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At least 19 recordsLinked to original sources
PubMed1977

Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.

The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly.

Adenosine Triphosphatases↗
PubMed1986

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus.

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein (Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high Mr polypeptides of 20 S dynein. This antiserum reacted in "Western" immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.

Adenosine Triphosphatases↗
PubMed1985

Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms.

Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24-32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.

Adenosine Triphosphatases↗
PubMed1985

Alloaffinity filtration: a general approach to the purification of dynein and dynein-like molecules.

Alloaffinity filtration simply and specifically separates certain axonemal dyneins and dynein arm components from crude mixtures on the basis of their ability to bind and decorate Tetrahymena axonemal microtubules on a filter in the absence of ATP and to detach and pass into the eluate when 0.5 mM ATP is added. The procedure, which may be performed repetitively, is successful in purifying a Tetrahymena dynein that has characteristics of 30 S dynein prepared by conventional methods, while other dyneins originally present in the mixture, e.g., 14 S Tetrahymena dynein, are not found in the ATP eluate. A relatively homogeneous population of dynein oligomers is obtained. Alloaffinity-purified 30 S Tetrahymena dynein consists of heavy-, intermediate-, and light-chain polypeptides that cosediment in a sucrose gradient in fixed molar ratios and that have structural features of in situ Tetrahymena arms. Dyneins from other species will bind to Tetrahymena microtubules and can be purified by this method. Alloaffinity-purified Chlamydomonas dynein is a set of polypeptides including the four heavy chains that characterize the outer arm.

Adenosine Triphosphatases↗
PubMed1987

Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles.

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.

Adenosine Triphosphatases↗
PubMed1977

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.

Adenosine Triphosphatases↗
PubMed1992

Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function.

In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.

Amino Acid Sequence↗
PubMed1975

Preparation of antiserum against a tryptic fragment (fragment A) of dynein and an immunological approach to the subunit composition of dynein.

An improved method for purifying the tryptic fragment (Fragment A) of flagellar ATPase (dynein) from sea urchin spermatozoa is described. The preparation appears homogeneous as judged by ultracentrifugation, electrophoresis on polyacrylamide gels, and immunological techniques. The molecular weight of undenatured Fragment A was determined to be 400,000 and 370,000 by the two methods of disc electrophoresis on polyacrylamide gel and sedimentation equilibrium, respectively. The fragment dissociated into two principal polypeptide chains with molecular weights of 190,000 and 135,000 when heated in the presence of sodium dodecyl sulfate. Antiserum against dynein was prepared in rabbits using purified Fragment A from the sea urchin Anthocidaris crassispina as an antigen. The specificity of this serum toward Fragment A and toward dynein was determined by double diffusion in agarose, by inhibition of ATPase activity, and by sodium dodecyl sulfate-electrophoresis of the antigen-antibody complex. This antiserum also reacted with the enzymes from two other species of sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Analysis of the precipitated antigen-antibody complex showed that the antiserum reacted specifically with the "high molecular weight" polypeptide seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude dynein fractions. This finding supports previous reports that this band derives from dynein ATPase. In our preparations, this "high molecular weight" dynein band appeared single.

Adenosine Triphosphatases↗
PubMed1987

Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function.

Mutants with outer dynein arm defects or deficiencies all show a major reduction in beat frequency to about half the normal value; some of these mutants show an additional decrease in sliding velocity associated with reduced shear amplitude and an additional reduction in beat frequency, as well as other more minor modifications of the normal forward mode bending pattern. New mutants (ida98, pf30), which appear to be deficient in a subset of inner dynein arms show a reduction in sliding velocity that is primarily associated with a reduction in shear amplitude, with only a small reduction in beat frequency. These differences in motility phenotype between inner and outer dynein arm mutants suggest that inner and outer dynein arms may have distinct functions. The relatively large decrease in sliding velocity associated with partial loss of inner arms is consistent with earlier observations on pf23, a nonmotile mutant lacking inner arms, suggesting that inner arms may have an essential function in motility. The ability to generate reverse mode bending patterns is retained in some inner or outer dynein arm mutants, but appears to be decreased in those mutants which show reduced shear amplitude for the forward mode bending pattern.

Adenosine Triphosphatases↗
PubMed1989

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.

Adenosine Triphosphatases↗
PubMed1979

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.

Adenosine Triphosphatases↗
PubMed1987

Melanoma dynein: evidence that dynein is a general "motor" for microtubule-associated cell motilities.

Platyfish-swordtail hybrid melanoma cells exhibit pigment aggregation in response to adrenergic stimulation or melanophore-concentrating hormone. This translocation of pigment granules is thought to be related to radially arrayed microtubules. Very little is known about the molecular "motor" that powers the translocation. We present evidence that dynein is located on these microtubules and is a candidate for the "motor". Vanadate and erythro-9-[3-(2-hydroxynonyl)]adenine, which are potent inhibitors of dynein ATPase, prevent the transport of melanosome granules in Brij-treated melanoma cells. Direct identification of dynein in melanoma cells and tissues is demonstrated by immunofluorescence microscopy and immunoblotting using anti-fragment A (tryptic fragment of sea urchin sperm dynein) serum. The cytoplasm of melanoma cells is stained with the antiserum and gives rise to a pattern similar to the distribution of microtubules. Western blotting shows that the molecular weight of an immunoreactive polypeptide in melanoma tissues coincides with that of the heavy chain of sea urchin sperm dynein.

Adenosine Triphosphatases↗
PubMed1985

Pathway of the microtubule-dynein ATPase and the structure of dynein: a comparison with actomyosin.

Dynein and myosin show several important similarities in design as well as some interesting differences in detail. Both ATPases function as crossbridges that undergo microscopic movements to drive the sliding of filaments, which results in macroscopic movements. They share a common design employing globular heads attached to flexible strands. Each head contains one ATP-binding site and one filament-binding site, and the binding of ATP induces an extremely rapid dissociation of the crossbridge-filament "rigor" complex. Following ATP hydrolysis, which is readily reversible, the crossbridge reassociates with the filament and returns to its original state with the release of products. Thus, the nucleotide-induced changes in conformation are effectively used to couple the hydrolysis of ATP to the dissociation and reassociation of the crossbridge in order to produce a force for net movement according to the Lymn-Taylor-Eisenberg model. The utilization of nucleotide-binding energy to induce a change in conformation can be rationalized in terms of our understanding of enzyme catalysis in general, whereby substrate binding energy is used to induce a change in conformation that stabilizes the transition state for catalysis. In these crossbridge ATPases, the substrate-induced change in conformation also serves to weaken the crossbridge-filament interaction. The pathway is symmetrical, with a return to the tight (filament) binding state coupled to product release. The ball on a string design may provide a reasonable basis to explain how a unidirectional force is obtained from a symmetrical cycle; opposite changes in conformation with the binding and release of the nucleotide produce a significant force only when pulling on the flexible strand. Moreover, the very rapid dissociation of the crossbridge following ATP binding limits the time that a negative force is in effect and also prevents a rigor crossbridge from retarding the sliding movements generated by other crossbridges. Myosin and dynein exhibit nearly identical kinetic constants governing ATP binding and the ATP-induced dissociation of the crossbridge. These appear as invariant steps that may reflect the basic principles of enzyme catalysis as applied to the mechanochemical cycle. The rates of ATP hydrolysis and synthesis by myosin and dynein differ slightly, but in each case the reactions are readily reversible with an equilibrium constant less than one. Steps involving the loss and rebinding of products occur at rates two to three orders of magnitude faster for dynein than for myosin.(ABSTRACT TRUNCATED AT 400 WORDS)

Actomyosin↗
PubMed1985

Binding of Tetrahymena dynein to axonemes of Marsilea vestita lacking the outer dynein arm.

Axonemes from the heterosporous water fern Marsilea vestita were fixed in the presence of tannic acid and examined by thin-section electron microscopy. Transverse sections revealed the normal 9+2 configuration except for the absence of the outer of the two dynein arms. Both arms were normally preserved in parallel preparations of Chlamydomonas axonemes. Isolated dynein from the ciliated protozoon Tetrahymena bound to Marsilea axonemes at the site normally occupied by the outer arm. Dynein binding was partially reversed by ATP as judged by both electron microscopy and polyacrylamide gel electrophoresis. This system should provide a valuable insight into the biochemistry and function of the inner dynein arm and the relationship of the two arms to motility in more conventionally equipped axonemes.

Adenosine Triphosphatases↗
PubMed1986

Kinetic properties of microtubule-activated 13 S and 21 S dynein ATPases. Evidence for allosteric behaviour associated with the inner row and outer row dynein arms.

The 13 S and 21 S dynein ATPases from Tetrahymena cilia rebind to extracted doublet microtubules as inner row and outer row arms. Rebinding is accompanied by four- to ninefold activation of the ATPase activity. The soluble (microtubule-free) forms of the two dyneins exhibit simple saturation kinetics (h = 1.0) with Vmax much less than mumol Pi mg-1 min-1 and Km = 20-40 microM-ATP. Mixing a fixed quantity of free dynein with increasing concentrations of extracted doublets results in systematic increases in all three kinetic parameters for each dynein. At infinite concentrations of doublets and ATP, each enzyme undergoes a significant shift to sigmoid saturation kinetics (h = 2-3), Vmax increases to a turnover rate of about 90 mol ATP per mol Es-1 and the Michaelis constant increases to much greater than 100 microM-ATP. These data suggest that both enzymes are allosteric and can be interpreted in terms of positive cooperativity relative to a minimum of two or three interacting sites. It is less clear whether this cooperativity is related to subunit interactions within the 21 S or 13 S particles, or to subunit interactions between adjacent particles (arms) on the microtubule lattice.

Adenosine Triphosphatases↗
PubMed1987

Chymotryptic digestion of Tetrahymena ciliary dynein. II. Pathway of the degradation of 22S dynein heavy chains.

As shown in the preceding paper (Toyoshima, Y. Y., 1987, J. Cell Biol., 105:887-895) three-headed Tetrahymena 22S dynein consists of three heavy chains (HCs) and is decomposed into two-headed (H) and one-headed (L) fragments by chymotryptic digestion. To accurately determine the presence of multiple ATPases and ultimately the location of various domains, it is necessary to determine the identity of each HC fragment relative to the original HCs in 22S dynein. The degradation pathway of each HC was determined by peptide mapping and immunoblotting. The three HCs (A alpha, A beta, and A gamma) were immunologically different; although SDS-urea gel electrophoresis showed that A gamma HC was apparently resistant to the digestion, actually three distinct HCs contributed to the same band alternately. H fragment was derived from A beta and A gamma HCs, whereas L fragment originated from A alpha HC. Since both fragments were associated with ATPase activity, these results directly demonstrate the presence of multiple ATPase sites in Tetrahymena 22S dynein.

Adenosine Triphosphatases↗
PubMed1992

Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein.

We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.

Adenosine Triphosphate↗
PubMed1992

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.

Actins↗