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1.
J C Seidel 《Archives of biochemistry and biophysics》1973,157(2):588-596
Myosin and heavy meromyosin have been spin labeled at either the S1 or S2 thiol groups, and their interaction with F-actin has been studied by electron spin resonance, both in the absence of substrate and during the hydrolysis of ATP. The spectrum of myosin labeled at either group indicates strong immobilization of the label. In the absence of substrate, actin added to S1-labeled myosin slightly increases the separation of the outer spectral peaks, indicating a decrease in the mobility of the spin label. Actin also reduces the microwave power required to saturate the esr signal of S1-labeled myosin or heavy meromyosin. The latter phenomenon is a more sensitive measure of the actin-myosin interaction than the spectral change seen in the absence of saturation. This suggests that saturation measurements may provide a more sensitive method of detecting changes in the environment of slowly tumbling nitroxide radicals than spectral measurements carried out in the absence of saturation. The decrease in the amplitude of the spectrum on adding actin at saturating microwave power was used to determine the stoichiometry of the interaction between actin and heavy meromyosin. This decrease is maximal when 2 moles of actin monomer are added per mole of heavy meromyosin and is reversed when actin and myosin are dissociated by ATP. During the steady state hydrolysis of ATP, actin had no detectable effect on the spectrum of S1-labeled myosin. It can be concluded that spin labels bound to the S1 groups are in a region of the myosin molecule that is affected by the interaction with actin. Actin does not affect the rate at which the bound spin label is reduced by dithiothreitol nor does the spin labeling of S1 groups affect the activation by actin of the ATPase activity of myosin. These findings suggest that the most likely mechanism by which actin alters the mobility of labels on S1 groups involves a change in the conformation of myosin. If a spin label is bound to the S2 thiol groups rather than the S1 groups, then actin has no detectable effect on the spectrum either in the presence or absence of ATP. 相似文献
2.
(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg2+). I50 values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg2+ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg2+ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg2+. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg2+ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg2+ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed. 相似文献
3.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km 0.25 μM, Vmax 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献
4.
The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin 总被引:3,自引:0,他引:3
Evidence is presented that both myosin and actomyosin in presence of Mg2+ and KCl catalyze an incorporation of 32Pi into ATP. The rate with actomyosin is about the rate of ATP hydrolysis; the rate with myosin is less than of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of 32Pi incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [32P]-ATP present and the rate constant, k?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔGo for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔGo for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage. 相似文献
5.
ATP, in the presence of 0.05–0.15 m KCl and greater than 50 μm Mg2+, induces dissociation (clearing) followed by superprecipitation of skeletal muscle actomyosin. Superprecipitation has been studied as a model of muscle contraction, and ATP depletion has been associated with the onset of superprecipitation. Recent studies [Puszkin and Rubin (1975) Science188, 1319–1320] indicate that ADP stimulates superprecipitation without increasing the rate of ATP hydrolysis. We confirm that ADP stimulates superprecipitation; however, contrary to the experience of these investigators, ADP does stimulate ATP hydrolysis in the system studied here. We present evidence that superprecipitation is associated with generation of a critical ADP:ATP ratio but it appears that this ratio is an indirect measure of an associated but uncharacterized phenomenon which signals the onset of superprecipitation. Added ADP decreased the extent and duration of clearing, increased the rate of ATP hydrolysis, and increased the extent of superprecipitation of rat skeletal muscle actomyosin in the presence of excess Mg2+. The ADP effect was not mimicked by EDTA or AMP. The duration of clearing was related not to the time required to attain a specific level of any nucleotide phosphate, but to the time required to generate an ADP:ATP ratio of approximately 3.6. Apparently only that ADP generated in the system by ATP hydrolysis was involved in the critical ADP:ATP ratio. Added ADP stimulated myosin ATPase activity in 1.6 or 3.2 mm Mg2+. This effect was not mimicked by EDTA or AMP. The results are used to relate studies by others of myosin sulfhydryl modification to a recent model [Burke et al. (1973) Proc. Nat. Acad. Sci. USA70, 3793–3796] in which myosin MgATPase activity is inhibited by formation of a stable cyclic complex of MgATP and the S1 and S2 sites of heavy meromyosin. 相似文献
6.
Matthew C. Clifton Michael J. Simon Satchal K. Erramilli Huide Zhang Jelena Zaitseva Mark A. Hermodson Cynthia V. Stauffacher 《The Journal of biological chemistry》2015,290(9):5555-5565
Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. 相似文献
7.
Gianfranco Liguri Massimo Stefani Andrea Berti Paolo Nassi Giampietro Ramponi 《Archives of biochemistry and biophysics》1980,200(2):357-363
The data presented in this paper concern a kinetic study of the calcium uptake by sarcoplasmic reticulum vesicles and of the hydrolysis of the substrates which support the process. The results show that substrates which are different from ATP, acetylphosphate, and carbamylphosphate are able to support calcium transport. The technique used to follow the process allows us to detect continuously the changes in the concentration of the calcium present in the external medium. In our experimental conditions the calcium uptake supported by all the high energy substrates tested proceeds for several seconds at a constant rate, presumably corresponding to the “steady state” of the process; furthermore the calcium transport is clearly Ca2+ and Mg2+ dependent: the lowering of the Ca+ concentration in the medium from 10?4 to 10?5m causes a remarkable reduction of the V of the calcium transport and an apparent increase of the affinity of the sarcoplasmic reticulum vesicles for the acylphosphates; in the absence of Mg2+, none of the substrates is able to support the calcium uptake which increases in the presence of rising amounts of Mg2+ in the reaction medium. Furthermore, both the calcium transport and the substrate hydrolysis appear to follow the Michaelis-Menten kinetics in the presence of acylphosphates but not in the presence of ATP. The hydrolytic activity of sarcoplasmic reticulum vesicles on ATP and acylphosphates reveals a clear Mg2+ dependence; furthermore, in the absence of free Ca2+ and in the presence of 5 mm Mg2+, the high energy substrates tested reveal a different susceptibility to the hydrolitic attack by sarcoplasmic reticulum vesicles. 相似文献
8.
Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca2+-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg2+-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg2+-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi. 相似文献
9.
This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of Pi. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-Pi subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, Pi release is reversible, which allows the regulation of filament dynamics by cellular Pi. The nature of the divalent metal ion — Ca2+ or Mg2+ — tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins. 相似文献
10.
Sato O White HD Inoue A Belknap B Ikebe R Ikebe M 《The Journal of biological chemistry》2004,279(28):28844-28854
The missense mutation of Cys(442) to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20-40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase ( approximately 10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (<10(-7) m) calcium but is calcium-sensitive with a 1.6-fold increase at high ( approximately 10(-4) m) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI. 相似文献
11.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km = 0.25 μM, Vmax = 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献
12.
Horacio Guimarães-Motta Leopoldo De Meis 《Archives of biochemistry and biophysics》1980,203(1):395-403
Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by Pi of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca2+ prior to the addition of Pi and Mg2+ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca2+ prior to the addition to the reaction mixture of Pi, Mg2+, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca2+ or, in the absence of Ca2+, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive Pi. In the presence of Ca2+, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca2+. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca2+-dependent ATPase were used. A transient phosphorylation by Pi was observed when the enzyme was preincubated in the absence of Ca2+ and then added to a medium containing Pi, Mg2+, and excess of Ca2+. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by Pi was observed when the enzyme was preincubated with Ca2+. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca2+ and then added to a medium containing Pi, ADP, Mg2+, and 20 mm CaCl2. This was not observed when the enzyme was preincubated in the presence of Ca2+. 相似文献
13.
Jean-Pierre Caillé Marie Pigeon-Gosselin Michel Pézolet 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,758(2):121-127
Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca2+, Mg2+ and ATP. The comparaison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg2+ and Ca2+ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg2+ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca2+ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the α-helical content, or of the orientation, of some of the contractile proteins. 相似文献
14.
The K+-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na+ + K+)-ATPase from pig kidney shows substrate inhibition (Ki about 9.5 mM at 2.1 mM Mg2+). Potassium antagonizes and sodium favours this inhibition. In addition, K+ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K+ activation. In the absence of Mg2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb+ from the Rb+ occluded unphosphorylated enzyme. With no Mg2+ and with 0.5 mM KCl, trypsin inactivation of (Na+ + K+)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E1 enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E1 state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na+ + K+)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site. 相似文献
15.
Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis 总被引:6,自引:0,他引:6
We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle. 相似文献
16.
J C Seidel 《The Journal of biological chemistry》1975,250(14):5681-5687
The ATP-induced enhancement of the intrinsic fluorescence of myosin and heavy meromyosin (HMM) that persists during the steady state of hydrolysis has been investigated. To compare the substrate-induced changes in fluorescence with those in the electron spin resonance spectrum of the spin-labeled enzyme, we studied the influence of temperature, pH, and ionic strength, as well as the effect of chemical modification (spin labeling) of the SH-1 sulfhydryl groups. Changing the pH between 6 and 9 does not affect the enhancement of fluorescence of myosin or HMM; changing the ionic strength, which could be studied only with HMM, also has no effect; and decreasing the temperature from 20 to 5 degrees slightly diminishes the enhancement with both myosin and HMM. Chemical modification with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, which blocks the SH-1 thiol groups, reduces the enhancement of fluorescence, induces a strong dependence on ionic strength and pH, and substantially increases the dependence on temperature. The enhancement with labeled myosin or labeled HMM increases with increasing pH, ionic strength, and temperature, closely paralleling the effects of these parameters on the electron spin resonance spectrum of spin-labeled myosin (SEIDEL, J.C. and GERGELY, J. (1973) Arch. Biochem. Biophys. 158, 853), suggesting that the same molecular change, induced by ATP and associated with formation of the MADP-P1 complex, underlies both the change in fluorescence and the change in ESR spectrum. Those analogues of ATP that produce the maximal enhancement of fluorescence (WERBER, M., SZENT-GYORGYL, A.G., and FASMAN, G. (1972) Biochemistry 11, 2872) also produce the maximal change in the ESR spectra. Both an amino group at position 6 of the substrate and an unmodified triphosphate chain are required for maximal change in either fluorescence or ESR spectra. The smaller enhancement of fluorescence produced by spin labeling the SH-1 groups persists after the nitroxide has been chemically changed to a diamagnetic species. Thus the small enhancement cannot be attributed to paramagnetic quenching of tryptophan fluorescence by the spin label. An initial burst of phosphate liberation accompanies the hydrolysis of ATP, cytidine 5'-triphosphate, uridine 5'-triphosphate, guanosine 5'-tryphosphate, iosine 5'-triphosphate, 2'-deoxyadenosine 5'-tryphosphate, adenosine 5'-tetraphosphate, and tripolyphosphate. The presence or absence of the burst does not correlate with the extent of the spectral change. 相似文献
17.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP. 相似文献
18.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1983,736(1):73-78
(1) Contrary to what has usually been assumed, (Na+ + K+)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na+ + Mg2+ to ADP-NH2 and Pi. The activity is ouabain-sensitive and is not detected in the absence of either Mg2+ or Na2+. The specific activity of the Na+ + Mg2+ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K+. The activity is not stimulated by K+, nor can K+ replace Na+ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na+ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The Km value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH2]P are not detectably hydrolysed by (Na+ + K+)-ATPase in the presence of Na+ + Mg2+. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg2+ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na+ + Mg2+ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na+ + K+)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C. 相似文献
19.
The Mg2+ dependence of the kinetics of the phosphorylation and conformational changes of Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)3 state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg2+ to saturating values of 195 (± 6) s−1 and 54 (± 8) s−1 for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (Kd) of Mg2+ interaction with the E1ATP(Na+)3 state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the Kd of Mg2+-ATP interaction in solution. Within the enzyme-ATP-Mg2+ complex, the actual Kd for Mg2+ binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation. 相似文献
20.
The kinetics of the phosphorylation and subsequent conformational change of Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)3 state. On mixing with ATP in the presence of Mg2+, a fluorescence increase occurred, due to enzyme conversion into the E2P state. The fluorescence change accelerated with increasing ATP concentration until a saturating limit in the hundreds of micromolar range. The amplitude of the fluorescence change (ΔF/F0) increased to 0.98 at 50 μM ATP. ΔF/F0 then decreased to 0.82 at 500 μM. The decrease was attributed to an ATP-induced allosteric acceleration of the dephosphorylation reaction. The ATP concentration dependence of the time course and the amplitude of the fluorescence change could not be explained by either a one-site monomeric enzyme model or by a two-pool model. All of the data could be explained by an (αβ)2 dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one α-subunit or at a high rate with ATP hydrolysis by both α-subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na+,K+-ATPase. 相似文献