ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog.

Penningroth, Stephen; Olehnik, K; Cheung, Anne H.

doi:10.1016/s0021-9258(18)43425-4

Cited by 43 publications

(12 citation statements)

References 16 publications

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“…All remaining salts were from Mallinkrodt Inc. (St. Louis, MO). To eliminate contaminating ATP (34), AMP-PNP was treated with apyrase (10 U/ml) for I h before use. Taxol was kindly provided by Dr. M.…”

Section: Reagentsmentioning

confidence: 99%

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond.

Moss¹,

Gatti²,

Witman³

1992

The Journal of Cell Biology

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Abstract. We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its/3/IC1 subunit. Microtubules glided on glassabsorbed sea urchin dynein for a period of time directly proportional to the initial MgATt n-concentration and then detached when 70-95 % of the MgATP 2-was hydrolyzed. Detachment resulted from MgATP 2-depletion, because (a) perfusion with fresh buffer containing MgATP 2-reconstituted binding and gliding, (b) microtubules glided many minutes with an ATPregenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the/~/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP 2-was exhausted. Cosedimentation assays showed that/ktrahymena outer arm dynein sedimented with microtubules in an ATPsensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the/3/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATE Thus, this subunit, while capable of generating motility, lacks both structural and rigortype microtubule binding.

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Section: Reagentsmentioning

confidence: 99%

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond.

Moss¹,

Gatti²,

Witman³

1992

The Journal of Cell Biology

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“…Recent evidence also suggests that the binding ofAMP-PNP to acto-S 1 and myofibrils in solution is weak, again with reported Kd values lying in the 1-5-mM range (Johnson, 1986;Biosca et al, 1986;Sleep and Glyn, 1986). It is not clear whether the wide disparity of results with AMP-PNP has been due to the difficulty in measuring moderately weak binding or whether it is due partly to impurities in commercially available AMP-PNP (Penningroth et al ., 1980;Johnson, 1986). However, we feel that the recent evidence suggests that AMP-PNP and PP; bind with about the same strength in fibers and solution, and that this binding is weak.…”

Section: Nucleotide and Analogue Binding In Fibers And In Solutionmentioning

confidence: 99%

ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants.

Schoenberg¹,

Eisenberg²

1987

The Journal of General Physiology

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We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Crossbridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site . First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached . Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PP;) do, it was possible, by using ADP as a competitive inhibitor of PP ; or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges . We found that adding 175 AM ADP to 4 mM PP; or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM . This suggests that ADP is binding to attached cross-bridges with a dissociation constant of -60 AM . This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-Sl) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-Sl in solution (Johnson, R. E., and P. H. Adams. 1984 .

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“…1 chromatography paper, 20 cm x 15 cm) was attached to the top of a PEI-cellulose thin-layer sheet to ensure continuous solvent flow, and the chromatogram was developed overnight. ATP was resolved from AMP-PNP by development in 1 .2 M LiCl (22) . Nucleotides were visualized under ultraviolet light, and contaminant levels were estimated.…”

Section: Analysis Of Nucleotidesmentioning

confidence: 99%

“…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) by the potent dynein ATPase inhibitor vanadate THE JOURNAL OF CELL BIOLOGY " VOLUME 92 MARCH 1982 733-741 ©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) .…”

mentioning

confidence: 99%

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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ATP formation from adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analog.

Cited by 43 publications

References 16 publications

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond.

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond.

ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants.

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

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