The 3′-hydroxyl termini in yeast ribosomal RNA

1970 ◽  
Vol 48 (10) ◽  
pp. 1113-1121 ◽  
Author(s):  
K. M. Olver ◽  
B. G. Lane
Keyword(s):  
Gradient Centrifugation ◽  
Group Analysis ◽  
Ribosomal Subunit ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Selective Precipitation ◽  
Ribosomal Subunits ◽  
Hydroxyl Termini ◽  
End Group ◽  
Total Yeast

(1) A fraction of high molecular weight RNA was prepared by subjecting total yeast RNA to selective precipitation from aqueous 2.5 M sodium chloride solution at 0°. This "insoluble" RNA accounted for about 80% of the total yeast RNA.(2) The "insoluble" RNA and various of its subfractions obtained by preparative sucrose density-gradient centrifugation were subjected to electrophoretic and end-group analyses.(3) One subfraction, largely composed of 17 S RNA from the smaller ribosomal subunit, was found to contain adenosine as the principal 3′-hydroxyl terminal nucleoside residue. Based on the analysis of 3′-hydroxyl termini, RNA in this subfraction had a mean residue length of about 1600 nucleotides.(4) Another subfraction, largely composed of 26 S RNA from the larger ribosomal subunit, was found to contain uridine as the principal 3′-hydroxyl terminal nucleoside residue. Based on the analysis of 3′-hydroxyl termini, RNA in this subfraction had a mean residue length of about 2400 nucleotides.(5) Electrophoretic and end-group analyses of other subfractions have been reported and discussed in terms of the possible origin of the RNA in these fractions, and also in terms of the way in which the RNA of these fractions might tend to affect the end-group analysis of both the parent fraction and the principal subfractions, which contain RNA from the ribosomal subunits.

THE TERMINAL GROUPS OF RIBONUCLEATE CHAINS IN EACH OF THE 16S AND 23S COMPONENTS OF ESCHERICHIA COLI RIBOSOMAL RNA

1967 ◽  
Vol 45 (6) ◽  
pp. 937-948 ◽  
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Ribosomal Rna ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Quantitative Estimates ◽  
16S Rna ◽  
The Mean ◽  
End Group

Ribosomal ribonucleates from Escherichia coli have been resolved into 16S and 28S components by sucrose density-gradient centrifugation, and the chain termini in each of the 16S and 23S RNA components have been analyzed by hydrolysis with alkali. The principal 5′-linked end group of 16S RNA was found to be adenosine, and the principal 5′-linked end group of 23S RNA was found to be uridine. The principal 3′-linked end group of 16S RNA was also found to be adenosine, whereas the principal 3′-linked end group of 23S RNA was found to be guanosine. Quantitative estimates of chain length based on analyses for 5′-iinked terminals indicate that the mean chain length for 16S RNA is about 1.3 × 103nucleotide residues and the mean chain length for 23S RNA is about 2.1 × 103nucleotide residues.

scholarly journals Peptide-chain initiation in duodenal mucosa of rachitic chicks after 1α-,25-dihydroxycholecalciferol administration

1984 ◽  
Vol 219 (1) ◽  
pp. 99-106 ◽  
Author(s):  
G Mezzetti ◽  
M Moruzzi ◽  
B Barbiroli
Keyword(s):  
Complex Formation ◽  
Gradient Centrifugation ◽  
Active Form ◽  
Ribosomal Subunit ◽  
Sucrose Density Gradient ◽  
Duodenal Mucosa ◽  
Binding Activity ◽  
Initiation Factor ◽  
Experimental Conditions ◽  
Sucrose Density Gradient Centrifugation

The post-ribosomal fraction of chick duodenal mucosa contains Met-tRNAMetf-binding protein(s) that behaves like the eukaryotic initiation factor (eIF-2) in protein synthesis. The binding activity of cytosol protein can be measured by retention of the radioactive complex formed on a nitrocellulose membrane. Complex-formation requires Met-tRNAMetf and GTP or guanosine [beta, gamma-methylene] triphosphate, and is inhibited by aurintricarboxylic acid. The ternary initiation complex thus formed can bind to ribosomal particles from chick intestine. By sucrose-density-gradient centrifugation, [35S]Met-tRNAMetf was found to bind exclusively to 40S and not to 60S ribosomal subunit particles. In the duodenal mucosa of rachitic chicks the ability of the cytosol proteins to promote the binding of Met-tRNAMetf to ribosomal particles via ternary-complex formation is detectably increased by 3 h after injection of 1 alpha,25-dihydroxycholecalciferol, the active form of vitamin D. Cholecalciferol and ergocalciferol under the same experimental conditions failed to stimulate Met-tRNAMetf-binding activity.

scholarly journals Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid

1979 ◽  
Vol 177 (2) ◽  
pp. 707-719 ◽  
Author(s):  
Petr Pohlreich ◽  
Oldřich Kříž ◽  
Zdena Tuháčková ◽  
Zdeněk Dušek ◽  
Jan Hradec
Keyword(s):  
Ternary Complex ◽  
Gradient Centrifugation ◽  
Ribosomal Subunit ◽  
Sucrose Density Gradient ◽  
Regulatory Function ◽  
Stable Complex ◽  
Sucrose Density Gradient Centrifugation ◽  
Initiator Trna ◽  
Protein Factor ◽  
Ribosomal Complex

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNAfMet and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNAMet or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8–10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNAfMet is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNAfMet and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1–2mm). The ternary complex of the factor with methionyl-tRNAfMet and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNAfMet not required for translation.

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.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

scholarly journals A glycoprotein associated with the membrane fraction of human B but not T lymphocytes.

1975 ◽  
Vol 142 (6) ◽  
pp. 1416-1424 ◽  
Author(s):  
S Fujita ◽  
S D Litwin ◽  
N Hartman
Keyword(s):  
Membrane Fraction ◽  
Gradient Centrifugation ◽  
Human Lymphocytes ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Sucrose Density Gradient ◽  
Peripheral Blood Lymphocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Lymphoid Lines

A method is described which employs differential centrifugation and sucrose density gradient centrifugation to isolate a membrane fraction from human lymphocytes. Membrane preparations from long-term human cultured B- and T-lymphoid lines, peripheral blood lymphocytes, tonsillar lymphocytes, and thymocytes were analyzed on 0.5% sodium dodecyl sulfate-7.5% polyacrylamide gels stained for protein and carbohydrate. The most important finding was a major glycoprotein of approximately 30,000 daltons associated with the membrane preparations from B lymphocytes. T-lymphocyte preparations did not contain readily detectable amounts of this membrane-associated component. The T-cell lymphoid line MOLT-4 was unique in that it had a narrow protein band at approximately 30,000 daltons which did not contain carbohydrate.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

Sign in / Sign up

Export Citation Format

Share Document