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.

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 The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.

1989 ◽  
Vol 109 (4) ◽  
pp. 1529-1535 ◽  
Author(s):  
J H Sinard ◽  
T D Pollard
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Critical Concentration ◽  
Myosin Ii ◽  
Acidic Ph ◽  
Thick Filaments ◽  
Low Ionic Strength

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Regulation of glucose-6-phosphate dehydrogenase activity in sea urchin eggs by reversible association with cell structural elements.

1986 ◽  
Vol 103 (4) ◽  
pp. 1509-1515 ◽  
Author(s):  
R R Swezey ◽  
D Epel
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Time Course ◽  
Calcium Concentration ◽  
Metabolic Activation ◽  
Structural Elements ◽  
Ionic Detergent ◽  
Low Ionic Strength ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Substrate Concentrations

In unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, glucose-6-phosphate dehydrogenase (G6PDH) associates with the particulate elements remaining either after homogenization or extraction of eggs with non-ionic detergent in low ionic-strength media. At physiological ionic strength, the extent of G6PDH binding to these particulate elements is proportional to the total protein concentration in the extracts. In fertilized eggs this association is prevented by one or more low molecular weight solutes. The dissociation is reversible, and there are no permanent modifications of either G6PDH or its particulate binding site that affect binding. After fertilization, the time course of dissociation of G6PDH from particulate elements is too fast to be caused by a change in intracellular pH, but it could be triggered, but not maintained, by an increase in the intracellular calcium concentration. Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations. Therefore, release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.

scholarly journals Accessibility of epitopes on fibrin clots and fibrinogen gels

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1469-1475 ◽  
Author(s):  
R Procyk ◽  
B Kudryk ◽  
S Callender ◽  
B Blomback
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Trials ◽  
Ionic Strength ◽  
Factor Xiii ◽  
Imaging Agent ◽  
Low Ionic Strength ◽  
Clot Structure ◽  
Radiolabeled Antibodies ◽  
Fibrin Clots

Abstract Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.

scholarly journals Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein.

1987 ◽  
Vol 105 (2) ◽  
pp. 927-936 ◽  
Author(s):  
R Yabkowitz ◽  
D R Burgess
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Binding Protein ◽  
Affinity Column ◽  
Supramolecular Organization ◽  
Independent Manner ◽  
Myosin Binding Protein ◽  
Egg Extracts ◽  
Myosin Binding ◽  
Low Ionic Strength

We identify a novel myosin-binding protein, designated 53K, which appears to mediate the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein possesses a subunit molecular mass on SDS-PAGE of 53 kD, an S value of 7, may be organized into disulfide-linked oligomers, and is associated with myosin in egg extracts. Both myosin and 53K co-precipitate from extract upon the addition of nucleoside triphosphates and co-sediment with an S value of 24 by sedimentation velocity centrifugation. Myosin in extracts not associated with 53K has an S value of 10. Further, myosin can be immunoprecipitated from extract with antibody to 53K and the 53K in extracts binds to a myosin affinity column. When extract is depleted of 53K, a majority of the myosin precipitates out of extract in a nucleotide-independent manner. Whereas purified myosin precipitates in the absence of nucleotide when recombined with dialysis buffer or myosin-depleted extract, reconstituting 53K and myosin before addition to buffer or myosin-depleted extract partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts. The 53-kD protein may represent a new class of authentic myosin-binding proteins that may regulate the supramolecular organization of myosin in nonmuscle cells.

scholarly journals Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and P1

1979 ◽  
Vol 38 (1) ◽  
pp. 105-123
Author(s):  
M. Okuno ◽  
C.J. Brokaw
Keyword(s):  
Sea Urchin ◽  
Glass Surface ◽  
Beat Frequency ◽  
Bend Angle ◽  
Flagellar Motility ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Linear Relationships ◽  
Clinical Patterns ◽  
Sea Urchin Sperm

Three clinical patterns of inhibition of MgATP2—activated flagellar motility have been found by measuring the motility of Triton-demembranated sea-urchin spermatozoa beating with their heads attached to a glass surface. Inhibition of beat frequency by the reaction products, ADP and Pi, is competitive with the normal substrate, MgATP2-, and the inhibitory effects are similar to a reduction in MgATP2- concentration. Inhibition of beat frequency by ATP4- is competitive with MgATP2, but is accompanied by an inhibition of bending, as measured by the angle between the straight regions on either side of a bend, which is not seen when MgATP2- concentration is reduced. Inhibition of beat frequency by Mg2+ is not competitive with MgATP2-, and is accompanied by an increase in bend angle, so that there is no change in the rate of sliding between flagellar tubules. These differences suggest unexpected complexity of dynein ATPase action in flagella. The beat frequencies of both swimming and attached spermatozoa show a linear double reciprocal dependence on MgATP2- concentration, with identical slopes. The calculated sliding velocities between tubules also give linear relationships, but the slopes are different, suggesting that beat frequency may be the more fundamental dependent variable in this system.

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 Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection.

1993 ◽  
Vol 123 (3) ◽  
pp. 681-689 ◽  
Author(s):  
B D Wright ◽  
M Terasaki ◽  
J M Scholey
Keyword(s):  
Cell Division ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Mammalian Cells ◽  
Embryonic Cell ◽  
Mitotic Apparatus ◽  
Specific Antibodies ◽  
Sea Urchin Embryos ◽  
Mitotic Figures

Previous studies suggest that kinesin heavy chain (KHC) is associated with ER-derived membranes that accumulate in the mitotic apparatus in cells of early sea urchin embryos (Wright, B. D., J. H. Henson, K. P. Wedaman, P. J. Willy, J. N. Morand, and J. M. Scholey. 1991. J. Cell Biol. 113:817-833). Here, we report that the microinjection of KHC-specific antibodies into these cells has no effect on mitosis or ER membrane organization, even though one such antibody, SUK4, blocks kinesin-driven motility in vitro and in mammalian cells. Microinjected SUK4 was localized to early mitotic figures, suggesting that it is able to access kinesin in spindles. In contrast to KHC-specific antibodies, two antibodies that react with kinesin-like proteins (KLPs), namely CHO1 and HD, disrupted mitosis and prevented subsequent cell division. CHO1 is thought to exert this effect by blocking the activity of a 110-kD KLP. The relevant target of HD, which was raised against the KHC motor domain, is unknown; HD may disrupt mitosis by interfering with an essential spindle KLP but not with KHC itself, as preabsorption of HD with KHC did not alter its ability to block mitosis. These data indicate that some KLPs have essential mitotic functions in early sea urchin embryos but KHC itself does not.

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