Kinetic analysis of axoneme and dynein ATPase from sea urchin sperm

The behavior of the ATPase of axoneme (detergent-treated flagellum) and dynein from sea urchin sperm was investigated. The activation of the ATPase by divalent cations was attributed to formation of a complex of ATP and the divalent cation; the metal-ATP complex is an effective substrate. However, free ATP is a modifier of the ATPase. Free ATP markedly changes the affinity of the metal-ATP complex to the enzymes. Calcium-activated ATPase activity of axoneme decreased at high concentration of CaCl₂, but that of dynein did not decrease.     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP.

Sea urchin sperm flagellar ATPase (EC 3.6.1.3) has magnesium-ATP as an effective substrate and is inhibited by free ATP. The inhibition is prevented by high concentration of KCl or NaCl. 0.4 M KCl extracts 48% of ATPase activity from axoneme. The 0.4 M KCl extract and 0.4 M KCl-treated axoneme are also inhibited by free ATP and this inhibition is reversed by KCl. Dynein purified twice by sucrose density gradient centrifugation is also inhibited by free ATP; this inhibition is also reversed by KCl.     

Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP

Sea urchin sperm flagellar ATPase (EC 3.6.1.3) has magnesium-ATP as an effective substrate and is inhibited by free ATP. The inhibition is prevented by high concentration of KCl or NaCl. 0.4 M KCl extracts 48% of ATPase activity from axoneme. The 0.4 M KCl extract and 0.4 M KCl-treated axoneme are also inhibited by free ATP and this inhibition is reversed by KCl. Dynein purified twice by sucrose density gradient centrifugation is alsof inhibited by free ATP; this inhibition is also reversed by KCl.     

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.     

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     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

Polypeptide subunits of dynein 1 from sea urchin sperm flagella

A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000–350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12–14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000–122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000–24,000) cosediment with the 21 S peak. The heavy chain composition of the 12–14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.     

Inhibition by palmitoyl CoA of dynein ATPase from sea urchin spermatozoa

ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 microns, and was stimulated at concentrations between 2 microns and 10 microns. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1 mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF1 ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.     

Ciliary dynein from sea urchin embryos.

Axonemal dynein ATPase [EC 3.6.1.3] was extracted from cilia of sea urchin embryos for a study of its enzymatic properties. Sedimentation analysis on a sucrose density gradient revealed that ATPase activity was associated with the 12S particles. The partially purified 12S enzyme was characterized mainly with regard to the optimum pH, divalent cation and ionic strength requirments and substrate specificity. Comparative investigation of the data obtained indicates that the properties of the present dyneine ATPase resemble those of other dynein(-like) ATPase hitherto reported. In addition, the possible relationship among dyneins within a single species, in particular between the ciliary dynein and cytoplasmic dynein-like ATPase, is discussed.     

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.     

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

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".     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of mammalian dynein using antibodies against a polypeptides of sea urchin sperm flagellar dynein

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.     

Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs.

1. Dynein was extracted with 0.5 M KCl from Tetrahymena axonemes. SDS-gel electrophoresis of the extract indicated that about 50% of the extracted protein had a molecular weight of about 3.5 X 10(5), and that 90% of the proteins with this weight had been extracted. 2. The ATPase [EC 3.6.1.3] reaction of the KCl-extracted dynein fraction was enhanced by 60-80% by addition of the outer doublet fraction. It showed an initial burst of Pi liberation of about 1 mol per mol of proteins with a molecular weight of 3.5 X 10(5). 3. We examined the interaction of the dynein-tubulin system from Tetrahymena cilia with ten ATP analogs [2'-dATP, 3'-dATP, epsilonATP, FTP, 8-NH(CH3)-ATP, 8,3'-S-cyclo-ATP, 8-Br-ATP, 8-OCH3-ATP, 8-SCH3-ATP, and AMPPNP]. Among them, 2'-dATP and 3'-dATP were good substrates for dynein ATPase, as they induced the dissociation of dynein arms from the B-tubule of outer doublets, the sliding movement between outer doublets, and the bending movement of axonemes. The other analogs did not induce the dissociation or the sliding movement. 4. Among the ATP analogs tested, only 2'-dATP and 3'-dATP induced the reorientation of cilia on the Triton model of Tetrahymena; the reorientation rates were smaller than that induced by ATP.