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

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.105.2.887 ◽

1987 ◽

Vol 105 (2) ◽

pp. 887-895 ◽

Cited By ~ 35

Author(s):

Y Y Toyoshima

Keyword(s):

Electron Microscopy ◽

Gel Electrophoresis ◽

Gradient Centrifugation ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Heavy Chains ◽

Time Courses ◽

The One ◽

Dynein Heavy Chains

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.

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DEMONSTRATION OF A CYTOPLASMIC RECEPTOR PROTEIN FOR OESTROGEN IN THE CANINE PROSTATE GLAND

Journal of Endocrinology ◽

10.1677/joe.0.0780131 ◽

1978 ◽

Vol 78 (1) ◽

pp. 131-139 ◽

Cited By ~ 21

Author(s):

N. CHAISIRI ◽

Y. VALOTAIRE ◽

BRONWEN A. J. EVANS ◽

C. G. PIERREPOINT

Keyword(s):

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Prostate Gland ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Receptor Protein ◽

Receptor Complex ◽

Binding Properties ◽

Sucrose Density Gradient Centrifugation ◽

A Value

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid–receptor complex was found to have a sedimentation coefficient of 4–5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3·8 ± 1·53 (s.d.) × 10−10 mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

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Über die Spaltung physikalisch intakter Poliovirus-Teilchen in Nucleinsäure und leere Proteinhüllen durch Wärmebehandlung

Zeitschrift für Naturforschung B ◽

10.1515/znb-1965-0910 ◽

1965 ◽

Vol 20 (9) ◽

pp. 870-878 ◽

Cited By ~ 12

Author(s):

O. Drees ◽

Ch. Borna

Keyword(s):

Protein Concentration ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Virus Protein ◽

Analytical Ultracentrifuge ◽

Sucrose Density Gradient Centrifugation ◽

Virus Particles ◽

Phosphate Buffered Saline

Purified preparations of poliovirus devoid of contaminating nucleic acid and so-called “C antigen” released their particle-bound ribonucleic acid (RNA) almost quantitatively on heating at 40°C for 48 hours in phosphate-buffered saline (pH 7.6). This process occurred without disruption of the virus protein and left, in addition to the free RNA and traces of undegraded virus particles, empty protein shells in the reaction mixture.The liberated RNA sedimented in the analytical ultracentrifuge as a very diffuse boundary with sedimentation coefficients (s20, w) in the range from about 8 S to less than 1 S. The shell material which could be isolated by means of sucrose density-gradient centrifugation proved to be homogeneous in the analytical ultracentrifuge. Its sedimentation coefficient (s20,w) extrapolated to zero protein concentration was found to be 78 S.

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

The Journal of Cell Biology ◽

10.1083/jcb.101.4.1400 ◽

1985 ◽

Vol 101 (4) ◽

pp. 1400-1412 ◽

Cited By ~ 90

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.

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Biochemical and Structural Changes in Mitochondria and Other Cellular Components of Pea Cotyledons During Germination

Canadian Journal of Biochemistry ◽

10.1139/o72-101 ◽

1972 ◽

Vol 50 (7) ◽

pp. 725-737 ◽

Cited By ~ 31

Author(s):

T. Solomos ◽

S. S. Malhotra ◽

S. Prasad ◽

S. K. Malhotra ◽

Mary Spencer

Keyword(s):

Electron Microscopy ◽

Structural Changes ◽

Gradient Centrifugation ◽

Microscopic Analysis ◽

Sucrose Density Gradient ◽

Electron Microscopic ◽

Sucrose Density Gradient Centrifugation ◽

Cellular Components ◽

Further Development

Integrated studies comprising biochemical and electron microscopic analysis suggested that the increase in respiratory activity of pea cotyledon mitochondria during germination results from further development of the original mitochondria present in dormant seeds. Electron microscopy of isolated mitochondria as well as mitochondria in situ has revealed that membranes are scarce in the mitochondria present in dormant seeds. Mitochondrial cristae become well developed during the initial stages of germination. Crude mitochondrial preparations from pea cotyledons were fractionated by sucrose density gradient centrifugation and analyzed through electron microscopy. These studies showed that, at all stages of germination, "peroxisome"-like structures were present in the fractions of higher sucrose densities than that containing mitochondria. Biochemical studies revealed that the activities of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) and peroxidase (guaicol:H2O2 oxidoreductase, EC 1.11.1.7) were associated mainly with these fractions and their activities increased during germination.

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Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽

10.1242/dev.106.4.799 ◽

1989 ◽

Vol 106 (4) ◽

pp. 799-808

Author(s):

E.K. Shibuya ◽

Y. Masui

Keyword(s):

Density Gradient ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Molecular Characteristics ◽

Small Peptides ◽

Sucrose Density Gradient Centrifugation ◽

Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

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PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

Acta Endocrinologica ◽

10.1530/acta.0.0620153 ◽

1969 ◽

Vol 62 (1) ◽

pp. 153-164 ◽

Cited By ~ 49

Author(s):

Olav Unhjem ◽

Kjell J. Tveter ◽

Asbjørn Aakvaag

Keyword(s):

Proteolytic Enzymes ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Ventral Prostate ◽

Macromolecular Complex ◽

Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

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

The Journal of Cell Biology ◽

10.1083/jcb.106.5.1607 ◽

1988 ◽

Vol 106 (5) ◽

pp. 1607-1614 ◽

Cited By ~ 30

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.

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Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent

Biochemical Journal ◽

10.1042/bj1810201 ◽

1979 ◽

Vol 181 (1) ◽

pp. 201-213 ◽

Cited By ~ 33

Author(s):

M E Birnbaumer ◽

W T Schrader ◽

B W O'Malley

Keyword(s):

Progesterone Receptor ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Deae Cellulose ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Cross Linking ◽

Sucrose Density Gradient Centrifugation ◽

Receptor Proteins ◽

The Cross

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.

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The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos

Biochemical Journal ◽

10.1042/bj1210511 ◽

1971 ◽

Vol 121 (3) ◽

pp. 511-519 ◽

Cited By ~ 11

Author(s):

N. H. Carey ◽

Gail S. Read

Keyword(s):

Gradient Centrifugation ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Chick Embryos ◽

Sucrose Density Gradient Centrifugation ◽

Reversible Dissociation ◽

Standard Buffer ◽

K Concentration ◽

The Individual ◽

Low K

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg2+, Ca2+ and K+ studied. 2. Lowering of the Mg2+ concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca2+ replaced Mg2+ in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg2+ concentrations. 4. Ca2+ also caused an almost complete conversion of monomers into dimers in the presence of Mg2+. 5. The effect of Ca2+ on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K+ concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K+ concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K+ concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.

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