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PubMed1985

Effects of ATP-MgCl2 and ATP-Na2 administration on renal function and cellular metabolism following renal ischemia.

The present study compared the effects of ATP-MgCl2 or ATP-Na2 administration on renal function and cellular metabolism following renal ischemia in rabbits. Following 100 min of ischemia, the blood flow to the kidney was allowed to reestablish itself, and the rabbits received either saline, ATP-MgCl2 (17.5 mumole/kg each), or ATP-Na2 (17.5 mumole/kg) intravenously. ATP-MgCl2 administration following ischemia significantly accelerated the recovery of renal function. However, administration of ATP-Na2 failed to show any beneficial effect on the recovery process. The changes in renal cellular ketone body ratio (acetoacetate/beta-hydroxybutyrate), energy charge, and pyruvate/lactate indicated that ATP-MgCl2, but not ATP-Na2, reversed ischemically induced impairment in renal cellular metabolism. Reduction in the renal blood flow caused by ischemia was also improved with ATP-MgCl2 treatment. These results indicate that ATP should be given in the form of ATP-MgCl2 for it to be effective following renal ischemia. The results also suggest that the salutary effect of ATP-MgCl2 following renal ischemia could occur through the improvement of cellular metabolism and concomitant improvement in tissue blood flow.

Acetoacetates↗

PubMed1985

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer.

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.

Actins↗

PubMed1993

Myofilament sliding per ATP molecule in rabbit muscle fibres studied using laser flash photolysis of caged ATP.

1. To estimate the distance of myofilament sliding per ATP molecule hydrolysed during the actin-myosin interaction in muscle, single glycerinated fibres prepared from rabbit psoas muscle were made to shorten under very small external loads (< or = 0.0005 maximum isometric force (Po), at 20-22 degrees C) by the laser flash photolysis of caged ATP (P3-1-(2-nitro) phenylethyladenosine 5'-triphosphate), a biologically inert and photolabile precursor of ATP. The laser flash-induced fibre shortening was recorded with a high-speed video system at 200 frames s-1. 2. Following the photochemical release of 75-300 microM ATP, the fibres shortened uniformly along the fibre length not only at the level of fibre segments but also at the level of sarcomeres. The fibres did not shorten appreciably in response to 50 microM ATP. 3. The initial velocity of the laser flash-induced fibre shortening increased with increasing concentration of released ATP, being 0.05 +/- 0.01, 0.12 +/- 0.04, 0.23 +/- 0.04, 0.38 +/- 0.03 and 0.95 +/- 0.08 microns s-1 (half-sarcomere)-1 (means +/- S.E.M., n = 10) with 75, 100, 150, 200 and 300 microM ATP, respectively. 4. The distance of the laser flash-induced fibre shortening also increased with increasing concentration of released ATP, being 10 +/- 2, 25 +/- 5, 65 +/- 7, 100 +/- 10 and 180 +/- 20 nm (half-sarcomere)-1 (means +/- S.E.M., n = 10) with 75, 100, 150, 200 and 300 microM ATP, respectively. 5. Comparison of the initial shortening velocities of the laser flash-induced shortening with the force-velocity relation of maximally Ca(2+)-activated fibres indicated the presence of considerable internal resistance against myofilament sliding following release of ATP. The initial velocity of shortening following the release of 300, 150 and 75 microM ATP was equal to the shortening velocity of maximally Ca(2+)-activated fibres under an external load of 0.55, 0.93 and 0.98 Po respectively. 6. These results suggest that, under nearly isometric conditions, the distance of myofilament sliding per ATP molecule hydrolysed is about 10 nm in each half-sarcomere.

Actin Cytoskeleton↗

PubMed2006

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport.

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the gamma-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.

Adenosine Triphosphate↗

PubMed2005

ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation.

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport.

ATP-Binding Cassette Transporters↗

PubMed1991

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.

ATP-Binding Cassette Transporters↗

PubMed2006

Elucidation of ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes: ATP potentiate HSP60 and Cu/Zn SOD expression and stimulates pI shift of peroxiredoxin II.

ATP has been shown to mediate stress responses in the brain. The present study examined the ATP-stimulated stress protein expression of RBA-2 type-2 astrocytes. Our results revealed that ATP stimulated HSP60 expression in a dose- and time-dependent manner. The stimulation requires a minimal ATP concentration of 500 microM and high concentration of extracellular ATP (1 mM) stimulated a significant increase of HSP60 expression from 2 to 24 h. In addition, the ATP-stimulated HSP60 expressions were inhibited by inhibitors for protein kinase C (PKC) and phospholipase D (PLD), and by antioxidants, resveratrol, and catalase. Furthermore, ATP stimulated the expression of Cu/Zn superoxide dismutase (SOD). In addition, ATP and P2X7 receptor selective agonist BzATP also decreased mitochondria membrane potential measured by flow cytometry. To further examine the proteins involving in ATP-mediated stress responses, we conducted proteomic analysis. We found that RBA-2 astrocytes possess abundant peroxiredoxin II (Prx II), an antioxidant enzyme. ATP and exogenous H2O2 stimulated Prx II shifting from oxidized form to reduced form. Thus, we concluded that ATP potentiated the expression of HSP60 and Cu/Zn SOD, and decreased mitochondria membrane potential. In addition, RBA-2 astrocytes expressed Prx II that might also serve as a protective mechanism to control the concentration of reactive oxygen species.

Adenosine Triphosphate↗

PubMed1991

Skeletal muscle ATP-sensitive K+ channels recorded from sarcolemmal blebs of split fibers: ATP inhibition is reduced by magnesium and ADP.

A new, nonenzymatically treated preparation of amphibian sarcolemmal blebs has been used to study the regulation of skeletal muscle ATP-sensitive K+ [K(ATP)] channels. When a frog skeletal muscle fiber is split in half in a Ca(2+)-free relaxing solution, large hemispherical membrane blebs appear spontaneously within minutes without need for Ca(2+)-induced contraction or enzymatic treatment. These blebs readily formed gigaseals with patch pipettes, and excised inside-out patches were found to contain a variety of K+ channels. Most prominent were K(ATP) channels similar to those found in the surface membrane of other muscle and nonmuscle cells. These channels were highly selective for K+, had a conductance of approximately 53 pS in 140 mM K+, and were blocked by internal ATP. The presence of these channels in most patches implies that split-fiber blebs are made up, at least in large part, of sarcolemmal membrane. In this preparation, K(ATP) channels could be rapidly and reversibly blocked by glibenclamide (0.1-10 microM) in a dose-dependent manner. These channels were sensitive to ATP in the micromolar range in the absence of Mg. This sensitivity was noticeably reduced in the presence of millimolar Mg, most likely because of the ability of Mg2+ ions to bind ATP. Our data therefore suggest that free ATP is a much more potent inhibitor of these channels than MgATP. Channel sensitivity to ATP was significantly reduced by ADP in a manner consistent with a competition between ADP, a weak inhibitor, and ATP, a strong inhibitor, for the same inhibitory binding sites. These observations suggest that the mechanisms of nucleotide regulation of skeletal muscle and pancreatic K(ATP) channels are more analogous than previously thought.

Adenosine Diphosphate↗

PubMed1985

Effector mechanisms for alpha,beta-methylene ATP and ATP derivatives in guinea-pig taenia caeci.

The effects of the ATP analog alpha, beta-methylene ATP and ATP derivatives on smooth muscle cells of guinea-pig taenia caeci were studied by measuring potential changes. The preparations were superfused (1 ml/min) with Krebs solution or calcium-free solution containing atropine (10(-6) M), prazosin (10(-6) M) and propanolol (10(-6) M) using the sucrose-gap method at 22 degrees C. The ATP analog (10(-5) M to 4 X 10(-4) M) caused slowly developing hyperpolarization in the presence of calcium. Hyperpolarization also occurred on addition of the analog in calcium-free medium. The area of the response increased with the analog concentration (10(-5) M to 4 X 10(-4) M). The response evoked after repeated addition of the ATP analog had slightly lower amplitude and slower onset than the first response both in the presence and the absence of calcium. The area of the response to the ATP analog in the absence of calcium was also decreased when preceded by the ATP response. When the opposite experimental sequence was used the ATP response was strongly inhibited, when preceded by the analog response evoked by the same concentration. The ATP analog caused depolarization of the muscle cells in the presence of apamin (3 X 10(-7) M), which was also observed with ATP. The ATP derivatives (4 X 10(-4) M) caused hyperpolarization of the smooth muscle cell membrane, except hypoxanthine and inosine which were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1990

Free energy of ATP-hydrolysis fails to affect ATP-dependent potassium channels in isolated mouse ventricular cells.

The single channel recording technique has been used to study the adenosine-5'-triphosphate (ATP)-dependent K+ channel in isolated mouse ventricular cells. The aim of this work was to determine if the activity of the K+ channel depends on the free energy of ATP-hydrolytic reaction (phosphorylation potential) in addition to the well-studied direct block by ATP. When the phosphorylation potential was changed from 60 to 50, to 40 and back to 60 kJ/mol at a constant ATP-concentration of 10(-4) mol/l, the ATP-channel activity showed a run down that is best described by a linear function. Changing the ATP-concentration of the bath solution from 0.01 to 1 and back to 0.01 mmol/l by a factor of 10 at a constant phosphorylation potential of 50 kJ/mol, resulted in a run down of the mean average current by a reduction of the open state probability in a concentration dependent manner. A dissociation constant of about 0.14 mmol/l could be estimated. The amplitude of the single channel current was not affected by ATP-concentrations in contrast to changes in phosphorylation potential. A significant increase in the single channel current accompanying a decrease in the phosphorylation potential at constant ATP concentrations was observed. This effect might be due to a decrease of free Mg2(+)-concentration by an increase of the ADP concentration in solutions with lower phosphorylation potentials. An allosteric regulation of the ATP-dependent K+ channel dependent on the free ATP concentration together with the Mg2(+)-ADP concentration seems to be a more likely explanation than regulation by phosphorylation/dephosphorylation secondary to a breakdown of ATP.

Action Potentials↗

PubMed1989

Nephrotoxicity assessment by measuring cellular ATP content. I. Substrate specificities in the maintenance of ATP content in isolated rat nephron segments.

To clarify the characteristics of cellular ATP synthesis in individual nephron segments for assessing nephrotoxicity of chemicals, cellular ATP content was measured by the luciferin/luciferase system under various conditions using intact nephron segments isolated from male Sprague-Dawley rats. Increasing the duration of collagenase treatment of kidney slices significantly lowered the cellular levels of ATP newly synthesized from 2 mM glutamine in PST at 37 degrees C over 30 min (p less than 0.01). The tubular incubation time significantly affected the cellular ATP content in the early and middle portions (S2) of the proximal tubule (p less than 0.05 and p less than 0.01, respectively) over 20 min and in the late proximal tubule over 10 min. Among numerous substrates tested, such as D-glucose, glutamine, pyruvate, DL-lactate, and beta-hydroxybutyrate, the substrate utilization for maintaining cellular ATP content was entirely variable according to each nephron segment. Pyruvate and glutamine were the best substrates in the proximal tubule. On the other hand, ATP production from glutamine was less than that from the other substrates in the distally located nephron segments: medullary and cortical thick ascending limbs of Henle's loop (MAL and CAL, respectively), distal tubule, cortical and medullary collecting tubules (CCT and MCT, respectively). In general, glucose, pyruvate, and lactate appear to be equivalent in maintaining ATP content in the distal segments of renal tubules. A monovalent cation ionophore, monensin, at 10 micrograms/ml decreased the cellular ATP content in MAL, CAL, and MCT significantly. Mercuric chloride (HgCl2) was used as a model compound to study nephrotoxicity by investigating its effects on cellular ATP metabolism in microdissected nephron segments. HgCl2 at 1 x 10(-6) M significantly decreased ATP content only in S2 (p less than 0.05), clearly demonstrating S2 to be the most sensitive segment within the nephron. These results indicate that measurement of cellular ATP content would be a useful method forecasting the intrarenal toxic site and potency of possible nephrotoxic chemical compounds.

Adenosine Triphosphate↗

PubMed2006

Evidence for functional ATP-sensitive (K(ATP)) potassium channels in human and equine articular chondrocytes.

OBJECTIVE: Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity. METHODS: Immunohistochemistry was used to explore K(ATP) channel expression in equine and human chondrocytes. Biophysical properties of equine chondrocyte K(ATP) channels were investigated with patch-clamp electrophysiology. RESULTS: Polyclonal antibodies directed against the K(ATP) Kir6.1 subunit revealed high levels of expression in human and equine chondrocytes mainly in superficial and middle zones of normal cartilage. Kir6.1 was also detected in superficial chondrocytes in osteoarthritic (OA) cartilage. In single-channel electrophysiological studies of equine chondrocytes, we found K(ATP) channels to have a maximum unitary conductance of 47 +/- 9 pS (n=5) and a density of expression comparable to that seen in excitable cells. CONCLUSION: We have shown, for the first time, functional K(ATP) channels in chondrocytes. This suggests that K(ATP) channels are involved in coupling metabolic and electrical activities in chondrocytes through sensing of extracellular glucose and intracellular adenosine triphosphate (ATP) levels. Altered K(ATP) channel expression in OA chondrocytes may result in impaired intracellular ATP sensing and optimal metabolic regulation.

Adenosine Triphosphate↗

PubMed2000

The I182 region of k(ir)6.2 is closely associated with ligand binding in K(ATP) channel inhibition by ATP.

The ATP-inhibited potassium (K(ATP)) channel is assembled from four inward rectifier potassium (K(ir)6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K(ir)6.2. The G334D mutation of K(ir)6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K(ir)6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K(i) values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K(i), many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.

Adenosine Triphosphate↗

PubMed2002

ATP-independent anoxic activation of ATP-sensitive K+ channels in dorsal vagal neurons of juvenile mice in situ.

The role of ATP in anoxic activation of ATP-sensitive K+ (KATP) channels was studied in dorsal vagal neurons of mouse brainstem slices. In the whole-cell configuration, cyanide-induced chemical anoxia evoked within 10 s a 300-pA outward current that gave rise to a hyperpolarization of 24 mV. These responses were mimicked by nitrogen-aerated saline, rotenone or diazoxide and abolished by tolbutamide. The cyanide-induced hyperpolarization was due to activation of 70 pS K(ATP) channels that were half-maximally blocked by 5 microM internal ATP. Dialyzing the cells with either 1, 20 or 0 mM ATP did not, however, affect the time to onset, the kinetics or the magnitude of the cyanide-induced hyperpolarization. Impairment of ATP consumption by ouabain, vanadate or reduced temperature had no effect either. Thus, anoxia-induced activation of these KATP channels cannot be explained by a fall of cellular ATP or a concomitant rise of ADP. Anoxia-related changes of the actin cytoskeleton or the composition of the plasma membrane are also not likely to be involved, as cytochalasin D did not affect the cyanide-evoked hyperpolarization and phosphatidylinositol 4,5-bisphosphate failed to decrease the ATP sensitivity of single KATP channels. Finally, because of a lack of effects of reduced/oxidized glutathione and the oxidase blocker diphenyliodonium on the cyanide-induced hyperpolarization, cellular redox state does not appear to be involved. Our results indicate that despite a high sensitivity to ATP in excised patches, anoxic activation of KATP channels is independent of cellular ATP. Rather the ATP block seems to be removed as a consequence of impaired mitochondrial function.

Actin Cytoskeleton↗

PubMed2003

Involvement of 3Na+/2K+ ATP-ase and Pi-3 kinase in the response of skeletal muscle ATP-sensitive K+ channels to insulin.

The modulation of ATP-sensitive K+ channel (K(ATP)) by insulin plays a role in neuromuscular disorders associated to altered K+ homeostasis. However, the mechanisms by which insulin modulates K(ATP) channels are not known. Here, the insulin-dependent 3Na+/2K+ ATP-ase and Pi-3 kinase pathways were explored by using patch-clamp techniques. High and low affinity inhibition of K(ATP) channels by ouabain was observed in the insulin-stimulated and resting fibers, respectively. The 9A5 antibody directed against the alpha1-subunit of the pump inhibited the K(ATP) channel in the resting fibers but fails to inhibit it in the insulin-stimulated fibers. In contrast, the RT2NKATPabr, an alpha2-subunit specific antibody, inhibited the K(ATP) channels in the insulin-stimulated fibers failing to inhibit it in the resting fibers. The insulin-dependent stimulation of K(ATP) channel was prevented by Pi-3 kinase inhibitors Wortmannin and LY294002. In conclusion, insulin stimulating the 3Na+/2K+ ATP-ase activates K(ATP) channels through a membrane-delimited interaction thus controlling the K+ homeostasis. The Pi-3 kinase is the intracellular insulin signal linking the glucose homeostasis to the K(ATP) channel.

Androstadienes↗

PubMed1994

Inhibition of ATP binding to myofibrils and acto-myosin subfragment 1 by caged ATP.

The inhibitory effect of P3-[1-(2-nitrophenyl)ethyl]adenosine 5'-triphosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was investigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma-32P] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have an inhibition constant of 145 microM, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding with an inhibition constant of 1.6 mM. The inhibitory effect of these ligands on the binding of ATP to acto-myosin subfragment 1 was investigated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 microM, respectively. This inhibitory effect of caged ATP on ATP binding accounts for the lower rate of ATP binding to fibers, deduced from caged ATP [(0.5-1) x 10(6) M-1 s-1], than that reported for acto-S1 (3.5 x 10(6) M-1 s-1) [Goldman, Y. E., Hibberd, M. G., & Trentham, D. R. (1984) J. Physiol. (London) 354, 577].

Adenosine Diphosphate↗

PubMed2002

Gastric H/K-ATPase liberates two moles of Pi from one mole of phosphoenzyme formed from a high-affinity ATP binding site and one mole of enzyme-bound ATP at the low-affinity site during cross-talk between catalytic subunits.

The maximum amount of acid-stable phosphoenzyme (E32P)/mol of alpha chain of pig gastric H/K-ATPase from [gamma-32P]ATP (K(1/2) = 0.5 microM) was found to be approximately 0.5, which was half of that formed from 32P(i) (K(1/2) = 0.22 mM). The maximum 32P binding for the enzyme during turnover in the presence of [gamma-32P]ATP or [alpha-32P]ATP was due to 0.5 mol of E32P + 0.5 mol of an acid-labile enzyme-bound [gamma-32P]ATP (EATP) or 0.5 mol of an acid-labile enzyme-bound [alpha-32P]ATP, respectively. The K(1/2) for EATP formation in both cases was 0.12 approximately 0.14 mM. The turnover number of the enzyme (i.e., the H+-ATPase activity/(EP + EATP)) was very close to the apparent rate constants for EP breakdown and P(i) liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of P(i) liberated to that of EP that disappeared increased from 1 to approximately 2 with increasing concentrations of ATP (i.e., equal amounts of EP and EATP exist, both of which release phosphate in the presence of high concentrations of ATP). This represents the first direct evidence, for the case of a P-type ATPase, in which 2 mol of P(i) liberation occurs simultaneously from 1 mol of EP for half of the enzyme molecules and 1 mol of EATP for the other half during ATP hydrolysis. Each catalytic alpha chain is involved in cross-talk, thus maintaining half-site phosphorylation and half-site ATP binding which are induced by high- and low-affinity ATP binding, respectively, in the presence of Mg2+.

Adenosine Triphosphate↗

PubMed1993

ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.

The molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1-5). Members of the family of heat-shock proteins of M(r) 70K, Hsp70, bind to unfolded proteins and short peptides. Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg-ATP binding but not Mg-ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+ for the effect of Mg-ATP: only the combination of K+ and Mg-ATP will cause the conformational change in Hsp70 that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg-ATP favours complex formation. We consider these results in terms of a G-protein-like model.

Adenosine Triphosphatases↗