scholarly journals The structure of ribosomes as indicated by studies on tetramers from hypothermic chick embryos

1972 ◽  
Vol 130 (3) ◽  
pp. 871-877 ◽  
Author(s):  
N. H. Carey ◽  
J. R. W. Hobbs ◽  
E. A. Cook
Keyword(s):  
Electron Microscope ◽  
Density Gradient ◽  
Large Subunit ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Small Subunit ◽  
Outer Edge ◽  
Chick Embryos ◽  
Two Dimensional Image ◽  
Electron Microscope Grid

Ribosome tetramers induced in chick embryos by exposure to cold, and tetramers of large subunits derived from them, have been studied by electron microscopy and sucrose-density-gradient analysis. Individual ribosomes of the normal tetramer are elongated bean-shaped structures, 220–280Å by 195Å (1Å#x003D;10-1nm) with a cleft in the outer edge which divides the two-dimensional image into two unequal ends. Most of the tetramers appear to attach to the surface of the electron-microscope grid by one preferred face. The subunits of the large-subunit tetramers have a round outline and no cleft. About 25% of the subunits of these tetramers have a line running radially across the particle. The dissociation of tetramers into large-subunit tetramers and small subunits has been shown to be reversible. Mixtures of these particles from sucrose-density-gradient fractions were reassociated to give a tetramer with the same sedimentation coefficient as the original tetramer and with the same structure as viewed in the electron microscope. The results indicate that the cleft is a property of the complete ribosome, and that it marks the position of the small subunit. The reversibility of the dissociation also strengthens the view that no change in the large subunit occurs during dissociation or reassociation, i.e. that the sites of interaction between ribosomes in both types of tetramer are the same. The conclusions affect the interpretation of electron-micrograph images and an anomaly in the relationship between the two types of tetramer is discussed.

scholarly journals Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats

1982 ◽  
Vol 204 (1) ◽  
pp. 31-36 ◽  
Author(s):  
D Sömjen ◽  
G J Sömjen ◽  
Y Weisman ◽  
I Binderman
Keyword(s):  
Density Gradient ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Macromolecular Complex ◽  
Long Bones ◽  
Newborn Rats ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation ◽  
Binding Component

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.

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.

ON THE MOLECULAR WEIGHT OF PREGNANT MARE'S SERUM GONADOTROPHIN

1964 ◽  
Vol 45 (4_Suppl) ◽  
pp. S163-S171 ◽  
Author(s):  
C. J. O. R. Morris
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Chromatographic Behaviour ◽  
The Cross ◽  
Active Preparation

ABSTRACT The chromatographic behaviour of pregnant mare serum and of a purified preparation of the gonadotrophin from uterine endometrial cup secretion on the cross-linked dextran gel media Sephadex G-100 and G-200 has been examined, and is consistent with a molecular weight greater than the value of 28 000 usually accepted. The sedimentation coefficient of an active preparation has been determined by sedimentation in a sucrose density gradient and serial bioassay. The value of 3.2 S for SW20 is consistent with current values. The literature data are shown to be most consistent with a molecular weight of 68 000 for the hormone, and the mechanism of its chromatography on dextran gels is discussed.

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.

A Factor Capable of Dissociating Rat Liver Ribosomes

1971 ◽  
Vol 49 (12) ◽  
pp. 1301-1306 ◽  
Author(s):  
G. Ross Lawford ◽  
Jutta Kaiser ◽  
W. C. Hey
Keyword(s):  
Ionic Strength ◽  
Ammonium Sulfate ◽  
Density Gradient ◽  
Rat Liver ◽  
Messenger Rna ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Small Subunit ◽  
Temperature Dependent ◽  
Liver Ribosomes

A factor capable of dissociating rat liver monomeric ribosomes into 60 S and 40 S subunits has been partially purified and characterized.The factor was prepared by extracting a fraction of rat liver enriched in its content of native subunits with 0.05 M triethanolamine–HCl, 1.0 M KCl, 0.01 M MgSO4, and 2 mM dithiothreitol. The activity of the preparation was assayed by testing its ability to dissociate monomeric ribosomes into subunits which were detected by sucrose density gradient analysis. The ribosomes used as substrate were prepared by dissociating polysomes in the presence of puromycin, 0.5 M KCl, and 3 mM MgSO4 and subsequently reassociating the subunits into monomers by lowering the ionic strength. The factor acts only on ribosomes freed of both messenger RNA and nascent protein by associating with the small subunit. The activity was time and temperature dependent, reaching a plateau after 30 min at 30 °C.The factor has been partially purified by ammonium sulfate fractionation between 35% and 65% saturation and by treatment at 40 °C for 15 min to precipitate ribosome-aggregating substances.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

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.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 541-551 ◽  
Author(s):  
N. E. COLLINS ◽  
B. A. ALLSOPP
Keyword(s):  
Genetic Recombination ◽  
Large Subunit ◽  
Small Subunit ◽  
Internal Transcribed Spacers ◽  
Rrna Genes ◽  
Gene Pools ◽  
Theileria Parva ◽  
Causative Agents ◽  
Internal Transcribed Spacer Sequences

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5·8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93·5% of T. p. parva isolates examined were detected by probe TPP1 and 81·8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

scholarly journals Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 477-484
Author(s):  
W F Wu ◽  
S Christiansen ◽  
M Feiss
Keyword(s):  
Protein Interactions ◽  
Ternary Complex ◽  
Large Subunit ◽  
Small Subunit ◽  
Functional Domain ◽  
Binary Complex ◽  
Protein Protein Interactions ◽  
Related Phage ◽  
Carboxy Terminal ◽  
Lambda Dna

Abstract The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

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