scholarly journals EVIDENCE FOR REGULATION OF PROTEIN SYNTHESIS AT THE TRANSLATION LEVEL IN RESPONSE TO DIETARY ALTERATIONS

1967 ◽  
Vol 33 (3) ◽  
pp. 657-663 ◽  
Author(s):  
Alexandra von der Decken
Keyword(s):  
Amino Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Amino Acid Incorporation ◽  
Sucrose Density Gradient Centrifugation ◽  
Protein Ratio ◽  
Acid Incorporation ◽  
Protein Free Diet ◽  
Protein Rich

Rats were maintained for several days on a protein-free diet which, approximately 14 hr before decapitation, was changed to a protein-rich diet. Microsomal subfractions of liver were obtained by sucrose density gradient centrifugation. The sedimentation patterns of the microsomal subfractions and of the polysomes were found to be unchanged after the alteration of the diet whereas the ability to incorporate amino acid was markedly enhanced. No significant differences in the RNA/protein ratio of the microsomal subfractions were observed. The changes in amino acid incorporation which were unrelated to concurrent changes in the amount of polysomes are discussed.

Interaction of DNA with ribosomes in cell-free protein synthetizing systems of Chlorella pyrenoidosa

1969 ◽  
Vol 24 (3) ◽  
pp. 321-327 ◽  
Author(s):  
G. Galling
Keyword(s):  
Actinomycin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Chlorella Pyrenoidosa ◽  
Sucrose Density Gradient ◽  
Amino Acid Incorporation ◽  
Sucrose Density Gradient Centrifugation ◽  
Free Protein ◽  
Green Alga Chlorella ◽  
Kinetics Of

1. In cell-free protein synthetizing systems of the green alga Chlorella pyrenoidosa, DNA from various sources enhances the amino acid incorporation.2. This stimulation is neither inhibited by actinomycin D nor by chloramphenicol or cycloheximide (actidione).3. In the presence of ribonuclease, some precipitable polypeptide is formed with DNA, although the endogenous incorporation is completely inhibited by ribonuclease.4. After sucrose density gradient centrifugation, polysomal aggregates of ribosomes with DNA are found. Electron micrographs of such polysomes show a direct association of the DNA molecule with several ribosomes.5. In Chlorella, direct translation of DNA can be obtained also in the presence of neomycin. The kinetics of this reaction are different from those of endogenous m-RNA mediated and of DNA stimulated polypeptide synthesis.

scholarly journals Inhibition of Hemoglobin Synthesis by Cyanate In Vitro

Blood ◽  
1974 ◽  
Vol 43 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Blanche P. Alter ◽  
Yuet Wai Kan ◽  
David G. Nathan
Keyword(s):  
Amino Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Hemoglobin Synthesis ◽  
Radioactive Amino Acid ◽  
A Cell ◽  
Red Cell Survival

Abstract Cyanate inhibits sickling and prolongs red cell survival in sickle cell anemia. However, cyanate markedly inhibits hemoglobin synthesis in vitro. Incorporation of radioactive amino acid into hemoglobin by human sickle reticulocytes or bone marrow and by rabbit reticulocytes (whole cell or cell-free lysate) was inhibited by as little as 2 mM cyanate and abolished by 50 mM. Both alpha- and beta-S chains were equally affected. The inhibition was only partially reversible by washing the cells after exposure to cyanate. Transport of radioactive amino acid into the cell was not impaired, and free intracellular amino acid was not carbamylated. Aminoacylation of transfer RNA was not inhibited; the acylated amino acid was not carbamylated. Examination of polysome patterns by sucrose density gradient centrifugation revealed degradation of polysomes to monosomes, suggesting inhibition of initiation of protein synthesis by cyanate. In a cell-free lysate, cyanate prevented hemin stimulation of initiation. We conclude that cyanate profoundly inhibits initiation of hemoglobin synthesis in vitro.

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 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.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

scholarly journals Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

1980 ◽  
Vol 185 (3) ◽  
pp. 667-677 ◽  
Author(s):  
J Elliott ◽  
S G Blanchard ◽  
W Wu ◽  
J Miller ◽  
C D Strader ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Selective Extraction ◽  
Synaptic Membranes ◽  
Detergent Solution ◽  
Major Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Torpedo Californica

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

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