spectrum induced by binding TNP-ATP to MF 1 had a distinct peak at 410 nm and a deep trough at 395 nm, which were similar to those induced when TNP-ATP bound to the isolated β subunit of the thermophilic F 1-ATPase. The magnitude of difference spectra as a function of TNP-ATP concentration suggested the presence of at least two types of binding sites on the MF 1 molecule. The first site, where substoichiometric TNP-ATP was hydrolyzed, had a very high affinity for TNP-ATP. TNP-AT(D)P bound to this site did not dissociate even in the presence of excess ATP. TNP-AT(D)P bound to the second site dissociated slowly when excess ATP was added. The steady state ATPase activity at 100 μM ATP was linearly suppressed as pre-loaded TNP-ATP increased. The binding of 2 mol of TNP-ATP per mol of MF 1 was required to abolish ATPase activity. A model which assumes mutually-activating two catalytic sites is presented to explain these results.

Regulation of Ca(2+)-dependent ATPase activity in detergent-skinned vascular smooth muscle

We measured force, actin-activated myosin adenosinetriphosphatase (ATPase) activity, and myosin light-chain (MLC) phosphorylation levels in Triton X-100 detergent-skinned media of swine carotid arteries. Pseudo-ATPase activity composed of MLC kinase and phosphatase activities contributed maximally 12% to steady-state tissue ATPase activity. An increase in the Ca2+ concentration ([Ca2+]) induced an increase in force, MLC phosphorylation, and actin-activated myosin ATPase activity; this protocol was defined as the force development phase of contraction. Force maintenance was defined as the state induced by decreasing the [Ca2+] after a maximal contraction. Lowering the [Ca2+] decreased MLC phosphorylation to levels similar to those measured during force development at each [Ca2+]. In contrast, force remained at elevated levels while actin-activated myosin ATPase activity fell to significantly lower levels than those measured during the development phase for each [Ca2+]. We suggest that the significantly lower actin-activated myosin ATPase activity observed during a state of elevated force, compared with the development phase of a contraction, is evidence of slowly cycling latch bridges.

Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.

The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases > or = 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-32P]ATP is included in the medium, incorporation of 32P label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the tight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.

Spatial precision of a catalytic carboxylate of F 1-ATPase β subunit probed by introducing different carboxylate-containing side chains

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S-carboxymethylcysteine (Cax) and S-carbamoylmethylcysteine (Cam) into the positions of Glu 190 and Glu 201 of the β subunit of F 1-ATPase from the thermophilic Bacillus PS3. The steady-state ATPase activities of α 3β 3γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), <1% (E190C), <1% (E190Q), and 73% (E201D), 40% (E201Cax), 25% (E201C), 20% (E201Q), 4% (E201Cam), of that of the wild-type α 3β 3γ complex. For the complexes containing E190C or E190Q, even the ability of single-site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu 190 and Glu 201 are interacting in the F 1-ATPase.

Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.

Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.

Kinetic Parameters of the Translocation of Bacteriophage T4 Gene 41 Protein Helicase on Single-stranded DNA

The bacteriophage T4 gene 41 helicase protein (gp41) carries a single stranded DNA-dependent ATPase activity that is essential to its helicase activity. This ATPase activity can be stimulated by a wide variety of single-stranded DNA cofactors, including homo-oligomers and homopolymers 8 to ∼ 10,000 nucleotide residues in length, and by natural single-stranded DNA, such as bacteriophage M13 DNA. The steady state ATPase activity of gp41 on single-stranded homopolymeric cofactors is dependent on the length of the cofactor, in that the kinetic parameters Vmax and Kact (or KDNAm) have a characteristic length dependence. Vmax values for different DNA lengths show a hyperbolic dependence on DNA length, while Kact values are independent of DNA lengths exceeding ∼ 20 nucleotide residues. Use of the detailed theoretical analysis developed in the preceding paper reveals that: (1) these results support the earlier proposal that gp41 translocates on single-stranded DNA in an ATP dependent manner; (2) translocation is undirectional; (3) translocation is processive to an extent that depends on the base composition of the DNA employed, with the average distance translocated per binding event ranging from 60 to 700 nucleotide residues; and (4) the detailed translocation mechanism of gp41 includes an obligatory slow step before or after the ATP-driven translocation process. Defined lengths of natural and homopolymer single-stranded DNA have also been created as gaps of known length distribution between clusters of gene 32 protein (gp32) bound along long single stranded DNA molecules. ATPase data obtained with cofactors of this type also show unidirectional ATP-driven translocation of gp41 on both natural and homopolymeric single-stranded DNA. Direct binding studies of gp41 to short dT oligomers reveal two further features of the interaction of gp41 to single stranded DNA: (1) nucleoside triphosphate binding is necessary for the formation of stable gp41-ssDNA complexes; and (2) the DNA binding site size of gp41 is between 12 and 20 nucleotide residues per protein monomer. Possible translocation mechanisms for gp41 are discussed within the context of these results.

Escherichia coli transcription termination factor rho. I. ATPase activation by oligonucleotide cofactors

Rho protein is required to bring about RNA release from Escherichia coli transcription complexes paused at specific (rho-dependent) termination sites. Rho functions in termination as a hexamer of identical subunits arranged in D3 symmetry, with each rho subunit carrying an RNA- and an ATP-binding site. The detailed mechanism of rho-catalyzed transcript release remains to be determined, but it is clear that the RNA-dependent ATPase activity that is stimulated by interaction with the nascent transcript is essential to the termination function of rho. In this study, we have used short (8-10 nucleotide residues) synthetic ribo-oligonucleotides to model the interaction of segments of the RNA cofactor with rho. A poly(dC) enhancement procedure was used to permit the measurement of steady state ATPase parameters. We show that (i) ATPase activation is cofactor composition- and sequence-dependent; (ii) at least 60% of the residues of these short RNA cofactors must be cytosine to produce maximal rho ATPase activation; (iii) oligo(rU,rC) cofactors with the rU residues located at the 5' termini of the oligomer are much better ATPase cofactors than oligomers containing rC residues only; (iv) this enhanced stimulation is not observed if the rU residues are replaced by rA residues; (v) this cofactor activity relative to oligo(rC) is reversed if the rU residues are placed at the 3' terminus of RNA oligomer; and (vi) these nucleotide sequence and composition effects do not appear to be functions of K+ or Mg2+ concentration. These ATPase activation results are correlated with the binding to rho of oligonucleotide cofactors in the accompanying paper (Wang, Y., and von Hippel, P. H. (1993) J. Biol. Chem. 268, 13947-13955).

Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis.

We have used time-resolved electron paramagnetic resonance spectroscopy and caged ATP to detect nucleotide-induced changes in the conformational state of spin-labeled myosin heads (IASL-S1). Changes in the internal rotational dynamics of IASL-S1 were monitored with millisecond time resolution during the pre-steady-state phase of ATP hydrolysis. The changes in the internal protein dynamics were rigorously correlated with specific biochemical kinetic transitions, allowing us to observe directly the dynamic sequence of structural changes in IASL-S1 during the binding and hydrolysis of ATP. When caged ATP was photolyzed (producing 500 microM ATP) in the presence of 100 microM IASL-S1, the EPR signal intensity rose transiently to the steady-state ATPase level, indicating increased rotational motion about the SH1 region of the myosin head. Kinetic and spectral analyses have resolved two phases of this transient, one representing the population of the M*.ATP state and the other representing the population of the M**.ADP.Pi state. We conclude that two motionally distinct states of the myosin head are present during ATP hydrolysis and that these states represent distinct conformational states that can be correlated with specific biochemical intermediates. Since specific labeling of myosin heads with IASL has been achieved in skinned muscle fibers, this study establishes the feasibility for the first direct detection and resolution of myosin's conformational transients during muscle contraction.

Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli

The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat. Active-site measurements using [alpha-32P]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotational dynamics of actin-bound myosin heads in active myofibrils

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.

Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

The "homology A" ("glycine-rich" or "P-loop") consensus sequence occurs in the catalytic sites of F1F0 ATP synthase enzymes. The conserved lysine of this motif is beta-subunit Lys-155 in Escherichia coli F1. The role of this lysine in binding and catalysis at the high affinity ATP binding site was studied with the mutants beta K155Q and beta K155E by measuring the rates of ATP binding/release, ATP hydrolysis/synthesis, and Pi release as a function of pH varied from 5.5 to 9.5. In wild type, protonated beta Lys-155 appears to contribute significantly to high affinity binding of ATP, probably through hydrogen bonding to the gamma-phosphate. ATP hydrolysis and synthesis were impaired strongly in the mutants, and the reaction equilibrium constant, which was pH-independent in wild type, was highly pH-dependent in beta K155Q and beta K155E. Studies of steady-state ATPase turnover showed that positive catalytic cooperativity was virtually absent and the pH-dependent component of positive catalytic cooperativity was abolished or reversed in the mutants. The data demonstrate that residue beta K155 is a critical catalytic residue in F1 ATPase.

Cooperative effects on filament stability in actin modified at the C-terminus by substitution or truncation.

We have studied the contribution of the C-terminus of actin to filament stability by chemical modification and limited proteolysis. Formation of mixed disulfides of the penultimate C-terminal cysteine residue 374 with various low-molecular-mass thiols resulted in filament destabilization, as reflected by an increase in critical concentration and steady-state ATPase activity. These effects were fully reversed by the addition of phalloidin. Both the destabilization by glutathionylation and the reversal of it by phalloidin exhibited a high degree of cooperativity; half-maximal destabilization required the modification of four out of five actin subunits, and half-maximal restabilization by phalloidin was already reached when only one out of 20 actin subunits was complexed. C-terminal truncation by limited trypsinolysis of filamentous actin resulted in a similar destabilization of the polymer, as shown by a 2-3-fold increase in the steady-state ATPase activity. This effect was likewise cooperative and could be reversed by phalloidin. Since truncation of the C-terminus of actin has an effect on stability similar to that of chemical modification with bulky substituents, the possibility can be excluded that, in the latter case, destabilization was caused by steric hindrance. Rather, it seems that the highly conserved C-terminal part of actin plays an active role in establishing a tight contact between neighbouring subunits.

Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation.

Serotonin, an activator of adenylate cyclase, stimulates motility in molluscan gill cilia and sperm flagella. To determine and compare potential targets of cAMP action, dynein was prepared from the lateral gill.cilia and sperm flagella of the mussel Mytilus edulis and the clam Spisula solidissima. In the flagella of both species, high-salt extraction removes about half of the ATPase activity, half of the alpha and beta heavy chains, and the outer arms. The dynein from both species sediments at 18-20 S, contains two or three intermediate chains, and three light chains. High-salt plus detergent removes most of the remaining dynein ATPase, alpha and beta heavy chains, and inner arms, also yielding a stable 18-20 S particle. In gill cilia of both species, high-salt extraction removes only 12-18% of the ATPase, up to 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and a subset of the outer arms. The dynein sediments at 18-20 S and, in Spisula, the heavy, intermediate, and light chains precisely co-sediment. High-salt plus detergent removes another 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and the remaining outer arms. The ATPase sediments mainly as a 13-14 S form showing considerable dissociation of co-sedimenting intermediate and light chains. The inner arms and at least half of the ciliary dynein ATPase activity remain unextractable, corresponding in mass mainly to an apparent beta heavy chain that is vanadate-cleavable. Cyclic AMP-dependent, calcium-independent phosphorylation takes place on specific dynein light chains in cilia but on only the dynein alpha heavy chain in flagella. Pre-activation of the flagella prevents subsequent addition of labeled phosphate. Phosphorylation has no effect on the steady-state ATPase properties. The single phosphate added to the flagellar alpha chain is located within the LUV1 vanadate photocleavage fragment. Considering the probable locus of the light chains and the site of the alpha heavy chain phosphorylation, both beyond the active site and toward the base of the molecule, these distinct phosphorylations may regulate dynein action by modulating arm flexibility or interaction.

F 1-ATPase with cysteine instead of serine at residue 373 of the α subunit

Escherichia coli strain AN718 contains the αS373F mutation in F 1F 0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F 1-ATPase activity. The revertant strain AN718SS2 containing the mutation αC373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F 1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in αS373F mutant F 1, were regained in αC373 F 1. N-Ethylmaleimide (NEM) inactivated αC373 f 1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of αC373 f 1 steady-state ATPase corresponded to incorporation of one NEM per f 1 (mol/mol), in just one of the three α subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue α373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F 1. The fact that NEM inhibits steady-state ATPase when only one α subunit of three is reacted suggests a cyclical catalytic mechanism.

Catalytic properties of Escherichia coli F 1-ATPase depleted of endogenous nucleotides

Nucleotide-depleted Escherichia coli F 1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343–350). This enzyme had high rates of steady-state ATPase and GTPase activity. When “unisite” ATP hydrolysis was measured using an F 1 ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 × 10 5 m −1 s −1 and the K d ATP was 7.9 × 10 −10 m, as compared to values of 3.8 × 10 5 m −1 s −1 and 1.9 × 10 −10 m, respectively, in native (i.e., nucleotide-replete) F 1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P i release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F 1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F 1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F 1. Such an entity has been reported for chloroplast and mitochondrial F 1. However, our experiments gave no indication for inhibitory MgADP in E. coli f 1.

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.

A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems.

A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. The Michaelis-Menten constants of the two substrates with the enzyme are 70 microM for the nucleoside and 26 microM for P(i); the kcat is 40 s-1 (25 degrees C). The assay was shown to quantitate P(i) in solution at concentrations at least down to 2 microM. It can be used to measure the kinetics of P(i) release from phosphatases, such as GTPases and ATPases, by coupling the two enzymic reactions. The utility of this assay was shown by three test systems: glycerol kinase plus D-glyceraldehyde acting as an ATPase and actin-activated myosin ATPase, and myosin subfragment 1, hydrolyzing a single turnover of ATP, releasing P(i) with a rate constant the same as the steady-state ATPase activity.

Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin.

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca(2+)-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between E1 and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by P(i) and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.