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.     

Effects of sulfhydryl reagents on the ATPase activity of solubilized 14S and 30S dyneins and on whole ciliary axonemes as a function of pH

The effects of five sulfhydryl (SH) reagents – N-ethylmaleimide (NEM), a spin-labeled maleimide (SLM), N-N′-phenylenedimaleimide (PPDM), bis(4-fluoro-3-nitrophenyl)sulfone (FNS), and carboxypyridine disulfide (CPDS) – on glycerol-treated, Triton X-100-demembranated ciliary axonemes of Tetrahymena, on the 30S and 14S dyneins extracted from such axonemes, and on the residual ATPase activity remaining associated with axonemes that have been extracted twice with Tris-EDTA have been examined as a function of pH in the range 6.9–8.6. Preincubation of axonemes and of solubilized 30S dynein with low concentrations of each of the five SH reagents, at 0°C and at 25°C, caused enhancement of the latent ATPase activity. PPDM was the most effective reagent, causing half-maximal enhancement (after 18 h at 0°C) at ～ 0.5 μM, corresponding to 0.19 moles/10⁵ g axonemal protein. The rate constants, k_a, for the enhancement reaction at 0°C depended on whether the 30S dynein was in situ or solubilized; the ratio k_a (in situ) /k_a (solubilized) was > 1 for NEM, ～ 1 for PPDM, and < 1 for FNS. For each SH reagent except CPDS, k_a (at 0°C) increased markedly with increasing pH in the range pH 6.9–8.6; for CPDS k_a increased only about fourfold. At long times of preincubation and high concentrations of NEM, SLM, PPDM, and CPDS, the enhancement of ATPase activity was followed by a loss of activity. The values of k_L, the rate constants for loss of ATPase activity from the peak enhanced level, were much lower than the corresponding values for k_a, and increased with increasing pH. With SLM and PPDM, inhibition continued until the ATPase activity was almost completely inhibited. With NEM, however, the initial rate of loss from the peak enhanced value decreased as the ATPase activity returned toward the control (unmodified) level, and further inhibition was very slow. The differences in degree of inhibition obtained with SLM as compared to NEM suggest that there are at least two classes of inhibitory SH groups on 30S dynein. The ATPase activity of 14S dynein was only inhibited by preincubation with NEM, SLM, PPDM, and, to a lesser extent, CPDS; k_L increased with increasing pH. Preincubation of 14S dynein with FNS yielded conflicting results when the reaction was “stopped” by adding dithiothreitol. When 14S dynein was preincubated at 0 C with FNS and the ATPase activity was then assayed at 25°C, a biphasic pattern of enhancement followed by inhibition was obtained. The residual ATPase activity of twice-extracted axomenes was relatively insensitive to each of the SH reagents studied; an initial rapid loss of some 20–40% of the ATPase activity occurred, followed by a very slow further loss of activity. Increasing the pH increased this slow rate of inhibition. The residual ATPase activity of unmodified twice-extracted axonemes decreased slightly with increasing pH, in contrast to the slight increase observed with increasing pH for the ATPase activity of axonemes and of solubilized 30S and 14S dyneins. The presence of ATP during preincubation of axonemes with PPDM at O°C prevented the enhancement of ATPase activity; only a slow loss of ATPase activity was observed. This rate of loss of ATPase activity was slower than the rate of loss observed (after peak enhancement of activity was reached) when PPDM reacted with axonemes in the absence of ATP. In these properties the SH groups of 30s dynein responsible for the enhancement of latent ATPase activity and for the inhibition of ATPase activity do not resemble the SH1 and SH2 groups of myosin, respectively, since the presence of ATP increases the rates of reaction of SH1 and SH₂ of myosin with SH reagents.     

A high-affinity ATP-binding site on 30S dynein

The enhancing effect of low concentrations (eg, 8 μM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1–3 μM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while β,γ-methylene-adenosine triphosphate (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of a high-affinity ATP-binding site on 30S dynein.     

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.     

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.     

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.     

On the role of sulfhydryl groups in the ATPase activity and pellet height response of Tetrahymena cilia

The effects of N-ethylmaleimide and p-hydroxymercuribenzoate on the ATPase activity of glycerinated Tetrahymena cilia, of 30 S dynein extracted from the cilia, and on the residual ATPase remaining after extraction were studied and correlated with the effects of these reagents on the pellet height response of these cilia. Simultaneous addition of N-ethylmaleimide and ATP to cilia caused a slight inhibition of ATPase activity. Preincubation of the cilia with low N-ethylmaleimide in the absence of ATP, however, enhanced the ATPase activity; the enhancement decreased with increasing time of preincubation. Preincubation of cilia with high N-ethylmaleimide caused increasing inhibition. p-Hydroxymercuribenzoate was more potent than N-ethylmaleimide, usually causing only an inhibition which increased if the cilia were preincubated with p-hydroxymercuribenzoate in the absence of ATP.The pellet height response of these cilia, which serves as a convenient assay of some events related to ciliary motility, was inhibited about 50% by high concentrations of N-ethylmaleimide in the presence of ATP. Preincubation of the cilia with low concentrations of N-ethylmaleimide led to complete loss of the pellet height response. p-Hydroxymercuribenzoate was a more potent inhibitor of the pellet height response than N-ethylmaleimide; complete inhibition was attained even in the presence of ATP, while preincubation with a low concentration of p-hydroxymercuri-benzoate caused a very rapid loss of pellet height response.The ATPase activity of the crude dynein obtained by extraction of cilia and removal of the axonemes was approximately doubled by preincubation with N-ethylmaleimide. 30 S Dynein, obtained from the crude dynein by sedimentation on a sucrose density gradient, was slightly inhibited by N-ethylmaleimide; p-hydroxy-mercuribenzoate was more potent. The residual ATPase activity remaining on the axonemes after two extractions was also only inhibited by N-ethylmaleimide and by p-hydroxymercuribenzoate.These results demonstrated that SH groups influence both the ATPase activity of dynein and the pellet height response of glycerinated cilia. The possible significance of the similarity in enhancing effect of N-ethylmaleimide on cilia ATPase and on myosin ATPase was 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.     

Effects of Organic Solvents on the Coupling Between ATP Hydrolysis and Sliding of Microtubules in Axonemes from Chlamydomonas Flagella

ABSTRACT. The effects of organic solvents on the ATPase activity and the sliding disintegration of axonemes from Chlamydomonas were investigated. The axonemal ATPase was markedly activated by methanol accompanying with marked inhibition of the sliding disintegration of axonemes. On the contrary, glycerol inhibited the ATPase activity without serious inhibition of the sliding disintegration. As far as the axonemes are not irreversibly denatured by extremely high concentration of solvents, the effects of solvents both on the ATPase and the ability of sliding are reversible. Therefore, the inhibition of sliding accompanied by the activation of ATPase is probably due to an inability to couple the hydrolysis of ATP to sliding between dynein and microtubule in the presence of methanol. The axonemal ATPase was less sensitive to vanadate inhibition after exposure to methanol. This indicates that methanol makes the dyneinADP.Pi complex unstable and increases product release. On the other hand, glycerol and ethylene glycol seem to stabilize the force generation responsible for the sliding through stabilizing the dynein.ADP.Pi complex.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.     

Biochemical Analysis of a Mutant Tetrahymena Lacking Outer Dynein Arms

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms. Electrophoretic analyses of axonemes isolated from nonmotile mutants ( oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild-type cells (wild-type axonemes) contain. The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild-type levels of ATPase activity. Two-dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein. This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein.     

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.     

Strikingly low ATPase activities in flagellar axonemes of a Chlamydomonas mutant missing outer dynein arms

The ATPase activities in Chlamydomonas axonemes were compared between wild type and a mutant (oda) that lacks entire outer dynein arms, at various ionic strengths and pH values, and in the presence of different concentrations of high-molecular-mass dextran. Over a 0-0.2 M KCl concentration range, the ATPase activity of oda axonemes was found to be 5-12 times lower than that of the wild-type axonemes. The low activity in oda is surprising since outer arm-depleted axonemes of sea urchin sperm have been reported to retain about 50% of the normal activity. In both wild type and oda, the ATPase activity of dynein was higher when contained within the axoneme than when released from it with 0.6 M KCl. The ATPase activation within the wild-type axoneme was inhibited by high ionic strengths or by the presence of dextran. The activation in oda axonemes, on the other hand, was not inhibited by these factors. These significantly different ATPase properties suggest that the inner and outer dynein arms perform somewhat different functions in this organism.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

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.     

Identification of an ATPase activity associated with the high molecular weight proteins of the human spermatozoon axonemes

The high salt extract obtained from demembranated human spermatozoa contains high molecular weight proteins. These proteins are associated with an ATPase activity inhibited by sodium orthovanadate. In association with lower molecular weight proteins, they constitute a 20 S particle and are probably localized in the dynein arms (and in the radial spokes) of the human spermatozoon axonemes. Evidence is shown for a biochemical analogy between the dynein ATPases extracted from the invertebrate axonemes and the human dynein-like ATPase described in this study.     

A comparison of the effects of gentle heating,acetone, and the sulfhydryl reagent bis (4-fluoro-3-nitrophenyl) sulfone on the ATPase activity and pellet height response of tetrahymena cilia

Incubation of glycerol-extracted, Triton X-100 demembranated Tetrahymena cilia with 2–10 vol % acetone caused an enhancement of ATPase activity by 2- to 3- fold, depending on concentration and time of incubation. Axonemal ATPase activity was also increased upon incubation with bis (4-fluoro-3-nitrophenyl) sulfone (FNS). Acetone and FNS enhanced the activity of solubilized 30S dynein, but slightly inhibited that of 14S dynein. Heating at 38°C, incubation with FNS, and incubation with acetone activated axonemal ATPase to the same extent. Subsequent studies of (1) the effect of time of preincubation with a spin-labeled maleimide (SLM) at 25°C as a function of pH on the ATPase activity, (2) the concentration dependence of the inhibition of ATPase activity by N-ethylmaleimide or SLM, (3) the ratio of ATPase activity assayed at 25°C to that assayed at 0°C, and (4) the ratio of ATPase activity at pH 8.6 to that at pH 6.9 did not reveal any difference in the properties of the axonemal ATPase after near maximal enhancement by the heat, acetone, or FNS treatments. It was concluded that enhancement of ATPase activity by gentle heat treatment, by incubation with acetone (or other organic solvents), or by FNS results from a conformation change of 30S dynein. The effect of acetone and of FNS on the pellet height response (a measure of the increase in height of the pellet of cilia precipitated by brief centrifugation in the presence of ATP as compared to the absence of ATP) was also determined. Enhancement of ATPase by these reagents did not lead to a decrease in pellet height response. This observation, in conjunction with other data, indicates that there are at least 3 states of the cross-bridge cycle of dynein arms in cilia.     

Some properties of bound and soluble dynein from sea urchin sperm flagella

Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 x 10¹⁵ g protein per µm of their length. Treatment of the axonemes with 0 5 M KCl for 30 min extracted 50–70% of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doublet tubules. Almost all of the dynein (85–95%) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12–14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KCl- and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KCl, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtained in the absence of KCl, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K_m and V_max increased with increasing concentrations of KCl up to 0.5 M When bound dynein was assayed under conditions that would induce motility in reactivated sperm (0 15 M KCl with Mg⁺⁺ activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynein, may result from its interactions with tubulin and other axonemal proteins     

Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms

Milligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37 degrees C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300-330 K molecular weight region, as well as tubulin at 51-54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross-section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three- to four-fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5-30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.     

Rhamnolipid from Pseudomonas aeruginosa inactivates mammalian tracheal ciliary axonemes

Isolated ciliary axonemes from pig trachea were exposed to increasing concentrations of purified Pseudomonas aeruginosa rhamnolipid. This is a defined ciliary system allowing observation of direct impairment of functional axonemes. Axonemal motility and ATPase activity were decreased in proportion to rhamnolipid concentrations. ATPase-associated proteins observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dynein arms seen in ultra-structural cross sections progressively disappeared from axonemes with exposure to rhamnolipid. These four independent measures establish that the rhamnolipid removes the ATPase-containing outer dynein arms from the ciliary axoneme, thereby rendering the axoneme immotile.