scholarly journals The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1969 ◽  
Vol 113 (1) ◽  
pp. 117-121 ◽  
Author(s):  
L. Stevens
Keyword(s):  
Ionic Strength ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Ribosomal Ribonucleic Acid ◽  
Number Of Binding Sites ◽  
Filtration Technique

1. The total intracellular concentrations of Na+, K+, Mg2+, spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4–60°. 5. Optimum binding occurred at about pH7·0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0·10–0·13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg2+ on equal terms for sites on the ribosomes.

scholarly journals Identical 3′-terminal octanucleotide sequence in 18S ribosomal ribonucleic acid from different eukaryotes. A proposed role for this sequence in the recognition of terminator codons

1974 ◽  
Vol 141 (3) ◽  
pp. 609-615 ◽  
Author(s):  
John Shine ◽  
Lynn Dalgarno
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Drosophila Melanogaster ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
18S Rrna ◽  
Base Sequence ◽  
Terminal Sequence ◽  
Ribosomal Ribonucleic Acid ◽  
Rrna Species

The 3′-terminal sequence of 18S ribosomal RNA from Drosophila melanogaster and Saccharomyces cerevisiae was determined by stepwise degradation from the 3′-terminus and labelling with [3H]isoniazid. The sequence G-A-U-C-A-U-U-AOH was found at the 3′-terminus of both 18S rRNA species. Less extensive data for 18S RNA from a number of other eukaryotes are consistent with the same 3′-terminal sequence, and an identical sequence has previously been reported for the 3′-end of rabbit reticulocyte 18S rRNA (Hunt, 1970). These results suggest that the base sequence in this region is strongly conserved and may be identical in all eukaryotes. As the 3′-terminal hexanucleotide is complementary to eukaryotic terminator codons we discuss the possibility that the 3′-end of 18S rRNA may have a direct base-pairing role in the termination of protein synthesis.

Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ischemia Reperfusion ◽  
Age Groups ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Site Model ◽  
Number Of Binding Sites ◽  
Competitive Binding Assays

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

scholarly journals Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

1981 ◽  
Vol 1 (1) ◽  
pp. 75-81 ◽  
Author(s):  
M Sameshima ◽  
S A Liebhaber ◽  
D Schlessinger
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Protein Turnover ◽  
Rna Turnover ◽  
Turnover Rates ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Cytoplasmic Rna ◽  
Ribosomal Ribonucleic Acid ◽  
I Cells

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).

scholarly journals Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose

1979 ◽  
Vol 178 (3) ◽  
pp. 581-587 ◽  
Author(s):  
A Rotondi ◽  
F Auricchio
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Large Scale ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Receptor Aggregation ◽  
Low Salt ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In ‘low-salt’ buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

scholarly journals Deoxyribonucleic acid–ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids

1969 ◽  
Vol 115 (2) ◽  
pp. 287-294 ◽  
Author(s):  
Michael Fry ◽  
Michael Artman
Keyword(s):  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Deoxyribonucleic Acid ◽  
Cellulose Nitrate ◽  
Absolute Size ◽  
Rna Molecules ◽  
Membrane Filters ◽  
Ribosomal Ribonucleic Acid

A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide–0·33m-potassium chloride–10mm-tris–hydrochloric acid buffer, pH7·4, at 33° for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.

scholarly journals Characterization of the vitamin D-dependent Ca2+-binding sites in rat intestinal Golgi-enriched membrane fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 347-354 ◽  
Author(s):  
J R F Walters ◽  
M M Weiser
Keyword(s):  
Vitamin D ◽  
Ionic Strength ◽  
Vitamin D Deficiency ◽  
Binding Sites ◽  
Control Diet ◽  
Specific Protein ◽  
High Affinity ◽  
Equilibrium Binding ◽  
Number Of Binding Sites

Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules.

Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

1985 ◽  
Vol 63 (2) ◽  
pp. 128-136
Author(s):  
Deborah A. Brown ◽  
Robert A. Cook
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Binding Sites ◽  
Malic Enzyme ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Regulatory Effects ◽  
Metal Cofactors

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

scholarly journals Changes in the number of binding sites for ribonucleic acid polymerase in chromatin of WI-38 fibroblasts stimulated to proliferate

1974 ◽  
Vol 141 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Bridget T. Hill ◽  
Renato Baserga
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Template Activity ◽  
E Coli ◽  
Human Diploid Fibroblasts ◽  
Chromatin Template ◽  
Number Of Binding Sites

1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [3H]UTP into RNA in the absence of reinitiation, and incorporation of [γ-32P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.

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