Measurement Of ATPase Activity Research Articles

Arginine 383 is a crucial residue in ABCG2 biogenesis

ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.

Measurement Of ATPase Activity During Ramped Stretches In Contracting Skeletal Muscle Fibers Of The Rabbit

Skeletal muscle force response to small amplitude and low velocity ramp stretches is biphasic. An initial fast increase in force, where myosin heads are forcibly detached is followed by a slower increase in force with net re-attachment of myosin heads to actin. The average ATPase rate is very low during stretch (Curtin & Davies,1973), but due to lack of time resolution the two phases to match force changes have never been resolved. We therefore examined and modeled tension and ATPase responses to ramp stretches (5% and 1% of fiber length, Lo) at low velocities (0.1 and 0.5 Lo/s) in permeabilised fiber bundles of rabbit psoas at 12 and 20°C. We show that ATPase activity drops to near zero during the initial fast phase of the stretch and increases slightly but still remains lower than isometric during the second part of the stretch phase, returning to normal post-stretch. The response was not as marked at 12 as at 20°C, although ATPase rate was still reduced in both the fast initial and slow secondary phase of the force response. During the initial phase the myosin heads are forcibly removed from actin, the cross-bridge cycle is not complete and release of hydrolysis products is interrupted. In the second phase, myosin heads re-attach whilst the muscle is still lengthened and the cross-bridge cycle is truncated for a fraction of attached heads, leading to slower Pi release than during isometric conditions. These effects are less marked at 12 than at 20°C because the fraction of strongly bound cross-bridges is reduced at the lower temperature.Curtin, N.A. & Davies, R.E. (1973). Cold Spring Harbor Symp. on Q. Biol., 37, 619-626.

Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.

Functional Impact of TnT Mutations Responsible for Hypertrophic Cardiomyopathy on Tn-Exchanged Single Myofibrils

Hypertrophic Cardiomyopathy (HCM) is an autosomal dominant cardiac disease resulting from mutations in genes encoding contractile proteins, including troponin (Tn; the complex of TnT, TnI and TnC). From measurements of ATPase activity of myofibrils, into which TnT mutants were exchanged, we showed that two HCM-associated mutations of TnT, E244D and K247R, increase the maximum ATPase activity without any change in Ca2+ sensitivity (Matsumoto et al., BSJ 2007, 1P150). It is, however, still not clear if the mutations have direct effects on the mechanical properties of muscle because ATPase activity and tension development are not necessarily coupled. We thus examined the effects of the mutations on maximal isometric tension and kinetics of Tn-exchanged myofibrils. Measurements were made in single myofibrils activated and relaxed by a fast solution switching device, with a dead time < 10ms (Tesi et al., Biophys. J., 2002, 83, 2142). Maximal isometric tension values of the mutant TnT exchanged myofibrils were significantly higher (E244D) or tended to be higher (K247R) than those of the wild type TnT exchanged myofibrils while Ca2+ sensitivity was unchanged. These results indicate that the effects of the mutations on myofibril mechanics are directly coupled with the effects on the ATPase activity. Measurements of the rate constants of force generation showed that the mutant TnT exchanged myofibrils had similar kinetics as the wild type TnT exchanged myofibrils. The result suggests that kinetics of cross bridge transitions are not influenced by these mutants.

GP10 of Bacteriophage Phi29 Exhibits ATPase Activity

Previously thought to function only as a passive portal for the phi29 DNA packaging motor, we demonstrate that the connector protein gene product 10 (gp10) exhibits significant ATPase activity. Bioluminescence assays have shown a concentration dependent decrease in available ATP after the addition of gp10. Also, enzyme-linked inorganic phosphate assays (ELIPA) have confirmed a time-dependent increase of phosphate in the presence of gp10 and ATP. Alone, gp10 acts as a slow ATPase with optimal hydrolysis of ATP occurring above room temperature at conditions native to the phi29 DNA motor. Overall increase of ATP hydrolysis in the system has been observed upon the addition of other phi29 motor components to gp10. Compared to gp16, a known ATPase, gp10 consumes ATP at a notably slower rate. Moreover, pRNA has shown no measurable ATPase activity. Combined, the phi29 motor assembly (i.e. gp10, gp16, and pRNA) has exhibited greater rates of ATP hydrolysis than any of these components independently.

Identification and Function of a Cytoplasmic K+ Site of the Na+, K+-ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.

Molecular cloning and functional characterization of cynomolgus monkey multidrug resistance-associated protein 2 (MRP2)

The monkey is an important experimental model in the pharmacological evaluation of new drugs. We isolated monkey multidrug resistance-associated protein 2 (MRP2) cDNA to examine expression profiles among various tissues and measured ATPase activity to assess substrate specificity. The amino acid sequence encoded by monkey MRP2 cDNA was very similar (96% identity) to the reported human MRP2 cDNA (GenBank accession no. NM_000392 ). The tissue distribution of MRP2 in monkeys was partially different from that in humans. We found relatively high expression of MRP2 in the monkey kidney and small intestine using Northern blotting. Substrate specificity was compared between human and monkey MRP2. The affinity of 17β-estradiol 17-(β- d-glucuronide), methotrexate, vinblastine, and probenecid to monkey MRP2 was higher than that to human MRP2. Functional and expression differences between human and monkey MRP2 should be incorporated into the evaluation of candidate drugs.

Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.

Interaction of Fish Myoglobin and Myofibrillar Proteins

Interactions between fish myoglobin (Mb) and myofibrillar proteins were investigated in a Mb-natural actomyosin (NAM) model at 4 degrees C. Increases in metmyoglobin (MetMb) formation and the relative content of bound Mb were observed, as were decreases in whiteness and Ca(2+)-ATPase activity (P < 0.05). During the first 6 h of incubation, Mb bound preferably to myosin at domains other than the head portion, as evidenced by measurable ATPase activity. The potential binding of Mb to myosin heads occurred after 24-h incubation as evidenced by the marked decrease in Ca(2+)-ATPase activity of the NAM-Mb mixture when compared to that of NAM alone (P < 0.05). The interaction between fish Mb and myofibrillar proteins was more pronounced with increased storage time; formation of high-molecular-weight aggregates (> 206 kDa) also increased with time. Electrophoretic study revealed that disulfide bonds were not involved in Mb-NAM interactions.

Effects of K-201 on the calcium pump and calcium release channel of rat skeletal muscle

The benzothiazepine derivative K-201 has been suggested as a potential therapeutic agent due to its antiarrhythmogenic action. To understand how the drug alters calcium release from the sarcoplasmic reticulum (SR), we investigated its effects on the SR calcium channel and calcium pump by single channel electrophysiology, whole-cell confocal microscopy, and ATPase activity measurements on control and post-myocardial infarcted (PMI) rat skeletal muscle. In bilayers, K-201 induced two subconductance states corresponding to approximately 24% (S(1)) and approximately 13% (S(2)) of the maximum conductance. Dependence of event frequency and of time spent in S(1) and S(2) on the drug concentration was biphasic both in control and in PMI rats, with a maximum at 50 microM. At this concentration, the channel spends 26 +/- 4% and 24 +/- 4%, respectively, of the total time in these subconductance states at positive potentials, while no subconductances are observed at negative potentials. K-201 altered the frequency of elementary calcium release events: spark frequency decreased from 0.039 +/- 0.001 to 0.023 +/- 0.001 s(-1) sarcomere(-1), while the frequency of embers increased from 0.011 +/- 0.001 to 0.023 +/- 0.001 s(-1) sarcomere(-1). Embers with different amplitude levels were observed after the addition of the drug. K-201 inhibited the Ca(2+) ATPase characterized by IC(50,contr) = 119 +/- 21 muM and n (Hill,contr) = 1.84 +/- 0.48 for control and IC(50,PMI) = 122 +/- 18 microM and n (Hill,PMI) = 1.97 +/- 0.24 for PMI animals. These results suggest that although K-201 would increase the appearance of subconductance states, the overall calcium release is reduced by the drug. In addition, the effect of K-201 is identical on calcium release channels from control and PMI rats.

Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.

Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites

Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.

Functional Characterization of the Atypical Hsp70 Subunit of Yeast Ribosome-associated Complex

Eukaryotic ribosomes carry a stable chaperone complex termed ribosome-associated complex consisting of the J-domain protein Zuo1 and the Hsp70 Ssz1. Zuo1 and Ssz1 together with the Hsp70 homolog Ssb1/2 form a functional triad involved in translation and early polypeptide folding processes. Strains lacking one of these components display slow growth, cold sensitivity, and defects in translational fidelity. Ssz1 diverges from canonical Hsp70s insofar that neither the ability to hydrolyze ATP nor binding to peptide substrates is essential in vivo. The exact role within the chaperone triad and whether or not Ssz1 can hydrolyze ATP has remained unclear. We now find that Ssz1 is not an ATPase in vitro, and even its ability to bind ATP is dispensable in vivo. Furthermore, Ssz1 function was independent of ribosome-associated complex formation, indicating that Ssz1 is not merely a structural scaffold for Zuo1. Finally, Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. The two domains of this protein thus cooperate in a way that allows for severe interference in either but not in both of them.

Jaw-opening muscle contracts more economically than jaw-closing muscle in rat

Mandibular movement is achieved by coordinated actions of the jaw muscles. To understand the assigned functional role ( e.g., motor or postural role) of each jaw muscle, we characterised not only their contractile and energy-consumption properties but also their compositions of myosin heavy chain (MHC) isoforms. The Ca 2+-dependent isometric tension development and ATPase activity were simultaneously measured in chemically skinned fibers harvested from rat jaw-closing (masseter and temporalis) and jaw-opening (digastric) muscles. After the measurements of isometric tension development and ATPase activity, the MHC compositions in each preparation were determined by SDS-gel electrophoresis. The Ca 2+-sensitivity of isometric tension development and ATPase activity was significantly ( P < 0.001) higher in the digastric fibers than in the masseter and the temporalis fibers. The tension cost (ATPase activity/tension) was significantly ( P < 0.0001) lower in the digastric fibers than in the masseter and the temporalis fibers. The MHCs in the digastric fibers consisted of a mixture of slow type I and fast type II isoforms, while mostly fast type II isoforms in the masseter and temporalis fibers. These results suggest that in rat the jaw-opening muscle contracts more efficiently in terms of the energy use ( i.e., more efficient ATP consumption for tension generation) than the jaw-closing muscle.

Entamoeba histolytica: Ouabain-insensitive Na +-ATPase activity

Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na ++K +)ATPase. On the other hand, we observed a Na +-ATPase with the following characteristics: (1) stimulated by Na + or K +, but these effects are not addictive; (2) the apparent affinity is similar for Na + and K + ( K 0.5 = 13.3 ± 3.7 and 15.4 ± 3.1 mM, respectively), as well as the V max (24.9 ± 1.5 or 27.5 ± 1.6 nmol Pi mg −1 min −1, respectively); (3) insensitive up to 2 mM ouabain; and (4) inhibited by furosemide with an IC 50 of 0.12 ± 0.004 mM. Furthermore, this enzyme forms a Na +- or K +-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.

Heterologous Expression and ATPase Activity of Mutant versus Wild Type PfMDR1 Protein

Mutation of the P. falciparum chloroquine resistance transporter (PfCRT) causes resistance to chloroquine (CQ) and other antimalarial drugs. Mutation and/or overexpression of one of the multidrug resistance protein homologues found in this malarial parasite (PfMDR1) may further modify or tailor the degree of multidrug resistance. However, considerable controversy surrounds the precise contribution of PfMDR1, in part because no direct biochemical studies of PfMDR1 have yet been possible. Using codon optimization and other principles, we have designed and constructed a yeast optimized version of the wild type pfmdr1 gene and have successfully overexpressed PfMDR1 protein in P. pastoris yeast. The protein is well expressed in either full length form or as two separate half transporters, is well localized to the yeast plasma membrane and is fully functional as evidenced by ATPase activity measurements. We have also expressed mutants that have previously been hypothesized to influence drug resistance in parasites. Using purified plasma membrane fractions, we have analyzed antimalarial drug effects on ATPase activity for wild type versus mutant proteins. Relative to other ABCB transporters involved in drug resistance, PfMDR1 is unusual. It has similar pH, [ATP], and Mg++ dependencies for ATP hydrolysis, yet relatively high Km and Vmax values for ATP hydrolysis, and ATPase activity is only mildly stimulated by antimalarial drugs. The largest measured drug effect is for CQ (to which PfMDR1 is not believed to confer resistance), and it is strongly inhibitory for WT PfMDR1. Drug resistance associated PfMDR1 mutants show either elevated (Dd2 allele encoded) or reduced (7G8 allele) basal ATPase activity and different patterns of drug stimulation or inhibition, relative to WT PfMDR1. The Dd2 PfMDR1 isoform also shows a slightly more alkaline pH optimum. Surprisingly, verapamil alone (1-300 microM) does not significantly affect WT ATPase activity but inhibits the Dd2 isoform at 1 microM. These data should assist ongoing analysis of the contribution of PfMDR1 to antimalarial drug resistance.

Three Mechanistically Distinct Kinase Assays Compared: Measurement of Intrinsic ATPase Activity Identified the Most Comprehensive Set of ITK Inhibitors

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.

The Presence of Sarcolipin Results in Increased Heat Production by Ca2+-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.

Potassium as an Intrinsic Uncoupler of the Plasma Membrane H+-ATPase

The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic phosphorylation domain, from where it is unlikely to be transported. Binding of K(+) to this site can induce dephosphorylation of the phosphorylated E(1)P reaction cycle intermediate by a mechanism involving Glu(184) in the conserved TGES motif of the pump actuator domain. Our data identify K(+) as an intrinsic uncoupler of the proton pump and suggest a mechanism for control of the H(+)/ATP coupling ratio. K(+)-induced dephosphorylation of E(1)P may serve regulatory purposes and play a role in negative regulation of the transmembrane electrochemical gradient under cellular conditions where E(1)P is accumulating.