scholarly journals The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

1975 ◽  
Vol 146 (1) ◽  
pp. 1-12 ◽  
Author(s):  
G Ragnotti ◽  
M G Aletti
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Protein Biosynthesis ◽  
Liver Weight ◽  
Subcellular Fractions ◽  
Intrinsic Defect ◽  
Preliminary Treatment ◽  
Membrane Bound ◽  
Stimulation Of

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.

scholarly journals Biotin-mediated protein biosynthesis

1974 ◽  
Vol 140 (3) ◽  
pp. 549-556 ◽  
Author(s):  
R. L. Boeckx ◽  
K. Dakshinamurti
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Liver ◽  
Intestinal Mucosa ◽  
Protein Biosynthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.

Protein synthesis is activated in primed neutrophils: a possible role in inflammation

1987 ◽  
Vol 7 (11) ◽  
pp. 881-890 ◽  
Author(s):  
Valerie Hughes ◽  
John M. Humphreys ◽  
Steven W. Edwards
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Protein Biosynthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Inflammatory Agent ◽  
Maximal Stimulation ◽  
Stimulation Of ◽  
Primed Neutrophil

Circulating human neutrophils exhibited low rates of protein biosynthesis, as determined by their ability to incorporate [35S]methionine into TCA-precipitable material. Exposure of cells to the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased their rate of protein synthesis, and the maximal stimulation of biosynthesis by this inflammatory agent was observed at 0.1 μM: this concentration of chemotactic peptide “primed” neutrophil activity and only activated the oxidase of these cells by 8% of maximum. The newly-synthesized proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those synthesized in control cells. Two classes of proteins were observed in “primed” cells. The first of these comprised proteins whose rate of biosynthesis changed very little upon “priming” whereas the second class comprised proteins whose rate of synthesis increased greatly after exposure to chemotactic peptide. The fMet-Leu-Phe stimulated protein synthesis was inhibited by actinomycin D and cycloheximide showing that this phenomenon required both transcription and translation. We propose that these fMet-Leu-Phe regulated proteins play an important role in the function of neutrophils during an inflammatory response.

scholarly journals Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1975 ◽  
Vol 152 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Subcellular Fractions ◽  
Anterior Lens Capsule ◽  
Cartilage Cells ◽  
Membrane Bound ◽  
And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

scholarly journals Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver

1992 ◽  
Vol 288 (2) ◽  
pp. 413-419 ◽  
Author(s):  
J Wilkinson ◽  
J A Higgins ◽  
P H E Groot ◽  
E Gherardi ◽  
D E Bowyer
Keyword(s):  
Endoplasmic Reticulum ◽  
Apolipoprotein B ◽  
Intracellular Distribution ◽  
Subcellular Fractions ◽  
Rabbit Liver ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Golgi Region ◽  
Membrane Bound ◽  
Vldl Assembly

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

scholarly journals PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

1974 ◽  
Vol 62 (1) ◽  
pp. 20-31 ◽  
Author(s):  
Kou M. Hwang ◽  
Linda C. Yang ◽  
Christine K. Carrico ◽  
Rose A. Schulz ◽  
John B. Schenkman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Cytochrome C ◽  
Peptide Synthesis ◽  
Reductase Activity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Nascent Peptide ◽  
Membrane Bound ◽  
Nadph Cytochrome C Reductase

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [3H]leucine and of [3H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [3H]leucine incorporation into cytoplasmic membranes was inhibited, while [3H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.

scholarly journals Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma

1975 ◽  
Vol 152 (1) ◽  
pp. 143-151 ◽  
Author(s):  
C N Murty ◽  
E Verney ◽  
H Sidransky
Keyword(s):  
Protein Synthesis ◽  
Initiation Factors ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Triton X ◽  
Stimulation Of

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken

1978 ◽  
Vol 170 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Rosemary Spencer ◽  
Marilyn Charman ◽  
Peter W. Wilson ◽  
D. Eric M. Lawson
Keyword(s):  
Vitamin D ◽  
Protein Synthesis ◽  
Calcium Binding ◽  
Binding Protein ◽  
Protein Biosynthesis ◽  
Calcium Content ◽  
Calcium Binding Protein ◽  
Intracellular Protein ◽  
Maximum Increase ◽  
Stimulation Of

1. The rapid stimulation of intestinal Ca2+transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca2+transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca2+transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca2+transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25–25μg of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca2+transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca2+transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca2+transport, which is initiated by other factors.

Ultrastructural and Cytochemical Manifestations of Protein Synthesis in the Peripheral Sarcoplasm of Denervated and Newborn Skeletal Muscle Fibres

1974 ◽  
Vol 14 (1) ◽  
pp. 113-137
Author(s):  
GERALDINE F. GAUTHIER ◽  
SUSAN F. SCHAEFFER
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Cell Surface ◽  
Phrenic Nerve ◽  
Muscle Fibres ◽  
Preliminary Treatment ◽  
Rat Diaphragm ◽  
Newborn Rat ◽  
Skeletal Muscle Fibres

In muscle fibres of the rat diaphragm, there is widespread accumulation of free ribosomes and rough-surfaced endoplasmic reticulum in the subsarcolemmal sarcoplasm at 28 days following section of the phrenic nerve. A corresponding accumulation of basophilic material is visible at the periphery of the fibres when examined with the light microscope. Both the ribosomes and the basophilic material are removed by preliminary treatment with ribonuclease. Fibres comprising the diaphragm of the newborn rat display similar aggregations of subsarcolemmal ribosomes and peripheral basophilia. These ultrastructural and cytochemical manifestations of protein synthesis correspond closely in time to the well documented ‘supersensitivity’ to acetylcholine, which occurs along the entire length of muscle fibres in the newborn and denervated rat diaphragm. It is suggested, therefore, that this protein-synthetic machinery, which is concentrated at the cell surface, is involved in the formation of new receptors.

Further evidence that protein synthesis can be decreased in vivo following hormonal stimulation in the rat pancreas

1976 ◽  
Vol 54 (3) ◽  
pp. 305-313 ◽  
Author(s):  
R. Mongeau ◽  
J. C. Dagorn ◽  
J. Morisset
Keyword(s):  
Protein Synthesis ◽  
Single Injection ◽  
Protein Biosynthesis ◽  
Specific Radioactivity ◽  
Hormonal Stimulation ◽  
In Vitro System ◽  
Precursor Pool ◽  
Stimulation Of

The present study has been undertaken to determine in the rat the influence of exocrine secretory stimulation on pancreatic protein synthesis. This stimulant consisted of a single injection of cholecystokinin–pancreozymin (8 Ivy units/kg) plus secretin (5 clinical units/kg). The rate of [14C]phenylalanine incorporation into total proteins was measured 5, 11, 17, 30, 45 and 60 min later. Incorporation was significantly decreased after 5 min, then significantly increased at 17 min, and finally returned to control values at 45 min. This biphasic evolution was shown not to be caused by variations in the precursor pool specific radioactivity. We concluded that secretory stimulation of the pancreas can induce a decrease in the rate of protein biosynthesis. This decrease is nevertheless a transient phenomenon, since the rate of biosynthesis was increased at 17 min. These results, obtained from a totally in vivo system, confirm previous data obtained from an in vivo – in vitro system.

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