Kinetic evidence for multiple dynein ATPase sites

We have examined the kinetics of ATP-induced dissociation of the microtubule-dynein complex at low ATP concentrations in the presence of vanadate, which inhibits the enzyme after the binding and hydrolysis of a single ATP per site (Shimizu, T., and Johnson, K. A. (1983) J. Biol. Chem. 258, 13833-13840). Four aspects of the dissociation reaction could not be explained by a model of dynein with a single ATP-sensitive microtubule binding site. First, titration of the light-scattering amplitude versus ATP concentration in the presence of vanadate gave Mr = 720,000/ATP binding site, indicating approximately 2.8 sites/2 million molecular weight particle. Second, the dissociation reaction was incomplete at concentrations of less than 2 microM ATP in the absence of vanadate, while the addition of vanadate led to complete dissociation at an increased rate. Third, the time course of dissociation induced by less than or equal to 1 microM ATP in the presence of vanadate was biphasic, with a small but distinct lag. Fourth, the ATP concentration dependence of the rate of dissociation in the absence of vanadate was concave upward at concentrations of ATP less than 5 microM, whereas the plot was linear in the presence of vanadate. These data suggest that dynein has three ATP-sensitive microtubule binding sites and each site must bind ATP for dynein to detach from the microtubule.     

The pathway of ATP hydrolysis by dynein. Kinetics of a presteady state phosphate burst

The kinetics of ATP binding and hydrolysis (formation of acid-labile phosphate) by the Tetrahymena 30 S dynein ATPase has been measured by chemical quench flow methods. The amplitude of the ATP-binding transient gave a molecular weight per ATP-binding site of approximately 750,000, suggesting nearly 3 ATP binding sites/2 million Mr dynein molecule (Johnson, K. A., and Wall, J.S. (1983) J. Cell Biol. 96, 669-678). ATP binding occurred at the rate predicted from the apparent second order rate constant of 4.7 X 10(6) M-1 S-1 measured by analysis of the ATP-induced dissociation of the microtubule-dynein complex (Porter, M. E., and Johnson, K. A. (1983) J. Biol. Chem. 258, 6582-6587). Hydrolysis was slower than binding and occurred at a rate of 55 S-1, at 30 and 50 microM ATP. The rate limiting step for steady state turnover (product release) occurred with a rate constant of 8 S-1. These data show that the first two steps of the pathway of coupling ATP hydrolysis to the microtubule-dynein cross-bridge cycle are the same as those described by Lymn and Taylor for actomyosin (Lymn, R. W., and Taylor, E. W. (1971) Biochemistry 10, 4617-4624). Namely, ATP binding induces the very rapid dissociation of dynein from the microtubule and ATP hydrolysis occurs more slowly following dissociation. Moreover, in spite of rather gross structural differences, the kinetic constants for dynein and myosin are quite similar.     

Microtubules accelerate ADP release by dynein

The effects of microtubules on the phosphate-water oxygen exchange reactions catalyzed by dynein were examined in order to determine the mechanism by which microtubules activate the ATPase. Microtubules inhibited the rate of medium exchange observed during net ATP hydrolysis. Inhibition of the exchange reaction was proportional to the extent of microtubule activation of ATP turnover with no effect on the partition coefficient. These data argue that microtubules do not increase the rate of release of phosphate from dynein; rather, they increase the rate of ADP release. Microtubules markedly inhibited medium phosphate-water exchange reactions observed in the presence of ADP and Pi. With increasing concentrations of ADP, the rate of exchange increased in parallel to the dissociation of dynein from the microtubules, suggesting that only free dynein and not the microtubule-dynein complex catalyzes the exchange reaction. The rates of dynein binding to microtubules in the absence and presence of saturating ADP were 1.6 X 10(6) and 9.8 X 10(5) M-1 s-1, respectively. ADP inhibited the rate of the ATP-induced dissociation of the microtubule-dynein complex with an apparent Kd = 0.37 mM for the binding of ADP to the microtubule-dynein complex. However, the rate of dissociation of ADP from the M.D.ADP complex was quite fast (approximately 1000 s-1). These data support the postulate of a high-energy dynein-ADP intermediate and indicate that microtubules activate the dynein ATPase by enhancing the rate of ADP release.     

Transient state kinetic analysis of the ATP-induced dissociation of the dynein-microtubule complex

The kinetics of ATP-induced dissociation of dynein from the dynein-microtubule complex has been investigated by stopped flow light scattering methods. The addition of ATP to the dynein-microtubule complex induced a large, rapid decrease in light scattering followed by a smaller and much slower decrease. The fast light scattering change was shown to be a measure of the ATP-induced dissociation of dynein from the dynein-microtubule complex and was distinguished from microtubule disassembly by several criteria. (i) The fast reaction occurred over a period of milliseconds and the rate was a function of the ATP concentration, whereas, the slow reaction occurred over a period of several seconds and was independent of ATP concentration; (ii) the amplitude of the fast reaction was directly proportional to the amount of dynein bound to the microtubule lattice; and (iii) only the slow phase was inhibited by the addition of the microtubule-stabilizing drug, taxol. The rate of ATP-induced dissociation of dynein from the microtubule increased linearly with increasing ATP concentration to give an apparent second order rate constant for ATP binding equal to k1 = 4.7 X 10(6) M-1 s-1 according to the following pathway: (formula; see text) where M X D represents the dynein-microtubule complex and D represents dynein. The loss of signal amplitude at high ATP concentration provided a minimum estimate for the rate of dissociation of the ternary complex (M X D X ATP) equal to kd greater than 1000 s-1. Thus, the dynein-microtubule system is similar to actomyosin in that ATP induces an extremely rapid dissociation of dynein from the microtubule.     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the dynein adenosinetriphosphatase by microtubules

Previous work has indicated that following the rapid adenosine 5'-triphosphate (ATP) induced dissociation of the microtubule-dynein complex, the rate-limiting step in the ATPase cycle is product release [Johnson, K. A. (1983) J. Biol. Chem. 258, 13825-13832], which occurs at a rate of approximately 2-6 s-1. In this report we complete the analysis of the ATPase cycle by examining the effect of microtubules on the rate of product release. For these studies we used repolymerized Tetrahymena axonemal microtubules and microtubule-associated protein (MAP) free bovine brain microtubules which were shown to be free of any measureable ATPase activity. Tetrahymena 22S dynein bound to these microtubules predominantly by the ATP-sensitive site and at a rate giving an apparent second-order rate constant of (0.2-1) X 10(6) M-1 s-1, which is 50-fold greater than the rate observed with brain microtubules containing MAPs. ATP induced the rapid dissociation of the microtubule-dynein complex with an apparent second-order rate constant vs. ATP concentration equal to 1.6 X 10(6) M-1 s-1; this value is only slightly lower than that observed in the presence of MAPs. After the ATP-induced dissociation, the dynein reassociated with the microtubules following a lag period due to the time required to hydrolyze the ATP. The duration of the lag time for reassociation decreased with increasing microtubule concentration, suggesting that microtubules increased the rate of ATP turnover. Direct measurements at steady state showed that the specific activity of the dynein increased with increasing microtubule concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.     

Kinetics processivity and the direction of motion of Ncd.

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.     

Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules

The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.     

Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

The kinetics of the increase in protein fluorescence following the addition of ATP to subfragment-1 (SF-1) and acto-SF-1 have been reinvestigated. The concentration dependence of the rate obtained with SF-1 did not fit a hyperbola and at high ATP concentration, approximately 40% of the signal amplitude was lost due to a fast phase at the beginning of the transient (20 degrees C). At lower temperature (less than or equal to 10 degrees C) the fluorescence transient was biphasic, with a fast phase observed at high ATP concentration. These results indicate that there are two steps in the SF-1 pathway in which there is a change in protein fluorescence. Measurements of ATP binding and hydrolysis by chemical quench-flow methods indicate that the rate of ATP binding is correlated with the fast fluorescence step and hydrolysis is correlated with the slow fluorescence change. The SF-1 mechanism can thus be described as: (formula: see text) where M represents SF-1 and states of enhanced fluorescence are given by M (16%) and M (36% enhancement, relative to SF-1). Step 1 is a rapid equilibrium with K1 approximately 10(3) M-1. Tight binding of ATP occurs in step 2 and the loss of signal amplitude requires k2 greater than or approximately 1500--2000 s-1. The maximum observed fluorescence rate defines the rate of hydrolysis, k3 + k-3 = 125 s-1 (20 degrees C, 0.1 M KCl, pH 7.0). The steps in the mechanism correspond to the Bagshaw--Trentham scheme, with the important difference that the assignment of rate constant is altered. Formation of the acto-SF-1 complex gave a fluorescence enhancement of approximately 14% relative to SF-1. Dissociation of acto-SF-1 by ATP produced a 20--22% enhancement in fluorescence. There was no detectable fluorescence change during dissociation as evidenced by a lag in the fluorescence transient which corresponded to the kinetics of dissociation. The fluorescence change occurred at the same maximum rate as for SF-1 but there was no loss in signal amplitude at high ATP concentration. The kinetics of the fluorescence change corresponded to the rate of ATP hydrolysis, whereas tight ATP binding occurred at a much faster rate in approximate agreement with the rate of dissociation. Thus the fluorescence change in the acto-SF-1 pathway corresponds to step 3 in the SF-1 mechanism. The complete scheme can be described as follows: (formula: see text) where AM represents acto-SF-1. The tight binding step in the SF-1 pathway (k2) is sufficiently fast so that a similar step (k2') in the acto-SF-1 pathway could precede dissociation but the AM-ATP intermediate has not been detected. Following hydrolysis on the free SF-1, actin recombines with M.ADP.Pi or possibly with a second SF-1 product intermediate as proposed by Chock et al. (1976) and the fluorescence returns to the original AM level with product release.     

ADP release is rate limiting in steady-state turnover by the dynein adenosinetriphosphatase

The kinetics of the product release steps in the pathway of ATP hydrolysis by dynein were investigated by examining the rate and partition coefficient of phosphate-water 18O exchange under equilibrium and steady-state conditions. Dynein catalyzed both medium and intermediate phosphate-water oxygen exchange with a partition coefficient of 0.30. The dependence of the rate of loss of the fully labeled phosphate species on the concentration of ADP was hyperbolic, with an apparent Kd for the binding of ADP to dynein of 0.085 mM. The apparent second-order rate constant for phosphate binding to the dynein-ADP complex was 8000 M-1 s-1. The time course of medium phosphate-water oxygen exchange during net ATP hydrolysis was examined in the presence of an ATP regeneration system. The observed rate of loss of P18O4 was comparable to the rate observed at saturating ADP which implies that ADP release is rate limiting for dynein in the steady state. Product inhibition of the dynein ATPase was also examined. ADP inhibited the enzyme competitively with a Ki of 0.4 mM. Phosphate was a linear noncompetitive mixed-type inhibitor with a Ki of 11 mM. These data were fit to a model in which phosphate release is fast and is followed by rate-limiting release of ADP, allowing us to define each rate constant in the pathway. A discrepancy between the total free energy calculated compared to the known free energy of ATP hydrolysis suggests that there is an additional step in the pathway, perhaps involving a change in conformation of the enzyme-ADP state preceding ADP release.     

Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubule--22S-dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5'-[alpha-thio]triphosphate (ATP[alpha S]), Sp or Rp, by light-scattering stopped-flow methods. The dissociation by (Sp)ATP[alpha S] was rapid and the rate of the light-scattering change by (Sp)ATP[alpha S] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (Rp)ATP[alpha S] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s-1). The adenosine 5'-[beta-thio]triphosphate (ATP[beta S]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol.mg protein-1.min-1)/apparent Km (microM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[alpha S] (Sp or Rp), ATP[beta S] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at beta phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[alpha S] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[alpha S] was one-quarter to one-half of that produced by ATP reactivation.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.     

Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion

Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.     

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.     

Two states of the conformation of 21S outer arm dynein coupled with ATP hydrolysis

Tryptic digestion of 21S outer arm dynein from sea urchin sperm flagella in the presence of ATP (or ADP) and vanadate produced quite different polypeptides from those obtained in the absence of ATP (ADP) and/or vanadate (Inaba and Mohri (1989) J. Biol. Chem. 264, 8384-8388). The 21S dynein heavy chains were consistently digested into 165- and 135-kDa polypeptides in the absence of both ATP (ADP) and vanadate. In the presence of 2 mM ADP and 100 microM vanadate, 300-kDa polypeptide, which appeared to be a precursor of 165- and 135-kDa polypeptides, became less accessible to trypsin, and 165- and 135-kDa polypeptides were digested into 150-/148-kDa and 96-kDa polypeptides, respectively. Quantitative analysis of the degradation of 165- and 135-kDa polypeptides showed that the conformations of these polypeptides change remarkably in the presence of ATP (ADP) and vanadate, and slightly in the presence of ATP gamma S. Photoaffinity labeling with 8-azidoadenosine 5'-triphosphate and vanadate-mediated photocleavage of dynein heavy chains revealed that both adenine- and gamma-Pi-binding sites were located on 165- and 150-/148-kDa polypeptides, but not on 135-kDa polypeptide. These results suggest that the conformational change occurring in the 165-kDa region on binding ATP spreads to the 135-kDa region and causes the conformational change of the 135-kDa region.     

Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP

ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme.     

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.     

Binding of dynein 21 S ATPase to microtubules. Effects of ionic conditions and substrate analogs

Binding of 21 S dynein ATPase isolated from Tetrahymena cilia to B subfibers of microtubule doublets was used as a model system to study dynein-tubulin interactions and their relationship to the microtubule-based sliding filament mechanism. Binding of 21 S dynein to both A and B microtubule subfibers is supported by monovalent as well as divalent ions. Monovalent cation chlorides support dynein binding to B subfibers with the specificity Li greater than Na congruent to K congruent to Rb congruent to Cs congruent to choline. The corresponding sodium or potassium halides follow the order F greater than Cl greater than Br greater than I. However, an optimal binding concentration of 40 mM KCl supports only 55% of the protein binding which takes place in 3 mM MgSO4 and does not stabilize dynein cross-bridges when whole axonemes are fixed for electron microscopy. Divalent metal ion chlorides (MgCl2, CaCl2, SrCl2, and BaCl2) have nearly equivalent effects at a concentration of 6 mM; all support about 140% of the binding observed in 6 mM MgSO4. The binding data suggest negative cooperativity or the presence of more than one class of dynein binding sites on the microtubule lattice. Low concentrations of MgATP2- induce dissociation of dynein bound to B subfibers in either 6 mM MgSO4 or 40 mM KCl. ADP, Pi, PPi, and AMP-PCH2P are unable to induce dynein dissociation, while AMP-PNHP and ATP4- both cause dynein release from B subfiber sites. The half-maximal sensitivities of the tubulin-dynein complex to MgATP2-, ATP4-, and adenylyl-imidodiphosphate (AMP.PNP) are 1.3 X 10(-8) M, 3.6 X 10(-5) M, and 4.7 X 10(-4) M respectively. Incubation of doublets or 21 S dynein in N-ethylmaleimide (NEM), which can inhibit active sliding, has no effect on either association of dynein with the B subfiber or on dissociation of the resulting dynein-B subfiber complex by MgATP2-.     

A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum.

Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude.