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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Ciliary dynein from sea urchin embryos. 1 78

Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.
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PMID:Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain. 183 Sep 24

Cytoplasmic microtubule-based motility in Paramecium was investigated using video-enhanced contrast microscopy, the quick-freeze, deep-etch technique, and biochemical isolations. Three distinct vesicle populations were found to be transported unidirectionally along the cytopharyngeal microtubular ribbons. This minus-end-directed movement exhibited unique in vivo features in that the vesicle transport was nonsaltatory, rapid, and predominantly along one side of the microtubular ribbons. To identify candidate motor proteins which may participate in vesicle transport, we prepared cytosolic extracts of Paramecium and used bovine brain microtubules as an affinity matrix. These preparations were found to contain a microtubule-stimulated ATPase which supported microtubule gliding in vitro. This protein was verified as a cytoplasmic dynein based upon its relative molecular mass, sedimentation coefficient of 16S, susceptibility to vanadate photocleavage, elevated CTPase/ATPase ratio, and its typical two-headed dynein morphology. This dynein was directly compared with the axonemal dyneins from Paramecium and found to differ by five criteria: morphology, sedimentation coefficient, CTPase/ATPase ratio, vanadate cleavage patterns, and polypeptide composition. The cytoplasmic dynein is therefore not an axonemal dynein precursor, but rather it represents a candidate for supporting the microtubule-based vesicle transport which proceeds along the microtubular ribbons.
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PMID:Vesicle transport along microtubular ribbons and isolation of cytoplasmic dynein from Paramecium. 214 40

A dynein-like ATPase activity has been isolated previously from soluble extracts of unfertilized sea urchin eggs. However, the use of non-quantitative isolation techniques, in particular affinity for microtubules or Ca2+/calmodulin, has precluded accurate estimates of dynein pool size. We have taken the unique approach of using dynein-like ATPase activity to quantitate the egg dynein pool. This approach is based on the isolation by anion-exchange chromatography on DEAE-Sephacel of a peak of dynein-like ATPase activity comprising 65% of soluble ATPase activity in the cytosolic extract. Identification of cytoplasmic dynein was based on dose-dependent inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine and orthovanadate, low GTPase activity and a sedimentation coefficient of 12 S. Two high molecular weight polypeptides corresponding to the A- and D-bands of axonemal dynein were shown to copurify with dynein-like ATPase activity and to undergo specific photocrosslinking with [alpha-32P]ATP, suggesting that they were egg dynein catalytic polypeptides. The specific ATPase activity of these putative catalytic polypeptides was determined to be 1.2 mumol.min-1.mg-1. The specific dynein-like ATPase activity of the crude soluble extract of unfertilized sea urchin eggs was determined to be 0.004 mumol.min-1.mg-1. The concentration of putative dynein catalytic polypeptides was therefore determined from the ratio of the specific activities of crude to pure cytoplasmic dynein catalytic polypeptide to be 0.33% of soluble protein, or 99 pg per egg. This is approximately 3-fold greater than the mass of dynein catalytic polypeptides estimated to be present in cilia at the blastula stage of sea urchin embryonic development. The large amount of cytoplasmic dynein in unfertilized eggs suggests that it could act as a precursor of embryonic ciliary dynein. Three minor peaks of ATPase activity were also resolved from cytosolic extracts and shown to be dynein-like. However, their GTPase activities were 2-4-fold higher than that of cytoplasmic dynein, raising the possibility that egg cytoplasm may contain several isoforms of dynein.
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PMID:Quantitation of the dynein pool in unfertilized sea urchin eggs. 252 62

Biochemical and immunological analysis of unfertilized sea urchin eggs has revealed the presence of at least two distinct isoforms of cytoplasmic dyneins, one soluble and the other microtubule-associated. The soluble enzyme is a 20 S particle with a MgATPase activity that can be activated 5-fold by nonionic detergents. It contains heavy chain polypeptides that 1) comigrate with the dynein heavy chains of embryonic cilia; 2) cross-react with antibodies against flagellar dynein; and 3) are cleaved by UV irradiation in the presence of MgATP and sodium vanadate into specific peptide fragments. The soluble egg dynein is, therefore, closely related to axonemal dynein and may be a ciliary precursor. Egg microtubule preparations contain a distinct dynein-like polypeptide, previously designated HMr-3 (Scholey, J.M., Neighbors, B., McIntosh, J.R., and Salmon, E.D. (1984) J. Biol Chem. 259, 6516-6525). HMr-3 binds microtubules in an ATP-sensitive fashion; it sediments at 20 S on sucrose density gradients, and it is susceptible to vanadate-sensitized UV cleavage. However, HMr-3 can be distinguished from the soluble cytoplasmic dynein on the basis of its weak cross-reactivity with antiflagellar dynein antibodies, its heavy chain composition on high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its low specific ATPase activity, and the molecular weight of its vanadate-induced UV cleavage fragments. HMr-3 may represent a dynein-like polypeptide that is distinct from the pool of ciliary dynein precursors.
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PMID:Dynein isoforms in sea urchin eggs. 289 99

Unfertilized sea urchin eggs contain a Mg2+-ATPase which shares physical and enzymatic characteristics with dynein, the enzyme which powers ciliary and flagellar movement. To further investigate the homology of the egg ATPase and axonemal dynein, ATP-binding subunits in preparations of each of the enzymes were identified using a photoaffinity probe of ATP, 8-azido-ATP (8-N3ATP), and three high molecular weight (HMW) polypeptide components of the two enzymes were compared by one-dimensional peptide mapping. Two heavy chains (A and B) of both the flagellar and egg ATPases bound [alpha-32P]8-N3ATP. The labeling of the HMW bands was specifically inhibited by ATP or ADP. Both the cytoplasmic ATPase and flagellar dynein utilized 8-N3ATP as a substrate indicating that the reagent binds to the active site. The two HMW ATP-binding polypeptides and one other HMW component of the egg ATPase were compared to flagellar dynein heavy chains by peptide mapping. Digestion of the egg versus flagellar HMW polypeptides with Staphylococcus V8 protease or alpha-chymotrypsin produced a highly similar group of peptides, and each pair of heavy chains was qualitatively estimated to be over 85% homologous. These data support the identification of the egg ATPase heavy chains as components of a cytoplasmic dynein and suggest that the HMW polypeptides form active enzymatic sites in flagellar and egg dynein which are substantially homologous.
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PMID:Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure. 293 92

Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 microM to 600 microM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 microM and 161 microM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 microM and 271 microM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 microM and 290 microM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins.
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PMID:Evidence for functional differences between two flagellar dynein ATPases. 294 77

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.
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PMID:MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. 295 82

The rapid, vectorial, microtubule-associated transport of organelles is believed to be mediated by specific mechanochemical transducers. Recent studies of various metazoan cells have allowed the identification of novel microtubule-dependent translocator molecules capable of promoting microtubule gliding across glass surfaces and translocation of inert beads along microtubules. These translocators could be involved in force generation for directional organelle movements in vivo. Here we report the identification of a microtubule-binding protein with characteristics expected for an organelle translocator in the giant freshwater amoeba Reticulomyxa. This factor has an apparent relative molecular mass (Mr) of 440,000 (440K) and sediments at 20-22S in sucrose-density gradients. It binds to microtubules under conditions of ATP depletion, possesses an ATPase activity and is sensitive to ultraviolet-induced, vanadate-dependent cleavage. Although its pharmacological properties differ from those of axonemal dynein, it can be considered to be a variant of cytoplasmic dynein. The Reticulomyxa high-molecular-weight protein (HMWP) promotes rapid, bidirectional movement of latex beads along Reticulomyxa microtubules in vitro at an average speed of 3.6 micron s-1. This protein, therefore, is a likely candidate for a microtubule-dependent motor.
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PMID:An ATPase with properties expected for the organelle motor of the giant amoeba, Reticulomyxa. 296 63

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.
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PMID:Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C). 297 Oct 69


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