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Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods. Through observing the effects of our probes on metabolism and performing confirmatory cellular and biochemical assays, we found that this chemical series inhibits ATP synthesis by uncoupling the mitochondrial potential. Affinity chemoproteomics experiments identified sarco(endo)plasmic reticulum Ca2+ -dependent ATPase (SERCA2) as a binding partner of the TDZ series, and subsequent validation studies suggest that the TDZ series can act as ionophores through SERCA2 toward Wnt pathway inhibition.

XPB helicase is the largest subunit of transcription factor IIH (TFIIH), a ten-subunit protein complex essential for transcription initiation and nucleotide excision repair (NER) in Eukarya. Two XPB homologues (XPBI and XPBII) are present in the genome of most crenarchaeota, one of the two major phyla of archaea; however, the biochemical properties have not been fully characterized and their cellular roles have not been clearly defined. Here, we report that XPBI from the hyperthermophilic crenarchaeon Sulfolobus tokodaii (StoXPBI) is able to destabilize double-stranded DNA (dsDNA) helix independent of ATP (designated as dsDNA melting activity). This activity is inhibited by single-stranded DNA (ssDNA) and relies on the unique N-terminal domain of StoXPBI, which is also likely responsible for the intrinsic strong ssDNA binding activity of StoXPBI as revealed by deletion analysis. We demonstrate that the ATPase activity of StoXPBII is remarkably stimulated by StoBax1, a nuclease partner of StoXPBII. The role of the unique dsDNA melting activity of XPBI in NER in archaea was discussed.

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.

1. 1. Ca 2+ uptake, Ca 2+ -dependent ATPase activity and halothane-induced Ca 2+ release from the heavy sarcoplasmic reticulum fraction of muscle from malignant hyperthermia susceptible individuals are similar to those of normal human muscle. 2. 2. Ca 2+ -induced Ca 2+ release from the diseased muscle was increased by 13%.

We provide a detailed procedure to determine the Ca2+-dependent ATPase activity in COS or HEK293 cells overexpressing a Ca2+ pump. The ATPase activity is determined by the Baginsky method, which allows measurement of the steady-state production of inorganic phosphate (Pi). We have adapted this widely applied method into a sensitive, fast, and semi-high-throughput protocol suitable for use in a 96-well plate format.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).

K + appears to decrease the affinity of the (Na + + K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, MG 2+ · ATP, and Mg 2+ · ATP, in turn, appears to decrease the affinity of the enzyme for K + . These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K + sites on the enzyme involved. K + increased the apparent K m for Mg 2+ · ATP, and effect that was antagonized competitively by Na + . The data can be fitted to a model in which Mg 2+ · ATP binding is prevented by occupancy of α-sites on the enzyme by K + (i.e. sites of moderate affinity for K + accessible on the “free” non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these α-sites by Na + has no effect on Mg 2+ · ATP binding to the enzyme. On the other hand, Mg 2+ · ATP decreased the apparent affinity of the enzyme for K + at the α-sites, in terms of (i) the K D for K + measured by K + -accelerated inactivation of the enzyme by F − , and (ii) the concentration of K + for half-maximal activation of the K + -dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K + from discharge sites, it is consistent with observations that K + can inhibit the enzyme at low substrate concentrations, and that Li + , which has poor efficacy when occupying these α-sites, can stimulate enzymatic activity at high K + concentrations by displacing the inhibitory K + .

The comparative kinetics of Na+-K+ dependent ATPase from ‘brains’ of Periplaneta americana were investigated in acclimated (16°C) and non-acclimated (24°C) specimens. K m and pH optima were determined over a range of 5°C to 37°C in both acclimated and non-acclimated specimens in order to estimate the acclimation potential of this species. In addition Mg+2 optima was determined both before and after the acclimation process. Low temperature acclimation was not found to alter the pH optima (pH 7.4). Apparent K m values decreased between 5°C and 24°C indicating a greater substrate affinity at lower temperatures in the acclimated specimens (non-acclimated K m = 0.24 mM ATP, acclimated K m = 0.04 mM ATP at 5°C; non-acclimated K m = 0.09 mM ATP, acclimated K m = 0.07 mM ATP at 24°C) and hence an increased mobilization of energy reserves during thermal stress. Mg+2 concentration optima was 1 mM in the non-acclimated specimens and remained unchanged after acclimation.

A study of the initial velocity kinetics of the Escherichia coli transcription termination protein rho, with respect to its poly(C)-dependent ATPase, indicates that this reaction occurs by an ordered sequential mechanism. Product inhibition and substrate analogue studies suggest that ATP binding must precede the binding of poly(C) and that the order of release of the products is ADP followed by Pi, then poly(C). A possible mechanism for relating the ATPase to the termination reaction of rho is discussed in relation to the model for rho proposed by Richardson (5).

F 0 F 1 , isolated from anaerobically grown Escherichia coli by sodium deoxycholate and ammonium sulphate [1], includes two additional proteins of molar mass 50–53 and 12–15 kDa. The ATPase activity of such preparations is strongly dependent on the K + content in the medium. The electrical conductivity of a BLM after incorporation of such preparations increases when ATP is added only in the presence of K + . In the F 0 F 1 preparations from aerobically grown cells, this phenomenon has not been observed. The results confirm our main hypothesis that two genetically independent membrane transport systems, F 0 F 1 and Trk, in anaerobically grown bacteria are structurally associated with the same mechanism and form a united supercomplex functioning as a H + —K pump. This association serves for structural and energetic economy in cells.

The Mg 2+ -dependent and Na + K + -dependent (Na + K + -activated or transport) ATPase (EC 3.6.1.3.) were prepared from human fundic gastric mucosa and the effects of prostaglandins E 1 and E 2 on their activity studied. Significant inhibitions of Mg 2+ -dependent, total (Mg 2+ -dependent and Na + K + -dependent) and Na + K + -dependent ATPase activities were caused by prostaglandins E 1 and E 2 , in concentrations of 10 −9 to 10 −6 M. A possible role of Mg 2+ Na + K + -dependent ATPase system is discussed in the inhibition of gastric secretion by type E prostaglandins.

A DNA-dependent ATPase has been isolated and purified from an Escherichia coli cell-free extract. The ATPase has the following characteristics: preferential dependence on single-stranded DNA, specificity for ATP hydrolysis, Km value of 1.4 X 10-4 M for ATP, and molecular weight of approximately 69,000. The ATPase can be shown to bind to single stranded DNA. The resemblance between this ATPase and that isolated from vaccinia cores is discussed.

The distribution of Na+-K+-activated, Mg2+ dependent ATPase (E.C. 3.6.1.3) was investigated in the alimentary tract of Locusta migratoria using biochemical and histochemical methods. Relatively high levels of Na+-K+-activated ATPase were present in microsomal preparations from the rectum, whereas preparations from the fore, midguts, ileum, and colon had low activity. The localisation of Na+-K+-activated ATPase in the epithelium of the rectal pads was examined but proved inconclusive.

We measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0 degrees C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg++-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg++-dependent ATPase and Na+, K+ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg++-dependet ATPase was dependent on the concentration of Mg++ but not of Ca++; maximum activity was obtained with 2 mM MgCl2. On the other hand, Mg++-dependent ATPase activity of SPM was dependent on both Mg++ and Ca++, activity being maximum using 2 mM MgCl2 and 10−5 M CaCl2. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.

The Relationship between ouabain-sensitive ATPase (Na-K ATPase) activity in erythrocytes and the thyroid status was studied in 36 patients with Graves' disease and 58 patients receiving L-thyroxine (T4) replacement therapy. Forty normal children served as control. Total ATPase activity in 4 untreated hypothyroid patients was significantly reduced (11.0 +/- 4.6 vs 17.3 +/- 4.1 micrograms-P/h/mg-protein, P less than 0.01), and Na-K ATPase was undetectable, both of which were normalized after 4 weeks of L-T4 therapy. Na-K ATPase in hyperthyroid patients was also decreased (0.9 +/- 0.8 vs. 4.0 +/- 2.7, P less than 0.01), but was gradually normalized after 3 months of euthyroid state. Clinically euthyroid children treated with L-T4 were divided into 2 groups with regard to Na-K ATPase activity, normal and low. Analysis of the possible factors producing this difference revealed that, in primary hypothyroidism, the factor appeared to be the endogenous T4 level, while in patients with dwarfism, the secretory capacity of TSH or TSH-releasing hormone (TRH) was contributory. Thus Na-K ATPase activity in red cells remains within the normal range after L-T4 replacement in the presence of a severe degree of primary hypothyroidism or in association with secondary or tertiary hypothyroidism. Other factors such as the L-T4 dose, duration of the therapy, serum T4 and T3 concentrations, were not significantly different in the two groups. These results indicate that (1) Na-K ATPase in red cells is decreased in hyper- or hypothyroid state, (2) restoration of normal activity requires 1-3 months of euthyroid period, and (3) it is a sensitive index of peripheral thyroid status over the preceding few months.

Summary This paper documents the existence of a very active bicarbonate-dependent ATPase which is located in the outer membrane of rat and frog liver mitochondria. The enzyme, which does not require Na + , is therefore not a Na + -K + type of ATPase and is inhibited by Ca ++ ; in contrast to others the rat liver enzyme is only slightly cold labile and the frog enzyme is not. Since in animals the bulk of bicarbonate is made during metabolism in mitochondria the bicarbonate-stimulated ATPase may have much biological significance.