C/A dynein isolated from sea urchin sperm flagellar axonemes. Enzymatic properties and interaction with microtubules

1994 ◽  
Vol 107 (2) ◽  
pp. 353-361 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Maximal Inhibition ◽  
Heavy Chains ◽  
Lower Molecular Mass ◽  
Cross Bridges ◽  
Polypeptide Chains ◽  
Brain Tubulin ◽  
Sea Urchin Sperm

C/A dynein is a novel dynein isolated from sea urchin sperm flagellar axonemes. It is composed of C and A heavy chains and some additional lower molecular mass polypeptide chains. The characterization of ATPase activity and the interaction of this dynein with microtubules polymerized from calf brain tubulin were investigated in this study. The ATPase activity of C/A dynein (0.3-0.4 mumol Pi/min per mg) was about one half that of outer arm 21 S dynein (0.6-0.8 mumol Pi/min per mg) at 25 degrees C. Vanadate inhibited the ATPase activity with a half-maximal inhibition at 1 microM. C/A dynein absorbed to the glass surface was able to translocate the microtubules towards its plus end. The velocity of the microtubule movement in the presence of 1 mM ATP was 4.0 to 4.5 microns/s at 22 degrees C. C/A dynein binds to and bundles the microtubules even in the presence of ATP. Cross-bridges were found between adjacent microtubules in the bundle with an axial periodicity of about 24 nm. The ATPase activity of C/A dynein was enhanced up to several-fold by the microtubules at concentration as low as 1 mg/ml. On the other hand, 21 S dynein bound to the microtubules with 24 nm axial periodicity only in the absence of ATP. Its ATPase activity was not activated by the microtubules. From these results, it is concluded that the manner of interaction with microtubules of C/A dynein is different from that of the outer arm dynein.

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

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

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.

scholarly journals PROPERTIES OF FLAGELLAR "RIGOR WAVES" FORMED BY ABRUPT REMOVAL OF ADENOSINE TRIPHOSPHATE FROM ACTIVELY SWIMMING SEA URCHIN SPERM

1974 ◽  
Vol 63 (3) ◽  
pp. 970-985 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Sea Urchin ◽  
Sperm Suspension ◽  
Complete Relaxation ◽  
Viscous Shear ◽  
Radial Spokes ◽  
Wave Forms ◽  
Cross Bridges ◽  
Triton X ◽  
The Waves ◽  
Sea Urchin Sperm

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

Isolation and Characterization of Low Density Detergent-Insoluble Membrane (LD-DIM) Fraction from Sea Urchin Sperm

1999 ◽  
Vol 258 (3) ◽  
pp. 616-623 ◽  
Author(s):  
Kaoru Ohta ◽  
Chihiro Sato ◽  
Tsukasa Matsuda ◽  
Masaru Toriyama ◽  
William J. Lennarz ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Low Density ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

scholarly journals Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis

1981 ◽  
Vol 195 (1) ◽  
pp. 171-176 ◽  
Author(s):  
V Giancotti ◽  
S Cosimi ◽  
P D Cary ◽  
C Crane-Robinson ◽  
G Geraci
Keyword(s):  
Secondary Structure ◽  
Sea Urchin ◽  
Fluorescence Spectra ◽  
Tertiary Structure ◽  
Histone H1 ◽  
Separation And Purification ◽  
Tertiary Structures ◽  
Calf Thymus ◽  
Sea Urchin Sperm

The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.

scholarly journals Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes

1982 ◽  
Vol 92 (3) ◽  
pp. 706-713 ◽  
Author(s):  
K Ogawa ◽  
S Negishi ◽  
M Obika
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Double Diffusion ◽  
Diffusion Test ◽  
Electron Microscopic ◽  
Antibody Method ◽  
Sea Urchin Sperm

The 0.5 M KCl-treatment solubilizes the outer arms from sea urchin sperm axonemes. Approximately 30 percent of A-polypeptide, corresponding to dynein 1 in SDS- polyacrylamide gel, was solubilized by this treatment (as SEA-dynein 1). Electron microscopic observation indicated that the extracted axonemes lacked the outer arms in various degrees. The DEA-dynein 1 was that the extracted axonemes lacked the outer arms in various degrees. The SEA-dyenin 1 was purified and an antiserum against it was prepared in rabbits. The specificity of antiserum to dynein 1 was determined by immunoelectrophoresis and ouchterlony's double-diffusion test. The anti-dynein 1 serum inhibited ATPase activity of purified SEA-dynein 1 by 95 percent. By the indirect peroxidase-conjugated antibody method, the loci of SEA-dynein 1 within the intact, salt- extracted and mechanically disrupted axonemes were determined to be the outer arms: deposition of electron-dense materials which represents their localization was detected at the distal ends of the outer arms, in the case of intact axonemes. The 5-6 cross- bridge was hardly decorated. No decoration was seen in the salt-extracted axonemes lacking all the outer arms. In disrupted axonemes, which consist of single to several peripheral doublets, electron-dense materials were deposited only on the outer arms. Approximately 73 percent of axonemal ATPase activity sensitive to antiserum was solubilized by repeated salt-extractions. One-half of A-polypeptide (SEA-dynein 1 located at the outer arms) was contained in the pooled extracts. The extracted axonemes contained another half of A-polypeptide (SUA-dynein 1 supposed to locate at the inner arms) and retained 31 percent of axonemal ATPase activity that was almost resistant to antiserum. Solubilized SUA-dynein 1 was immunologically the same as SEA-dynein 1. This result indicates that in situ SUA-dynein 1 did not receive anti-dynein 1 antibodies, coinciding with the result obtained for salt-extracted axonemes lacking all the outer arms by the enzyme-antibody method mentioned above. These observations suggest that immunological dissimilarity in dynein 1 between outer and inner arms but do not tell us that the inner arms do not contain dynein 1.

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 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.

Sign in / Sign up

Export Citation Format

Share Document