Conformational Changes of Dynein: Mapping and Sequence Analysis of ATP/Vanadate-Dependent Trypsin-Sensitive Sites on the Outer Arm Dynein   Heavy Chain from Sea Urchin Sperm Flagell

2000 ◽  
Vol 127 (6) ◽  
pp. 1115-1120 ◽  
Author(s):  
K. Inaba
Keyword(s):  
Sequence Analysis ◽  
Sea Urchin ◽  
Conformational Changes ◽  
Heavy Chain ◽  
Dynein Heavy Chain ◽  
Sea Urchin Sperm

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

1988 ◽  
Vol 107 (5) ◽  
pp. 1793-1797 ◽  
Author(s):  
W S Sale ◽  
L A Fox
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Glass Surface ◽  
Functional Assay ◽  
Atpase Subunits ◽  
Low Ionic Strength ◽  
Intermediate Chains ◽  
Sea Urchin Sperm

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

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes

1994 ◽  
Vol 107 (2) ◽  
pp. 345-351 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

scholarly journals The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules.

1992 ◽  
Vol 118 (5) ◽  
pp. 1189-1200 ◽  
Author(s):  
A G Moss ◽  
W S Sale ◽  
L A Fox ◽  
G B Witman
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Force Generation ◽  
Alpha Subunit ◽  
Dependent Manner ◽  
Independent Manner ◽  
Cross Sectional ◽  
Dynein Arms ◽  
Sea Urchin Sperm

Glass-adsorbed intact sea urchin outer arm dynein and its beta/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its alpha heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified alpha heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the beta/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the alpha heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine crossbridges. In contrast to the bridges formed by the intact arm, the links formed by the alpha subunit are irregularly spaced, suggesting that binding of the alpha heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the beta/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the alpha heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its alpha heavy chain. We conclude that whereas force-generation is a function of the beta/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the alpha subunit.

Speract-receptor interaction and the modulation of ion transport in Strongylocentrotus purpuratus sea urchin sperm

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S20-S21 ◽  
Author(s):  
Blanca-Estela Galindo ◽  
Takuya Nishigaki ◽  
Esmeralda Rodríguez ◽  
Daniel Sánchez ◽  
Camen Beltrán ◽  
...  
Keyword(s):  
Ion Transport ◽  
Sea Urchin ◽  
Conformational Changes ◽  
Sea Urchins ◽  
Strongylocentrotus Purpuratus ◽  
Dissociation Rate ◽  
Receptor Interaction ◽  
Jelly Coat ◽  
Dissociation Rate Constants ◽  
Sea Urchin Sperm

We are studying the regulation of ion transport in sperm physiology. Sperm ion permeability is modulated by components from the outer layer of the egg which, depending on the species, regulate sperm motility, Chemotaxis and the acrosome reaction (AR). This reaction is required for sperm to fertilise the egg in many species from sea urchins to man (Darszon et al., 1999).Speract, a decapeptide from the external layer of Strongylocentrotus purpuratus sea urchin eggs, influences sperm respiration, motility and possibly the AR. Signal transduction starts when speract binds to a protein of 77 kDa closely coupled to sperm guanylyl cyclase (Garbers, 1989). Our recent receptor binding experiments using fluorescent-labelled speract (fluorescein and rhodamine) have allowed estimates of the association (kon 2.4 × 107 M−1s−1) and dissociation rate constants (koff 1.3 × 10−4 s−1). Furthermore, studies with fluorescent speract analogues indicate that the receptor undergoes conformational changes that depend on intracellular pH (pHi). The overall results are consistent with the possibility that speract may induce in sea urchin sperm a hyperactivated-like flagellar movement inside the jelly coat to accelerate sperm penetration through this layer.

scholarly journals The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella.

1985 ◽  
Vol 101 (4) ◽  
pp. 1400-1412 ◽  
Author(s):  
W S Sale ◽  
U W Goodenough ◽  
J E Heuser
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Heavy Chains ◽  
Low Salt ◽  
Spherical Head ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Globular Head

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.

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

1992 ◽  
Vol 119 (6) ◽  
pp. 1597-1604 ◽  
Author(s):  
M P Koonce ◽  
P M Grissom ◽  
J R McIntosh
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Cellular Slime Mold ◽  
Reading Frame ◽  
Dynein Heavy Chain ◽  
Binding Domains

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.

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

1988 ◽  
Vol 106 (5) ◽  
pp. 1607-1614 ◽  
Author(s):  
G Mocz ◽  
W J Tang ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Heavy Chain ◽  
Terminal Region ◽  
Beta Chain ◽  
Phosphate Binding ◽  
Atp Binding Site ◽  
Heavy Chains ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Sea Urchin Sperm

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.

Sign in / Sign up

Export Citation Format

Share Document