Amino acid incorporation by a cell-free preparation from rat adipose tissue. Effects of fasting

Ribosomes were isolated from a postmitochondrial, detergent-treated supernatant fraction prepared either from adipose tissue passed through a tissue press, or from a collection of free fat cells. Optimum conditions for incorporation were established and the kinetics of the system denned with and without artificial messenger RNA. Studies were performed on ribosomes isolated from fed, 24-h fasted, and 48-h fasted animals. There was a progressive reduction in yield and activity (per milligram ribosomal RNA) of ribosomal material recovered from animals fasted up to 48 h. In the presence of poly-U, incorporation by ribosomes from fasted animals returned towards but did not reach the fed level. This return was less complete with ribosomes from 48-h than from 24-h fasted groups although as fasting proceeded, the percentage stimulation produced by poly-U progressively increased. Sucrose density gradient analysis revealed a greater proportion of light ribosome species and a smaller proportion of polysomes in fasted than in fed animals. These results suggest that fasting, in addition to producing a progressive reduction in ribosomal yield, also caused an early loss of messenger RNA or messenger function and later an alteration of ribosome function. The effect of fasting on messenger function could not be explained by alterations in ribonuclease activity.

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Regulation of Glucose Transporter Messenger RNA Levels in Rat Adipose Tissue by Insulin

Molecular Endocrinology ◽

10.1210/mend-4-4-583 ◽

1990 ◽

Vol 4 (4) ◽

pp. 583-588 ◽

Cited By ~ 20

Author(s):

William I. Sivitz ◽

Susan L. DeSautel ◽

Toshiaki Kayano ◽

Graeme I. Bell ◽

Jeffrey E. Pessin

Keyword(s):

Adipose Tissue ◽

Messenger Rna ◽

Glucose Transporter ◽

Rat Adipose Tissue ◽

Rna Levels

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RAPIDLY-LABELLED RNA IN THE MOUSE MAMMARY GLAND BEFORE AND DURING LACTATION

Journal of Endocrinology ◽

10.1677/joe.0.0560145 ◽

1973 ◽

Vol 56 (1) ◽

pp. 145-152 ◽

Cited By ~ 11

Author(s):

D. N. BANERJEE ◽

M. R. BANERJEE

Keyword(s):

Mammary Gland ◽

Ribosomal Rna ◽

Gradient Analysis ◽

Sucrose Density Gradient ◽

Mouse Mammary Gland ◽

Nuclear Fraction ◽

Cytoplasmic Rna ◽

Nuclear Rna ◽

Nuclear Synthesis

SUMMARY Linear sucrose density gradient analysis showed that the synthesis of rapidly-labelled high molecular weight RNA was virtually absent in the mammary glands of virgin mice. The rapidly-labelled RNA was first evident in the mammary gland in pregnancy and was also present during lactation. The bulk of this newly-made nuclear RNA sedimented as 45S and 32S fractions after a 15-min [3H]uridine pulse period in vivo. No labelled 18S RNA was detectable in the nuclear fraction after the 15-min pulse but it was present in the cytoplasm, suggesting that the 18S RNA migrates to the cytoplasm almost immediately after its formation. Thirty minutes after injection of [3H]uridine, the initial radioactivity of the 45S region migrated to the 32S fraction and a labelled 28S peak was also present in the cytoplasmic RNA at 60 min, suggesting that the processed 28S ribosomal RNA in the mammary gland began to migrate to the cytoplasm between 30 and 60 min after the nuclear synthesis of the precursor molecule.

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Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Biochemical Journal ◽

10.1042/bj1860791 ◽

1980 ◽

Vol 186 (3) ◽

pp. 791-798 ◽

Cited By ~ 9

Author(s):

John J. Lucas ◽

Helen Tepperman ◽

Jay Tepperman

Keyword(s):

Adipose Tissue ◽

High Glucose ◽

Alkaline Treatment ◽

The Other ◽

Mild Acid Hydrolysis ◽

Dolichyl Phosphate ◽

Solvent Systems ◽

Rat Adipose Tissue ◽

Kinetics Of ◽

Mild Acid

Microsomal preparations from rat adipose tissue catalyse the transfer of [14C]mannose from GDP-[14C]mannose to an endogenous acceptor forming a [14C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [14C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate. The kinetics of transfer of [14C]mannose from GDP-[14C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The Km and Vmax. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations. In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [14C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose. Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

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Characterization of Rapidly Labelled Nucleic Acids in Tissues of Plant Seedlings

Zeitschrift für Naturforschung B ◽

10.1515/znb-1966-1016 ◽

1966 ◽

Vol 21 (10) ◽

pp. 983-992 ◽

Cited By ~ 14

Author(s):

Vera Hemleben-Vielhaben

Keyword(s):

Nucleic Acids ◽

Ribosomal Rna ◽

Messenger Rna ◽

Specific Activity ◽

Supernatant Fraction ◽

Nucleic Acid Metabolism ◽

Tissue Homogenates ◽

Fraction I ◽

Plant Seedlings

The nucleic acid metabolism of plant tissues was examined by incubating seedlings of Phaseolus, Vicia, Pisum, and Soja or sections of them with 32P for short periods of time. The nucleic acids extracted from this material were fractionated on columns of methylated albumin coated on kieselgur, and the radioactivity and composition of the specific fractions were determined. Application of 32P to intact seedlings or to excised parts of seedlings resulted in the same pattern of labelled nucleic acids in all tissues, but the total amount of incorporated radioactivity was different. In all tissues investigated five rapidly labelled RNA fractions were found associated with the following components: (I) soluble RNAs, (II) DNA, (III—V) ribosomal RNA. Fraction I contained equally high amounts of CMP and GMP thus differing significantly from the soluble RNAs. The composition of fraction (II) which was probably bound to DNA in the form of a complex, was similar to that of fraction I provided the labelling period was short. Fractions III—V were of the ribosomal type. The rapidly labelled DNA fraction had a high guanine-cytosine content (60%) as compared with the bulk DNA (40%). Fractional centrifugation of the tissue homogenates revealed that the labelled RNA of the ribosomal type was partly associated with the ribosomes, partly with larger particles which were sedimented by low speed centrifugation. The RNA associated with the latter had a higher specific activity than the ribosomal RNA in the supernatant. Fraction I and the second component of the soluble RNA (s-2) were also confined to the sediment of larger particles. Actinomycin D (10 µg/ml) inhibited the incorporation of 32P in the nucleic acids. In chase experiments it caused a decrease in the specific activity of all RNA fractions, most prominently, however, in fraction I and II indicating their instability and resemblance to messenger RNA.

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Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

Biochemical Journal ◽

10.1042/bj1151063 ◽

1969 ◽

Vol 115 (5) ◽

pp. 1063-1069 ◽

Cited By ~ 28

Author(s):

T. Scott-Burden ◽

A. O. Hawtrey

Keyword(s):

Density Gradient ◽

Rat Liver ◽

Lithium Chloride ◽

Ribosomal Rna ◽

Gradient Analysis ◽

Extraction Procedure ◽

Liver Microsomes ◽

Sucrose Density Gradient ◽

Rat Liver Microsomes ◽

Phenol Extraction

1. Treatment of washed rat liver microsomes in a medium containing 0·12m-sucrose, 12·5mm-potassium chloride, 2·5mm-magnesium chloride and 25mm-tris–hydrochloric acid buffer, pH7·6, with 2m-lithium chloride at 5° for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting 14C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH–2,6-dichlorophenol-indophenol reductase, NADH–neo-tetrazolium reductase, NADH–cytochrome c reductase and ribonuclease activities. 5. 3H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of 3H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.

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A Factor Capable of Dissociating Rat Liver Ribosomes

Canadian Journal of Biochemistry ◽

10.1139/o71-189 ◽

1971 ◽

Vol 49 (12) ◽

pp. 1301-1306 ◽

Cited By ~ 15

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.

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