Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility

Kobayashi, Takaaki; Martensen, Todd M.; Nath, Jayasree; Flavin, Martin

doi:10.1016/0006-291x(78)91279-2

Cited by 160 publications

(115 citation statements)

References 22 publications

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“…Recently, vanadate has been found to be a potent inhibitor of dynein ATPase (l4, 19,27), as well as of the movement of the reactivated demembranated flagella (19). In the present paper, the effect of vanadate on flagellar stiffness was investigated, and correlated with observations on rigor bend relaxation and beat frequency of demembranated sea urchin sperm flagella .…”

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confidence: 73%

Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Okuno¹

1980

The Journal of Cell Biology

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Direct measurements of the stiffness (elastic bending resistance) of demembranated sea urchin sperm flagella were made in the presence of MgATP 2-and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of -0 .9 x 10-2 ' N m l, which is -5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP -'does not substitute for MgATP 2 " in producing the relaxed state . A progressive inhibition of movement is observed after addition of MgATP 2-to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP 2-and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP 2-that is not enough to induce spontaneous beating . Vanadate enhances competitive inhibition of beat frequency by MgADP -but not by ADP3-, ATP -, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dyneinnucleotide complexes induced by MgATP 2-and MgADP -. The state accessible by MgATP2-binding must be a state in which the cross-bridges are detached and the flagellum is relaxed . The state accessible by MgADP -binding must be a crossbridged state . Bound vanadate prevents the transition between these two states . Inhibition and relaxation by vanadate in the presence of MgATP2-results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the detached state .The sliding microtubule hypothesis for flagellar and ciliary movement has been firmly established by a series of experimental (42, 49, 52) and theoretical works (4,5,31, 40), since the first important observations by P. Satir (43,44) . This hypothesis predicts that the dynein arm plays a vital role in generating the bending wave of flagella, because the dynein arm is involved in the transduction of the chemical energy provided by ATP dephosphorylation into mechanical energy (15, 16) . The conformational change of the dynein arm is assumed to involve the cyclic change of its orientation, including association and dissociation with the adjacent microtubule, as in the actomyosin system (26). This mechanochemical behavior of the dynein arm is thought to be tightly coupled with the cyclic dephosphorylation of ATP, because the rate of ATP hydrolysis is tightly coupled with flagellar movement and beat frequency (7, 10, 11), and because dynein ATPase not only has a latency (I, 21, 32, 47, 48) but also can be increased in activity by microtubular protein (l, 39) and tubulin (25).The dynein microtubule coupling mediated with ATP has also been suggested by some physiologi-J . CELL . BIOLOGY

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confidence: 73%

Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Okuno¹

1980

The Journal of Cell Biology

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“…The contractile action of sodium vanadate on muscle proteins can hardly be considered, since it has been reported that vanadate inhibits myofibrilar ATPase of cardiac muscle (Solaro et al, 1980) and actomyosin ATPase of skeletal muscle (Kobayashi et al, 1978). It has also been reported that sodium vanadate caused the relaxation of skinned tracheal smooth muscle contracted in the presence of Ca (Nayler & Sparrow, 1983).…”

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confidence: 99%

Effects of soldium vanadate on the smooth muscle of the guinea-pig vas deferens.

Sunano

Shimada

Shimamura

1985

Jpn. J. Smooth Muscle Res.

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“…©The Rockefeller University Press -0021-9525/82/03/0733/09 $1 .00 (12,16), were reasonably accommodated by a model that interpreted flagellar relaxation as corresponding to the first step of the mechanochemical cross-bridge cycle of dynein (24). This mechanochemical interpretation of relaxation appears to have remained prevalent despite recent reports by several groups that AMP-PNP does not effect flagellar relaxation (19,20,22,25) .…”

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confidence: 91%

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.

Penningroth¹,

Cheung²,

Olehnik³

et al. 1982

The Journal of Cell Biology

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The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations, was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin ( Lytechinus pictus) spermatozoa into ATP-free buffer . Relaxation in response to nucleotide was quantitated by measuring G, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation . It was found that (a) the rate of flagellar relaxation induced by 4 x 10 -6 M ATP was inhibited 80% by vanadate concentrations of 3 x 10 -5 M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and E-ATP), were also capable of effecting relaxation ; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein arm in the course of flagellar relaxation ; and (d) the ratio of the rate of relaxation to the rate of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP concentration was raised from 2 x 10 -6 to 9 x 10 -6 M. It is concluded that (a) flagellar relaxation depends on ATP hydrolysis ; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein ; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon . A general model of relaxation is proposed based on active microtubule sliding.The relaxation (straightening) of rigor-wave sea urchin sperm flagella is currently favored as a model system for investigating the mechanochemical cycle of flagellar motility (18,19,23,24). This is due to the view that relaxation results from the release of dynein cross-bridges in the flagellar axoneme in response to ATP binding . A number of observations have contributed to this view . Dynein arms were shown to be in the cross-bridged configuration in rigor-wave flagella (11), and this configuration correlated with mechanical indices of flagellar rigidity (10, 11) and stiffness (18). Addition of micromolar concentrations of ATP induced rigor-wave flagella to relax (10). Straightening was accompanied by a 15-fold decrease in flagellar stiffness (18); images of dynein cross-bridges were not reported in relaxed flagella (11). The nonhydrolyzable ATP analogue, adenyl-5'-yl imidodiphosphate (AMP-PNP), was reported to act as a competitive inhibitor of the beat frequency of reactivated sea urchin sperm (19,23) and to effect the relaxation of flagellar rigor waves (23). This and other reports that AMP-PNP also caused the dissociation of dynein arms from isolated outer-doublet microtubules (26), and that relaxation was not blocked (24) (12, 16), were reasonably accommodated by a model that interpreted flagellar relaxation as corresponding to ...

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Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility

Cited by 160 publications

References 22 publications

Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Effects of soldium vanadate on the smooth muscle of the guinea-pig vas deferens.

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.

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