ATP-dependent activation of the atrial acetylcholine-induced K+ channel does not require nucleoside diphosphate kinase activity.

Prior reports by others have shown that cytoplasmically applied ATP can activate the acetylcholine-induced K+ channel in inside-out atrial membrane patches when no guanine nucleotides are present in the solution bathing the cytosolic face of the membrane. A nucleoside diphosphate kinase mechanism was proposed to explain the activation by ATP. We show in the present study that cytoplasmic adenylylimidodiphosphate mimics the activation by ATP. Unlike ATP, the activation by adenylylimidodiphosphate does not subside on washout. Although commercially available adenylylimidodiphosphate is contaminated by guanylylimidodiphosphate, the activation by adenylylimidodiphosphate still occurs after HPLC purification to remove guanine nucleotide contamination. Adenylylimidodiphosphate does not support phosphotransferase activity by nucleoside diphosphate kinase. Therefore, nucleoside diphosphate kinase activity cannot explain the activation of atrial acetylcholine-induced K+ current by ATP and adenylylimidodiphosphate. We hypothesize that the activation by millimolar concentrations of ATP is due to binding of adenine nucleotide to the guanine nucleotide binding site of the G protein(s) responsible for stimulating the acetylcholine-induced K+ current.

The mitochondrial protein import machinery. Role of ATP in dissociation of the Hsp70.Mim44 complex.

Interaction of preproteins with the heat shock protein Hsp70 in the mitochondrial matrix is required for driving protein transport across the mitochondrial inner membrane. Binding of mt-Hsp70 to the protein Mim44 of the inner membrane import site seems to be an essential part of an ATP-dependent reaction cycle. However, the available results on the role played by ATP are controversial. Here we demonstrate that the mt-Hsp70.Mim44 complex contains ADP and that a nonhydrolyzable analog of ATP dissociates the mt-Hsp70.Mim44 complex in the presence of potassium ions. The previously reported requirement of ATP hydrolysis for complex dissociation was due to the use of a nonphysiological concentration of sodium ions. In the presence of potassium ions, mt-Hsp70 undergoes a conformational change that is not observed with a mutant Hsp70 defective in binding to Mim44. The mutant Hsp70 is able to bind substrate proteins, differentiating binding to Mim44 from binding to substrate proteins. We conclude that binding of ATP, not hydrolysis, is required to dissociate the mt-Hsp70.Mim44 complex and that the reaction cycle includes an ATP-induced conformational change of mt-Hsp70.

Nucleotide interconversions in microtubule protein preparations, a significant complication for accurate measurement of GTP hydrolysis in the presence of adenosine 5'-(beta, gamma-imidotriphosphate).

Pursuing the observation of Carlier and Pantaloni [Carlier, M.-F., & Pantaloni, D. (1982) Biochemistry 21, 1215-1224] that adenosine 5'-(beta, gamma-imidotriphosphate) (pNHppA) strongly inhibited tubulin-independent phosphatases in microtubule protein preparations, we observed with a number of commercial preparations of pNHppA that a major proportion of the terminal phosphate of [gamma-32P]GTP added to microtubule protein preparations was rapidly converted into ATP. Initially postulating degradation of pNHppA to AMP followed by stepwise conversion of AMP to ATP, we isolated two nucleoside monophosphate kinase activities from microtubule protein capable of generating ATP from AMP + GTP. The amounts of these enzymes in microtubule protein preparations, however, are probably too low to account for rapid ATP formation. Instead, ATP formation most likely is caused by nucleoside diphosphate kinase acting on ADP contaminating commercial pNHppA preparations. Such ADP contamination was demonstrated by high-performance liquid chromatography, with the amount of ATP formed with different pNHppA preparations proportional to the amount of ADP contamination. Repurification of commercial pNHppA until it was free of contaminating ADP also resulted in the elimination of ATP formation. The repurified pNHppA potently inhibited GTP hydrolysis in microtubule protein preparations. In addition, especially when supplemented with equimolar Mg2+, the repurified pNHppA strongly inhibited GTP hydrolysis and microtubule assembly in reaction mixtures containing purified tubulin and heat-treated microtubule-associated proteins (which contain negligible amounts of tubulin-independent phosphatase activity). We conclude that studies of microtubule-dependent GTP hydrolysis which make use of pNHppA must be interpreted with extreme caution.

Interactions between citrate and nucleoside triphosphates in binding to phosphofructokinase.

Interrelationships between the binding by rabbit muscle phosphofructokinase of citrate, ATP, GTP, and adenyl-5'-yl imidodiphosphate (AMP-PNP) were investigated. To allow measurements at 25 degrees C, pyruvate kinase and phosphoenolpyruvate were included in the dialysis media to rephosphorylate ADP formed by the weak ATPase action of phosphofructokinase. Binding of citrate was enhanced by GTP nearly as much as by ATP, although GTP does not inhibit the catalytic activity of the enzyme. The results are consistent with the interpretation that binding of GTP, and, by analogy, ATP, at the catalytic site enhances the binding of citrate. AMP-PNP also enhanced citrate binding. Both ATP and GTP appear to bind at three sites per enzyme subunit, with the apparent third site binding relatively weakly. The estimated dissociation constants for the first two sites, about 33 microM for both for ATP compared with 3 and 280 microM for GTP, are consistent with kinetic results that imply lack of effective competition by GTP for the inhibitory site. When a compound binds at two or more sites on a macromolecule, the position and shape of the binding curve are sensitive to the geometric mean of the binding constants but quite insensitive to the magnitudes of the individual constants; thus, binding affinities cannot be estimated with confidence in such cases.

Adenylylimidodiphosphate: effect of contaminants on adenylate cyclase activity.

Commercially available adenylylimidodiphosphate, APP(NH)P, appears to be contaminated with a variety of compounds. The effects of unpurified APP(NH)P on adenylate cyclase activity closely resembled the effects of guanylylimidodiphosphate (GPP(NH)P); that is, APP(NH)P alone caused a time-dependent, quasi-irreversible activation, and the stimulation of adenylate cyclase by APP(NH)P in combination with epinephrine was synergistic, eliminating the need for GTP. The GPP(NH)P-like activity of APP(NH)P could be separated from APP(NH)P by purification of the analogue on DEAE cellulose columns or by paper chromatography. The purified APP(NH)P does not appear to interest at the GTP site.