P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.     

Effect of divalent cations on the inhibition of alkylsulfatase induction and the generation of ATP in Pseudomonas aeruginosa after exposure to exogenous uridine 5'-triphosphate.

In the absence of added Mg2+, alkylsulfatase induction in resting cells of Pseudomonas aeruginosa was inhibited 17% by exogenous 0.05 mM UTP. Under these conditions, the cells converted UTP to ATP and rapid degradation of these nucleotides did not occur. In the presence of 0.73 mM Mg2+, 0.05 mM UTP repressed the synthesis of the enzyme by 71%. Under these conditions, the cells rapidly degraded both ATP derived from UTP as well as residual UTP. In the presence of Mg2+ and 0.1 mM UTP, full repression of alkylsulfatase formation occurred whereas Mg2+-depleted cell suspensions were still capable of synthesizing 47% of the enzyme under these conditions compared with control levels. The inhibition of alkylsulfatase induction was highly specific for UTP. Some inhibition was observed with exogenous uracil, uridine, and pyrophosophate but only at concentrations greater than 1.0 mM. Exogenous UMP and UDP (2mM) had no effect.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] </= 0.1 mM or 100 microM ouabain) or when IK-ATP was completely inhibited by 10 mM ATP. In whole-cell configuration, ouabain inhibited up to 60% of inwardly rectifying IK-ATP at 1 mM ATP in the pipette but not at 10 mM ATP and 10 mM phosphocreatine when IK-ATP was always blocked. However, mathematical simulation of giant-patch experiments revealed that only 20% of ATP depletion may be attributed to the ATP concentration gradient in the bulk solution, and the remaining 80% probably occurs in the submembrane space.     

Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp.

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).     

Multiple effects of sulphydryl reagents on sugar transport by rat soleus muscle

Iodoacetate, over the range 0.2-2 mM, stimulated the uptake of D-xylose by rat soleus muscle and inhibited anaerobic lactate production by soleus muscle. Stimulation of sugar transport is considered to be due to the resultant fall in ATP. p-Chloromercuribenzene sulphonate (0.5-2 mM) stimulated xylose uptake to a lesser extent than iodoacetate and induced a proportionately smaller fall in ATP, consistent with the inhibitory effect of p-chloromercuribenzene sulphonate on lactate production. Under certain conditions, p-chloromercuribenzene sulphonate stimulated sugar transport without affecting the ATP level. This suggests that whereas p-chloromercuribenzene sulphonate can be expected to stimulate sugar transport through the lowering of muscle ATP, it may also act through some other mechanism. No stimulatory effect on xylose uptake was observed when muscles were exposed to N-ethylmaleimide (0.02-2 mM) either for brief (1 min) or more prolonged (30 min) periods. Because N-ethylmaleimide induced a marked fall in muscle ATP, it is surprising that N-ethylmaleimide did not stimulate sugar transport; in most experiments this inhibitor actually inhibited sugar transport. N-Ethylmaleimide inhibited the stimulation of sugar transport by 2,4-dinitrophenol and anoxia; this inhibitory effect appears to explain why N-ethylmaleimide itself did not stimulate sugar transport. p-Chloromercuribenzene sulphonate also inhibited 2,4-dinitrophenol-stimulated xylose uptake by a mechanism which seems similar to that of N-ethylmaleimide; this could explain in part the modest stimulatory effect of this inhibitor on muscle sugar transport.     

Cell-free stimulation of the insulin-sensitive cAMP phosphodiesterase by the joint actions of ATP and the soluble fraction from insulin-treated rat liver

The insulin-sensitive cAMP phosphodiesterase (PDE) from rat adipocytes was stimulated 60-70% upon incubation with 2 mM ATP and the soluble fraction (Fraction S-1) from insulin-treated rat liver. The effect of ATP was partially mimicked by ATP-gamma-S or GTP, but not by AMP-PNP. The PDE-stimulating activity in Fraction S-1 was preserved in the presence of 50 mM sodium phenyl phosphate, 50 mM sodium fluoride, and 0.1 mM sodium vanadate. The PDE-stimulating activity was not inhibited with either 0.5 mM H-7 or 5 microM PKI-(5-24)-peptide, but was blocked with 1 mM Kemptide. The active component in Fraction S-1 may be a phosphorylated compound, which, in the presence of ATP, may mediate the hormonal action on PDE.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.     

Involvement of anion channels in mediating elicitor-induced ATP efflux in Salvia miltiorrhiza hairy roots

This study examines the roles of anion channels and ATP binding cassette (ABC) protein transporters in mediating elicitor-induced ATP release in Salvia miltiorrhiza hairy root cultures. The elicitor-induced ATP release was effectively blocked by two putative membrane anion channel blockers, niflumic acid and Zn²⁺, but not by a specific Cl⁻ channel blocker, phenylanthranilic acid. The elicitor-induced ATP release was also significantly suppressed by two ABC inhibitors, glibenclamide and ethacrynic acid. Notable ATP release from the hairy roots was also induced by verapamil (2 mM), an ABC activator in animal cells. The verapamil-induced ATP release was effectively blocked by niflumic acid, but only slightly inhibited by the ABC inhibitors. Another notable effect of verapamil was the induction of exocytosis, the secretion of vesicle-like particles to the root surface. The verapamil-induced exocytosis was not inhibited by nifulumic acid and YE did not induce the exocytosis. Overall, the results suggest a significant role of anion channels, a possible involvement of ABC proteins and no significant involvement of exocytosis in mediating the ATP efflux in hairy root cells.     

Reconstitution of human hepatoma endosome-endosome fusion in vitro: potential roles for an endoprotease and a phosphoprotein phosphatase.

We developed a sensitive fluorometric assay to study in vitro fusion between early endosomes isolated from the human hepatoma, Hep G2. Biochemical characterization of this assay showed that fusion between endosomal vesicles was dependent on physiologic temperature, cytosol, and ATP. Fusion was inhibited by pretreatment of vesicles and cytosol with either 1 mM N-ethylmaleimide or 20 microM GTP gamma S. Neither 3 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid nor 1 mM CaCl2 significantly affected fusion. In addition, ATP gamma S neither inhibited fusion at 50 microM nor supported fusion at 5 mM. To further our understanding of the factors regulating fusion, inhibitors of endoprotease activity and phosphotyrosine phosphatase activity were assayed for their effect on fusion. The dipeptide inhibitor of endoprotease activity, Cbz-gly-phe-amide, inhibited fusion 70% at 3 mM whereas a dipeptide analogue, Cbz-gly-gly-amide, was without effect. Furthermore, orthovanadate, an inhibitor of phosphotyrosine phosphatase activity, stimulated fusion twofold at 0.5 mM. These results suggest that both tyrosine dephosphorylation and endoprotease activity contribute to the regulation of endosome fusion.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.     

Activation and inhibition of ATP synthesis in cell envelope vesicles of Halobacterium halobium

The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.     

Effects of potassium antimony tartrate on rat erythrocyte phosphofructokinase activity

In an earlier study, we observed a marked accumulation of antimony in erythrocytes of rats administered potassium antimony tartrate (Sb) in drinking water. This observation has raised concerns of possible adverse effects on the hematological systems. A study was therefore carried out to investigate the effects of Sb on phosphofructokinase (PFK), a rate-limiting enzyme of erythrocyte glycolysis. Preincubation of PFK with Sb caused a marked inhibition of the enzyme with 95% loss of activity at 5 mM. In comparison, 5 mM sodium arsenite, a known enzyme inhibitor, reduced PFK activity by only 38%. Increasing the concentrations of fructose-6-phosphate (F6P) or magnesium had no effects on the inhibitory potency of Sb. Varying the concentrations of ATP and Sb produced a complex effect on PFK activity. At 1 mM ATP, 0.2 mM Sb was required for 50% inhibition (IC₅₀) of PFK but only 0.05 mM Sb was required for the same inhibition when the concentration of ATP was reduced to 0.2 mM. Glutathione (2–10 mM) and hemoglobin (8–40 <μ > M) partially protected the enzyme from the Sb effect, with the protection being more effective at low antimony concentrations. When Sb was added to assay mixtures after initiation of a PFK reaction with physiological concentrations of ATP (0.2 mM) and F6P (0.1 mM), PFK activity was approximately 50% inhibited by 0.5 mM Sb and completely inhibited by 5 mM Sb. In contrast, glucose utilization in whole blood was only 16% lower over an 8 hour incubation period in the presence of 5 mM Sb. It is concluded that while PFK is markedly inhibited by Sb under in vitro assay conditions, glycolysis in erythrocytes is not significantly affected except at very high Sb concentrations. The weak effect of Sb on glycolysis in erythrocytes may be due in part to the protective effect of hemoglobin and, to a lesser extent, glutathione on PFK. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 227–233, 1998     

Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2+ and Ca2+. The inhibitors ouabain (0.01-1 mM), N-ethylmaleimide (0.01-4 mM), levamisole (0.10-0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03-30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50-300 microM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2+ and Ca2+ ATP/ADPase at 100 microM. Lanthanum decreased Mg2+ and Ca2+ ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach.     

Effects of cAMP-dependent protein kinase and ATP on N1-oxidation of 9-benzyladenine by animal hepatic microsomes

N1-Oxidation is a major metabolic pathway for 9-benzyladenine (BA) catalyzed by the cytochrome P450 system in animal hepatic microsomes. After normal hamster hepatic microsomes or phenobarbital induced rabbit hepatic microsomes were preincubated in the presence of cyclic AMP-dependent protein kinase catalytic subunit (PKA), MgCl2 and ATP, BA-N1-oxidation was significantly decreased. However, further investigation indicated that the decrease of BA-N1-oxidation seemed to be a combination of the effects of PKA and ATP, as ATP alone showed a biphasic regulatory effect on BA-N1-oxidation when microsomes were preincubated in the presence of various concentrations of ATP. In the lower ATP concentration range (0.5-2.5mM), BA-N1-oxidation increased along with the increase of ATP concentration; whereas BA-N1-oxidation decreased when the ATP concentration was higher (>5mM). The biphasic regulatory effects of ATP on BA-N1-oxidation seem dependent on the incubation process, as preincubation markedly strengthened the effects. When microsomes were incubated at 37 degrees C for different time lengths in the absence or presence of ATP (2.5 or 20mM), the activity of BA-N1-oxidase decreased at similar rates in all groups, but the activity levels of BA-N1-oxidase were different among the groups. The cytochrome P450 content was not changed parallel to the variation of BA-N1-oxidation when microsomes were incubated in the presence of ATP, indicating that the effects of ATP on BA-N1-oxidation were not mediated by affecting CYP stability. In addition, the activity of NADPH-cytochrome P450 reductase was not markedly affected by ATP without incubation. The result implied that ATP did not inhibit the reductase directly. After microsomes were incubated in the presence of low ATP concentration (2.5mM), the reductase was slightly inhibited, whilst high ATP concentration (20mM) showed marked inhibition (83% of control). This may partially contribute to the down-regulatory effect of ATP on BA-N1-oxidation. Furthermore, it was found that the presence of magnesium ions during preincubation weakened the up-regulatory effect of ATP (2.5mM) on BA-N1-oxidation, but showed no effect on the down-regulatory effect of ATP (20mM). Since these observed phenomena are not readily explained, a possible mechanism, i.e. phosphorylation and dephosphorylation of cytochrome P450, is suggested.     

Regulation of fatty acid oxidation in rat brain mitochondria: inhibition of high rates of palmitate oxidation by ADP

Regulation of oxidation of [1-14C]palmitate in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin prepared by use of a Ficoll/sucrose density gradient. The mitochondrial preparation contained considerable Mg2+-ATPase activity, but was virtually free of contamination with nonmitochondrial fractions. Palmitate oxidation was inhibited by increasing the concentration of ATP in the assay system to near-physiological levels (2 mM), and the inhibition at 2 or 4 mM ATP was analyzed by comparing it with palmitate oxidation at near-maximal rates with low levels of ATP (0.5 or 1 mM). Inhibition was increased by the addition of ADP or by increasing the concentration of Mg2+ in the assay system, whereas inhibition was decreased by decreasing the concentration of mitochondrial protein or L-carnitine in the assay system. Increasing CoA concentration also had a deinhibitory effect. With 0.5 or 1 mM ATP, however, neither inhibition by added ADP nor protein concentration-dependent inhibition was observed, and the rate of oxidation was saturated with increasing concentrations of Mg2+, L-carnitine, or CoA. These results indicated that ADP was involved in the inhibition of high rates of palmitate oxidation in the presence of sufficient ATP and L-carnitine. The inhibitory effect of increasing the concentration of mitochondrial protein could be explained by the enhanced amounts of ADP present in the preparation; similarly, increased concentrations of Mg2+ would provide higher levels of ADP by stimulating the Mg2+-ATPase reaction. We discuss the possibility that the transport of ADP across the inner membrane of brain mitochondria is coupled to the inhibition of palmitate oxidation.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions

Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump.