Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity.

The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1-2 mM for MgCl2 and 2 mM for CaCl2. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 degrees C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.     

Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity

The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1?2 mM for MgCl₂ and 2 mM for CaCl₂. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 °C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than in the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.     

Recombination of ciliary dynein of Tetrahymena with the outer fibers.

Recombination of ciliary dyneins of Tetrahymena pyriformis with the outer fibers was investigated using turbidimetry, co-sedimentation analysis and electron microscopy. As reported by Gibbons, 30S dynein could recombine with the outer fibers, while 14S dynein did to so a lesser extent. At acidic pH, however, most of the 14S dynein was also rebound to the outer fibers. When an excess of crude dynein fraction was added to the outer fiber fraction at pH 8.2, electron microscopic observations showed that the outer doublet microtubules were decorated not only with arms but also with other electron-dense materials. On the other hand, when crude dynein fraction was mixed with the outer fibers in an appropriate quantity, only arms were reconstituted at the regular positions of A-subfibers. ATP had an inhibitory effect on the recombination of dynein with the outer fibers.     

Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.     

A biochemical study of flagellar dynein from starfish spermatozoa: protein components of the arm structure.

Half of the adenosine triphosphatase (dynein) activity of starfish sperm tail axonemes was extracted with 0.6 m KCl-10 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (KCl-EDTA), while with 1 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (Tris-EDTA) around 90% of the activity was extracted. The main adenosine triphosphatase (ATPase) in the KCl-EDTA extract had a sedimentation coefficient of 20S and that in the Tris-EDTA extract had a sedimentation coefficient of 12S. The effects of divalent cations, pH, and an SH-blocking reagent and the K_m for ATP were different for the activities of the two forms of dynein ATPase. These two forms of dynein can interconvert to some extent when the ionic strength of the medium is changed. In a medium suitable for recombination of dynein to outer doublet microtubules (recombination buffer, 20 mm Tris-HCl (pH 7.6)-2 mm MgCl₂-0.5 mm dithiothreitol), the 20S ATPase converted to a 24S ATPase. Recombination of the ATPase activity from the KCl-EDTA extract was almost complete while that from the Tris-EDTA extract was around 50%. Outer arms disappeared preferentially by the treatment with KCl-EDTA, and the extracted arms could be reconstituted in the recombination buffer. In the case of the Tris-EDTA extraction, both the outer and inner arms disappeared and the reconstitution of the arms could not be confirmed. From the above results it can be considered that the 20 or 24S dynein represented the arm structure. The 20 or 24S ATPase fraction contained two large polypeptide chains as main components having electrophoretic mobilities in the presence of sodium dodecylsulfate similar to those of Tetrahymena ciliary dyneins and of sea urchin sperm flagellar dyneins. The relationship between these chains and dynein subunits is discussed.     

Effects of adenosine triphosphate on N-ethylmaleimide-induced modification of 30S dynein from Tetrahymena cilia.

Ciliary 30S dynein of Tetrahymena was investigated with regard to modification of the ATPase activity with N-ethylmaleimide (NEM) in the presence of ATP. The elevation of enzyme activity due to the modification was largely repressed by addition of ATP at a concentration of 1 mM or more during preincubation of 20 h at 0 degrees C. The repression was highly specific for ATP, though ADP and AMPPNP showed slight repressive effects. After complete hydrolysis of ATP added to the preincubation mixture, however, elevation of 30S dynein ATPase activity occurred. It is suggested that the repression by ATP of NEM-induced elevation of 30S dynein ATPase activity is simply due to a protecting effect of ATP on certain SH group(s) (probably SH1-type group(s)) around the active center of 30S dynein. When 30S dynein was maximally activated by modification with NEM, ATP or ADP did not significantly promote the inactivation of the modified enzyme upon further treatment with NEM, indicating that 30S dynein lacks the characteristics of SH2-type groups. On the other hand, ATP also showed a protective effect against inhibition of native 30S dynein by high concentrations of NEM. High concentrations of ADP and AMPPNP were inhibitory to 30S dynein ATPase activity but inorganic phosphate did not inhibit 14S or 30S dynein ATPase activities at all.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases

Cilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5-min extraction was generally insensitive to added calmodulin, whereas that obtained by 20-hr extraction of the 5-min extracted axonemes was activated by calmodulin, the activation being much larger in the “light” 14S fractions than in the “heavy” fractions. The 30S dynein ATPase obtained by a 5-min extraction was generally activated over 1.6-fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3-fold. After further purification of the 5-min extracted 30S dynein and of the 5-min to 20-hr-extracted 14S dynein on DEAE-Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro-9-[3-(2-hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms mat are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.     

Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes.

The effects of N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide(SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis have been studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. Electron spin resonance spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased twofold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. The results suggest that ATP causes a conformation change affecting a sulfhydryl group(s) involved in the mechanochemical system. It was also shown that beta,gamma-methylene ATP(AMP-PCP) is an inhibitor of dynein ATPase. This analogue of ATP is not hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+, the inhibition of 30S dynein is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. These results support the view that at least 2 SH groups are involved in ciliary motility and that their reactivity to SH reagents depends on whether the dyneins are in situ or have been extracted.     

Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules

Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.     

Interrelationships between thermal-, N-ethylmaleimide-, and Ca2+-calmodulin-mediated activation/inactivation of dynein ATPase activities

Interactive effects between calmodulin activation of 30 S dynein ATPase activity and activation by heat or N-ethylmaleimide (NEM) have been studied. Addition of calmodulin during the heat treatment caused a larger increment in ATPase activity (above that caused by heating alone) than did addition of calmodulin after the heat treatment. Similar results were obtained in experiments where activation was caused by NEM treatment. For both the heat and NEM treatments, the synergistic effect of calmodulin when present during the treatment was Ca²⁺ dependent although activation of ATPase activity by either treatment alone was not Ca²⁺ dependent. Heating 14 S dynein inhibited its ATPase activity and reduced the effectiveness of calmodulin as an activator. The activating effect of calmodulin added after heat or NEM treatments was about the same as if the calmodulin was present during the treatment, i.e., interactive effects were minimal. Concentrations of NEM that had little effect on the ATPase activity of 14 S dynein largely eliminated the ability of calmodulin to activate its ATPase activity. Chromatography of the heat-treated 14 S dynein on calmodulin-Sepharose 4B indicated that the loss of sensitivity of 14 S dynein ATPase to calmodulin was not due to loss of ability of the dynein to bind to calmodulin. Retention of calmodulin binding ability was also shown for heat-treated 30 S dynein. These results suggest that calmodulin and heat/NEM activate solubilized 30 S dynein ATPase by separate mechanisms which may include a common process.     

AN EFFECT OF ADENOSINE TRIPHOSPHATE ON THE LIGHT SCATTERED BY SUSPENSIONS OF CILIA

Cilia were isolated from Tetrahymena pyriformis by an ethanol-calcium method. Addition of adenosine triphosphate to a suspension of intact or digitonin-extracted cilia caused a decrease of about 20 per cent in turbidity. Study of fractionated cilia showed that the presence of two distinct axonemal components, the outer fibers and the 30S dynein (the axonemal ATPase protein), was necessary for this effect on turbidity to occur. The decrease in turbidity is interpreted as a result of a specific interaction of ATP with these protein components causing an effective increase in hydration. The high nucleotide specificity suggests that the change in hydration is closely related to the processes responsible for motility. The outer fibers themselves swell when suspended in media of very low ionic strength. The concentration of salt needed to prevent this swelling (2 mM MgSO₄ or 30 mM KCl) is about the same as that needed to keep dynein bound to the fibers. The recombination of purified 30S dynein with the outer fibers can be followed by the rise in turbidity resulting from increased dry mass of the particles.     

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.     

Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia

Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Effect of thiourea and substituted thioureas on dynein ATPase and on the turbidity response of tetrahymena cilia

The effects of thioura and of several substituted thioureas–phenylthiourea, α-naphtylthiourea, metiamide, and burimamide–on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of β-mercaptoethanol or dithiotheritol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (～ 1 μM) concentrations of ATP and, less effectively, by AMP-PNP, but not by AMP-PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP-PNP. The thioureas inhibit the ATP-induced decrease in turbidity (measured as ΔA₃₅₀) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of β-mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice extracted axonemes is not changed by treatment with phenylthiourea or metiamide. These observations indicate that the thioureas react with at least two sets of SH or S–S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.     

Stimulation of ATPase activity of 30-S dynein with microtubular protein

Microtubule protein and 30-S dynein (an axonemal ATPase protein) were prepared from cilia of Tetrahymena pyriformis and their mutual interaction was investigated. Microtubular protein stimulated the ATPase activity of 30-S dynein. These observations give some support to models involving the interaction between tubules and dynein arms in ciliary movement.     

Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".