The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.     

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. GTP-induced calcium accumulation and GTPase activity

We previously demonstrated that the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium and does not support the translocation of calcium and oxalate into the vesicular space. In response to GTP, however, calcium is accumulated into a compartment which is sensitive to pH and ionophore. In the present paper, we further explored the relationship between GTP hydrolysis and GTP-induced calcium accumulation. Both ATP- and GTP-induced calcium accumulation were prevented by the sulfhydryl reagent, N-ethylmaleimide (NEM; I50 = 0.2 mM). In contrast, the sensitivity of NTP hydrolysis to NEM differed markedly; GTPase activity was not affected by NEM, whereas ATPase activity was markedly inhibited. Conversely, although the GTPase was noncompetitively inhibited by the ATP analogue, adenylyl imidodiphosphate (Ki = 8 microM), and was competitively inhibited by the GTP analogue, guanylyl imidodiphosphate (Ki = 60 microM), GTP-induced calcium accumulation was not affected by the NTP analogues at any concentration. Therefore, the GTP-dependent accumulation of calcium into the pH- and ionophore-sensitive compartment of cardiac SR may not require GTP hydrolysis but may be dependent on GTP binding. The previously reported noncompetitive inhibition of the GTPase by ATP was also observed when the calcium-dependent hydrolysis of ATP was prevented by NEM (Ki = 1.2 microM). Along with the noncompetitive inhibition of the GTPase by adenylyl imidodiphosphate, the inhibition of the GTP by ATP in the presence of NEM suggests that ATP binding may be involved in the observed inhibition. The Ki for the noncompetitive inhibition of GTPase activity is compatible with ATP binding to the high affinity catalytic site of the ATPase. Thus, although GTP-induced calcium accumulation differs somewhat from ATP-dependent calcium translocation, the similarities between the two processes (i.e. similar time courses and sensitivity to pH, ionophore, and sulfhydryl modification) suggest that they may be related in some manner.     

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells.

The mechanisms by which nucleotides stimulate the activity of the ATP-regulated K(+)-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic beta-cells. ATP produces a concentration-dependent inhibition of channel activity with a Ki of 18 microns. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.     

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.     

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).     

Characterization of native and reconstituted hydrogen ion pumping adenosinetriphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.     

ATPase activity of the novocaine segregation zones isolated from the erythrocytes of the common frog

Novocaine segregation zones in frog's erythrocytes, isolated by differential centrifugation, were shown to be ATPase active. The enzyme displays half of its maximum activity at 0.18 Mm ATP concentration to be inhibited by high concentrations of ATP. ATPase is activated by both Mg2+ and Ca2+ (in a lesser degree), with the maximum activity being at pH 7.5. A 5 minutes heating without the substrate results in decreasing the enzyme activity at 30 degrees, and in the total inhibition at 50 degrees C. Along with ATP, the enzyme can hydrolyse GTP and, in a lesser degree, ADP and sodium pyrophosphate. The ATPase activity is not effected with oligomycin (0.5-1.5 mkg/ml) or ouabaine (0.1 mM). Oligomycin in concentration 5 micrograms/ml induced non-specific inhibition of ATPase. Uncouplers, like 2,4-dinitrophenol and carbonyl cyanid p-trifluorometoxyphenylhydrazone, stimulate the enzyme activity. The lack in the ATP-ase sensitivity to oligomycin (specific inhibitor of mitochondrial F1-ATPase) and ouabaine (specific inhibitor of Na+, K+-ATPase) may suggest that the ATPase activity of novocaine segregation zones in frog's erythrocytes is not associated with a random contamination with mitochondria or cytoplasmic membranes. The ATPase under study has much in common with the lysosomal +H-ATPase. The results obtained support a hypothesis that +H-ATPase may function as a course of protones for maintaining acidic medium in segregation zones and promote accumulation of weak bases by means of their protonation.     

Chemical modification of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Binding of N-ethylmaleimide to sarcoplasmic reticulum: evidence for sulfhydryl groups in the active site of ATPase and for conformational changes induced by adenosine tri- and diphosphate.

The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.     

Characterization of a microtubule-stimulated adenosinetriphosphatase activity associated with microtubule gelation-contraction

A microtubule-stimulated ATPase is associated with particles that are responsible for microtubule gelation-contraction in vitro. These particles have been proposed to be slow axonal transport, component a, particulates (SCAPs) [Weisenberg, R. C., Flynn, J. J., Gao, B., Awodi, S., Skee, F., Goodman, S., & Riederer, B. (1987) Science (Washington, D.C.) 238, 1119-1122]. The SCAP ATPase activity is stimulated approximately twofold by microtubules. The microtubule-stimulated ATPase activity correlates with the occurrence of microtubule gelation-contraction. Both microtubule-stimulated ATPase activity and microtubule gelation-contraction are inhibited by millimolar calcium, 0.3 M KCl plus 2 mM ethylenediaminetetraacetic acid (EDTA), 5 microM vanadate, and millimolar N-ethylmaleimide (NEM). Neither the ATPase activity nor microtubule gelation-contraction is affected by high magnesium concentrations (up to 8 mM) or by the anti-ATPase drugs ouabain, oligomycin, sodium azide, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Magnesium is required for both ATPase activity and microtubule gelation-contraction. Microtubule-stimulated hydrolysis of GTP, CTP, ITP, and UTP is less than 50% of ATP hydrolysis, and microtubule gelation-contraction is reduced in these nucleotides. On the basis of these results we propose that the microtubule-stimulated ATPase activity associated with SCAPs is a previously undescribed enzyme that is responsible for microtubule gelation-contraction in vitro and that is the likely motor for component a of slow axonal transport.     

Competitive inhibition of mouse brain gamma-aminobutyrate aminotransferase by ATP

The nucleotide ATP was shown to be a reversible inhibitor of partially purified gamma-aminobutyrate aminotransferase isolated from mouse brain. This inhibition was of the competitive type with respect to the substrate, gamma-aminobutyric acid (Ki = 3.7 +/- 0.6 mM), but was noncompetitive with respect to both the second substrate alpha-ketoglutarate and the cofactor pyridoxal 5'-phosphate. Two analogues of ATP, ADP and GTP, also gave rise to an inhibition gamma-aminobutyrate aminotransferase that was similar to that produced by ATP. These results are consistent with the view that mouse brain gamma-aminobutyric acid aminotransferase could be under regulatory control by ATP and certain other nucleotides within the mitochondria.     

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. kinetic properties of the basal and trypsin-stimulated activities

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.     

Cytosolic ADP enhances the sensitivity to tolbutamide of ATP-dependent K+ channels from pancreatic B-cells

The effects of intracellular purine nucleotides on tolbutamide-induced block of ATP-dependent K+ channels from mouse pancreatic B-cells were studied using the patch-clamp technique. When applied to the inside of excised patches, tolbutamide alone blocked channel activity half-maximally at 55 microM and the concentration-response curve for the inhibition of K+ channels by tolbutamide was flat. ADP (1 mM), but not other nucleotides (AMP, GTP or GDP) increased the steepness of the concentration-response curve and decreased the half-maximally effective tolbutamide concentration to 4.2 microM. It is suggested that the ATP-dependent K+ channel or a closely related structure contains a receptor which is accessible for cytosolic ADP and controls the sensitivity to tolbutamide.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Purification and characterization of a casein kinase from human erythrocyte cytosol

A casein kinase was extracted from human erythrocyte cytosol and purified by ammonium sulfate precipitation, chromatography on DEAE and phosphocellulose, and affinity chromatography on ATP-agarose. This enzyme did not use histone as a substrate; its activity was not stimulated by cyclic nucleotides. The pH of optimal activity was 6.5. The enzyme had an absolute requirement of Mg²⁺ ions at an optimal concentration of 30 mM; activity was stimulated by Na⁺ and K⁺ at a maximal concentration of 0.125 M and inhibited by Ca²⁺. Casein was used as a substrate with a Km of 0.25 mg/ml; ATP was the preferential phosphoryl donor with a Km of 14.7 μM; GTP may be used with a lower yield and a Km of 26.3 μM. ADP was a competitive inhibitor of ATP with a Ki of 14 μM. 2–3 DPG was an allosteric inhibitor of ATP with an apparent Ki of 4.6 mM and a Hill coefficient of 3.8. Kinetic data indicate that the reaction follows a coordinated mechanism with ATP as the first substrate and subsequent formation of a ternary complex with the protein. SDS-PAGE of the purified enzyme showed two different peptide chains of molecular weight 35 000 and 25 000.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

The effect of ATP analogues on ATPase and phosphatase activities of Na+, K+-ATPase for duck salt glands

ATP analogues are studied for their effect on phosphatase and ATPase activities of Na+, K+-ATPase with the aim to obtain data concerning properties and structure of sites of high and low affinity to ATP. The activating effect of nucleotides on K+-dependent phosphatase reflecting their ability to be bound with the centres of high affinity decreases in a series: ATP, N1-oxy-ATP, CTP, JTP. In the domain of high ATP concentrations, where low affinity site is saturated, ADP is a competitive inhibition of ATPase reaction with Ki of 300 microM. The analysis of N1-oxy-ATP inhibiting effect has shown that its affinity to this site is six times less than that of ADP. The absence of the inhibiting effect of CDP, JDP, GDP and UDP in concentrations up to 10 mM testifies to the fact that sites of both low and high affinity to ATP are characterized by high specificity with respect to the adenine part of the substrate molecule.