Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Alterations in the structure of the ribose moiety of ATP reduce its effectiveness as a substrate for the sarcoplasmic reticulum ATPase

The substrate specificity of the calcium ATPase of isolated sarcoplasmic reticulum vesicles was examined using a series of ribose-modified ATP analogs. Steady state hydrolytic rate measurements using analogs modified at the 2'- and 3'-positions demonstrate that both hydroxyl groups contribute to substrate specificity and high catalytic activity. The 3'-hydroxyl is especially significant in this regard since all analogs modified at the 3'-position were slowly hydrolyzed, if at all, and they did not produce the activation at millimolar concentrations characteristically observed with ATP. In contrast, ATP analogs modified only at the 2'-position were more rapidly hydrolyzed (although at rates less than for ATP), and they did produce activation at millimolar concentrations. These results suggest that neither the catalytic (high affinity) nor the regulatory (low affinity) site of the CaATPase tolerates changes in the hydroxyl substituent at the 3'-position of ATP, whereas steady state rates associated with substrate binding at both types of sites are less affected by changes in the 2'-hydroxyl of ATP.     

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.     

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.     

Principles for designing enzyme-like catalysts based on the rate-acceleration mechanisms of semisynthetic oxidases.

Combinations of substrate-binding sites and catalytic groups constitute various kinds of enzyme-like catalysts. The design of such catalysts can be evaluated by the enhancement of the overall catalytic activity by combining these parts into one catalyst. For a catalyst having one substrate-binding site and one catalytic group, an equation was obtained which shows the relationship between the rate-acceleration due to the combination, the affinity of the site (1/Kd), intrinsic effective concentration (kin/kex) and substrate concentration ([S]). The intrinsic effective concentration is the ratio of the first-order rate constant (kin) of the intramolecular reaction between the catalytic group and the bound substrate and the second-order rate constant (kex) of the intermolecular reaction between the catalytic group and the free substrate; the value depends on the method of linking the catalytic group and the binding site. This equation provides the following principles for designing catalysts of this type with a considerable grade of rate-acceleration: [S] less than or equal to kin/kex and (1/10)[S] less than or equal to Kd less than or equal to kin/kex. To increase kin/kex, the structure of the binding site is required not to reduce the reactivity of the bound substrate, and the linker connecting the binding site and the catalytic group is required to be flexible and to have an appropriate length. A subunit structure is also found to be effective to improve the catalytic activity: the activity of an n-mer is at most n2 times as high as that of the monomer. As for the substrate-binding sites, the sites of natural enzymes and antibodies are good candidates because various kinds of binding sites with high affinity and specificity to the corresponding substrates are available. In addition, the equation relating the rate-acceleration with Kd, kin/kex, and [S] is used for explaining the catalytic efficiency of enzymes energetically. The principle for designing a multifunctional catalyst having several kinds of binding sites for its substrates and intermediates and several kinds of catalytic groups was then investigated. In this case, the diffusion of the intermediates strongly affects the activity of the multifunctional catalyst, and such a diffusion process was also analyzed. On the basis of these analyses, the following principles were obtained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure and function of the two heads of the myosin molecule. IV. Physiological functions of various reaction intermediates in myosin adenosinetriphosphatase, studied by the interaction between actomyosin and 8-bromoadenosine triphosphate.

The kinetic properties of the hydrolyses of 8-Br ATP and 8-SCH3 ATP by myosin [EC 3.6.1.3] and actomyosin were compared with those of ATP, and the following results were obtained. The Ca-NTPase activities of myosin using these two ATP analogs as substrates were smaller than that of ATPase, and the NTPase activities toward these analogs were strongly suppressed by EDTA. The Mg-NTPase activities toward these analogs were higher in a medium of high ionic strength than in a medium of low ionic strength, in contrast to the activity of Mg-ATPase. These analogs did not produce any initial burst of Pi liberation, activation of myosin NTPase by F-actin, or superprecipitation of actomyosin. The interactions between 8-Br ATP and HMM, acto-HMM, actomyosin, and myofibrils were studied in detail in the presence of Mg2+ in medium of low ionic strength. The Michaelis constant, Km, and the maximum rate, Vm, of 8-Br ATPase of HMM were 27 muM and 21 min-1, respectively. The fluorescence change of HMM induced by 8-Br ATP also followed the Michaelis-Menten equation, and the Michaelis constant, Kf1, was as low as 4 muM. Acto-HMM and acto-S-1 were fully dissociated by the addition of 8-Br ATP. The relation between the extent of dissociation of acto-HMM and the concentration of 8-Br ATP followed the Michaelis-Menten equation, and the apparent dissociation constant, Kd, was 22 muM. This Kd value is almost equal to the Km value of 8-Br ATPase of HMM described above. Myofibrillar contraction was not supported by 8-Br ATP. It was concluded that in the myosin NTPase reaction with 8-Br ATP as a substrate, M2NTP but not MNDPP is formed in route (1), while MNTP is formed in route (2). It was also concluded that the key intermediate for the actomyosin NTPase reaction is MNDPP, and that dissociation of acto-HMM is induced by the formation of M2NTP and MNTP in routes (1) and (2), respectively.     

Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 1. Probing of the nucleotide sites by Naphthoyl-ATP, a fluorescent nontransportable analogue of ATP

The ADP/ATP carrier protein was extracted and purified from beef heart mitochondria, and its binding parameters with respect to 3'-O-naphthoyladenosine 5'-triphosphate (N-ATP), a fluorescent nontransportable analogue of ATP, were studied. The binding of N-ATP to the isolated carrier protein was accompanied by a decrease in fluorescence. Conversely, the release of bound N-ATP upon addition of carboxyatractyloside (CATR) or ATP resulted in a fluorescence increase. The bound N-ATP that was released upon addition of an excess of CATR or ATP was referred to as specifically bound N-ATP, i.e., N-ATP bound to the nucleotide sites of the carrier protein. Two classes of binding sites for N-ATP could be identified; the number of high-affinity sites (Kd less than 10 nM) was equal to the number of low-affinity sites (Kd = 0.45 microM). CATR behaved apparently as a noncompetitive inhibitor of the binding of N-ATP. The amount of N-ATP released increased linearly with the amount of CATR added, indicating an extremely high affinity of the carrier protein for CATR. The number of CATR binding sites was equal to half the total number of N-ATP binding sites (high- and low-affinity sites); at saturating concentrations of N-ATP, the binding of 1 mol of CATR resulted in the release of 2 mol of bound N-ATP, one from the high-affinity site and the other from the low-affinity site, showing unambiguously that each CATR site is interacting with a pair of probably interdependent N-ATP sites. A clear competition between N-ATP and ATP for binding to the carrier protein was demonstrated. The Kd values of the high- and low-affinity sites for ATP were less than 50 nM and 5 microM, respectively. In the presence of high concentrations of ATP, the two classes of N-ATP binding sites became indistinguishable, suggesting interconversion. It is proposed that the asymmetry in affinity for N-ATP binding is induced by the binding step itself, the carrier protein exhibiting a negative cooperativity for N-ATP binding.     

Interaction of ADP and ATP with noncatalytic sites of isolated and membrane-bound chloroplast coupling factor CF<Subscript>1</Subscript>

This study of ATP and ADP binding to noncatalytic sites of membrane-bound CF1 (ATP synthase) revealed two noncatalytic sites with different specificities and affinities for nucleotides. One of these is characterized by a high affinity and specificity to ADP (Kd=2.6+/-0.3 microM). However, a certain increase in ADP apparent dissociation constant at high ATP/ADP ratio in the medium allows a possibility that ATP binds to this site as well. The other site displays high specificity to ATP. When the ADP-binding site is vacant, it shows a comparatively low affinity for ATP, which greatly increases with increasing ADP concentration accompanied by filling of the ADP-binding site. The reported specificities of these two sites are independent of thylakoid membrane energization, since both in the dark and in the light the ratios of ATP/ADP tightly bound to the noncatalytic sites were very close. The difference in noncatalytic site affinity for ATP and ADP is shown to depend on the amount of delta subunit in a particular sample. Thylakoid membrane ATP synthase, with stoichiometric content of delta-subunit (one delta-subunit per CF1 molecule), showed the maximal difference in ADP and ATP affinities for the noncatalytic sites. For CF1, with substoichiometric delta subunit values, this difference was less, and after delta subunit removal it decreased still more.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Specificity of cooperative interactions between catalytic sites

Cooperative interactions between nucleotide binding sites on beef heart mitochondrial F1-ATPase have been studied by measuring substrate-promoted release of 5'adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) from a single high affinity site. The site is initially loaded by incubating F1 with an equimolar amount of the nonhydrolyzable ATP analog. When unbound [3H]AMP-PNP is removed and the complex diluted to a concentration below the Kd, release of ligand shows an apparent absolute requirement for medium ADP. Release is biphasic with the extent of release during the initial rapid phase dependent on the concentration of medium ADP. Although phosphate alone has no effect, it enhances the rapid phase of ADP-promoted release over 2-fold with a half-maximal effect at 60 micrometers P1. The binding of efrapeptin (A23871) to the F1.AMP-PNP complex completely prevents ADP-promoted dissociation. Although AMP-PNP release also occurs in the presence of medium ATP, the F1.AMP-PNP complex does not dissociate if an ATP-regenerating system of sufficient capacity to prevent accumulation of medium ADP is added. Consistent with an inability of nucleoside triphosphate to promote release is the failure of medium, nonradioactive AMP-PNP to affect retention of the 3H-labeled ligand. The stability of F1.AMP-PNP complex in the absence of medium nucleotide and the highly specific ability of ADP plus P1 to promote rapid release of the ATP analog are interpreted as support for an ATP synthesis mechanism that requires substrate binding at one catalytic site for product release from an adjacent interacting site.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.     

Binding of oxytocin to uterine cells in vitro. Occurrence of several binding site populations and reidentification of oxytocin receptors

Myometrial and endometrial cells of sheep, rat, and calf in monolayer cell culture display at least three populations of binding sites for oxytocin, with dissociation constants (Kd) of approximately 5 X 10(-9), 4 X 10(-7), and greater than 10(-5) mol/liter, respectively. Binding of the tritium-labeled oxytocin (concentration range, 10(-11) to 5 X 10(-4) M) to the first two sites is displaceable by cold oxytocin. The ratio of binding capacities of the high to medium affinity site appears to average 1:18. Dissociation rate constants for these sites (22 degrees C) are roughly 10(-4) and 2 X 10(-3) s-1, respectively. The capacity of the low affinity site varies in individual cell preparations and is between 5 and 66 times that of the medium affinity site. The low affinity binding sites may not be fully saturable and may follow a nonasymptotic binding isotherm. Logarithms of Kd and binding capacity for individual binding sites are linearly correlated. The coexistence of the three sites was also proven by cluster analysis based on similarities between Kd, binding capacity, and Hill coefficient. Only minor systematic species and cell type differences occur in these properties. The value of Kd for the oxytocin receptor in rat myometrium, derived recently from a stepwise irreversible inhibition of uterotonic response to oxytocin, is close to 2.5 X 10(-7) mol/liter. Additional pharmacological data (pA2 values of structural analogues of oxytocin acting as competitive inhibitors) also reveal a Kd value of 3 X 10(-7). It is, therefore, concluded that the receptors for oxytocin in rat myometrium are identical with the medium affinity site.     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.