scholarly journals The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos

1971 ◽  
Vol 121 (3) ◽  
pp. 511-519 ◽  
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.

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

1978 ◽  
Vol 78 (1) ◽  
pp. 131-139 ◽  
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.

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

1965 ◽  
Vol 20 (9) ◽  
pp. 870-878 ◽  
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.

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.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
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.

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

1969 ◽  
Vol 62 (1) ◽  
pp. 153-164 ◽  
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.

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

1979 ◽  
Vol 181 (1) ◽  
pp. 201-213 ◽  
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.

scholarly journals The intracellular localization of malate dehydrogenase isoenzymes in Pisum arvense roots

2014 ◽  
Vol 55 (4) ◽  
pp. 621-627
Author(s):  
Genowefa Kubik-Dorosz
Keyword(s):  
Malate Dehydrogenase ◽  
Isoelectric Focusing ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Root Cells ◽  
Pisum Arvense ◽  
The Individual

Mitochondria and plastids were isolated from <em>Pisum arvense</em> root cells by sucrose density gradient centrifugation. The individual subcellular fractions so obtained were subjected to isoelectric focusing on cellulose acetate strips. Mitochondria and plastids each contained one NAD -malate dehydrogenase, while three isoenzymes were associated with the supernatant.

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

1987 ◽  
Vol 105 (2) ◽  
pp. 887-895 ◽  
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.

scholarly journals Hydrodynamic and pharmacological characterization of putative α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive l-glutamate receptors solubilized from pig brain

1994 ◽  
Vol 300 (2) ◽  
pp. 365-371 ◽  
Author(s):  
T Y Wu ◽  
Y C Chang
Keyword(s):  
Binding Sites ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glutamate Binding ◽  
Stokes Radius ◽  
Triton X

L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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