Stimulation Of Phenylalanine Incorporation Research Articles

Beta-adrenoceptor stimulation attenuates the hypertrophic effect of alpha-adrenoceptor stimulation in adult rat ventricular cardiomyocytes

OBJECTIVESThe study investigated whether β-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine.BACKGROUNDIn adult ventricular cardiomyocytes, stimulation of α- but not β-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously α- and β-adrenoceptors. We investigated whether β-adrenoceptor stimulation interferes with the hypertrophic response caused by α-adrenoceptor stimulation.METHODSAdult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape.RESULTSNorepinephrine (0.01 to 10 μmol/liter) increased concentration-dependent phenylalanine incorporation; pEC50value was 5.9 ± 0.1 (n = 8). The α1-adrenoceptor antagonist prazosin (0.1 μmol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 μmol/liter) and the β1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 μmol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the β2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of β1-adrenoceptors increased the amount of protein kinase C-α and -δ isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of β1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3′, 5′-cyclic phosphorothiolate-Rp). The inhibitory effect of β1-adrenoceptor stimulation on the α-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding.CONCLUSIONSIn isolated ventricular cardiomyocytes from rats, β1-adrenoceptor stimulation attenuates the hypertrophic response evoked by α1-adrenoceptor stimulation.

Protein Synthesis in Cell-Free Extracts from a Thermoacidophilic Archaebacterium

An in vitro polypeptide synthesizing system was developed from the thermoacidophilic archaebacterium, Sulfolobus solfataricus. The stimulation of phenylalanine incorporation by poly U was entirely dependent upon the addition of polyamines or increasing the Mg++ concentration. The largest stimulation was observed at 3 mM spermine in the presence of 10 mM Mg++. The optimal temperature for poly U-directed phenylalaline incorporation was 75 °C. The ability of poly G to code for glycine could be demonstrated in assays incubated at elevated temperatures. When tested at 45 °C, streptomycin and neomycin did not inhibit the poly U-phenylalanine reaction except in the case of neomycin at the highest concentration tested. Neither aminoglycoside induced significant miscoding for leucine at any concentration tested. Chloramphenicol inhibited poly UG-directed phenylalanine incorporation to a small extent. Tetracycline, virginiamycin and cycloheximide were ineffective in inhibiting polyphenylalanine synthesis, but this reaction was slightly inhibited by anisomycin at the highest concentration tested

Changes in polysomes of the developing rat brain.

Abstract— Rat brain polysomes were prepared from a deoxycholate‐treated postmito‐chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation. The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6‐day‐old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes. The rate of [14C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals. The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals. The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid‐soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra‐acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals. The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence. Polysomal preparations also contained RNase, phosphomonoesterase, phospho‐diesterase and 5′‐nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

Effect of dihydrostreptomycin on protein synthesis in whole cells and in cell-free extracts of a streptomycin-dependent strain of Escherichia coli B.

The effect of dihydrostreptomycin on the incorporation of amino acids into protein in antibiotic-deprived cells of a streptomycin-dependent strain of Escherichia coli B has been compared with its effect on protein synthesis in extracts from cells of the same strain. Stimulation of phenylalanine incorporation into protein in whole cells occurred within 5 min of addition of dihydrostreptomycin to a deprived culture and was maximal at an antibiotic concentration of 20 mug/ml. Stimulation of protein synthesis in cell-free extracts from antibiotic-deprived cells was maximal at a dihydrostreptomycin concentration of 10 mug/ml in systems programmed with f2-ribonucleic acid and poly AGU, whereas extracts from cells grown on nonlimiting concentrations of dihydrostreptomycin were unaffected by the addition of antibiotic. These results indicate that protein synthesis is an antibiotic-requiring process in streptomycin-dependent E. coli B.

The response of rat liver polysomes to added homopolynucleotides: The removal of inactive ribosomes

The addition of poly U, poly I and poly A to rat liver polysomes promotes the incorporation of a number of amino acids not coded for either normally or ambiguously by the added polynucleotides. Part of the stimulation is due to protection by the added polynucleotides of the endogenous messenger from nuclease attack, but we suggest most of this stimulation is caused by the removal of inactive or inoperative ribosomes from the native polysomes. The active ribosomes left are then able to translate more of the endogenous messenger. Low concentrations of puromycin stimulate translation of polysomes that have stopped incorporating, probably by removing a block constituted by inoperative ribosomes that are unable to release their peptide chains. Stimulation of phenylalanine incorporation is observed on addition of poly U to rat liver polysomes in a cell-free system. To see whether the ribosomes present as polysomes or the monomeric ribosomes contaminating the polysomes were responsible for the stimulation, use was made of poly I to block the initiation of ribosomes prior to the addition of poly U. Evidence from this type of experiment and also experiments with fractionated polysomes indicates that the stimulation is partly due to active monomeric ribosomes present in the polysome preparation and partly due to active monomeric ribosomes derived from the polysomes during the process of translation.

Azaguanine inhibition of protein synthesis: II. Effects of poly (U) in Bacillus cereus

1. Synthetic exogenous messenger RNA (poly (U)) was added to extracts of control and azaguanine-treated Bacillus cereus to see if the inhibition of polypeptide synthesis would be abolished, thus indicating that azaguanine was inhibiting protein synthesis at the messenger RNA (mRNA) level. 2. Using [ 14C]phenylalanine as a substrate, azaguanine extracts incorporated 3–5-fold more label than control cells in the presence of poly (U). The site of this stimulation was found to be in the ribosome-free supernatant (S-150) of the azaguanine extract. When control [ 14C]phenylalanyl transfer RNA from B. cereus was used, the stimulation of phenylalanine incorporation by azaguanine extracts in presence of poly (U) was abolished. Dialysis or passage of the S-150 fractions through Sephadex G-25 also eliminated the stimulation. Finally, using control [ 14C]phenylalanyl-tRNA as substrate, azaguanine extracts showed 10 % incorporation of phenylalanine into proteins compared with control extracts; poly (U)-directed polyphenylalanine synthesis in azaguanine extracts was again unimpaired. 3. It is concluded that azaguanine inhibits protein synthesis by its effect on mRNA, since addition of exogenous messenger reverses inhibition of polypeptide synthesis. The stimulation of phenylalanine incorporation in azaguanine extracts when [ 14C]phenylalanine was the substrate appears to be due to a decrease in the endogenous pool of phenylalanine which diluted added [ 14C]phenylalanine less than in the control extract.

Stimulation of phenylalanine incorporation in hepatic polyribosome preparations by polyuridylic acid.

Stimulation of phenylalanine incorporation in hepatic polyribosome preparations by polyuridylic acid.

Polyuridylic acid stimulation of phenylalanine in human ribonucleoprotein particles.

THE discovery that synthetic polynucleotides can direct the incorporation into protein of specific amino-acids in cell-free bacterial systems1,2 has been extended to include sub-cellular systems in rat liver3, and mouse plasma cell tumour4, where coding units for amino-acid incorporation were found to be similar to the Escherichia coli system. Weinstein and Schechter4 also reported polyuridylic acid (poly U) stimulation of phenylalanine incorporation into crude cell-free fractions of normal mouse and rat livers, a rat liver tumour, rabbit reticulocytes and a human plasma cell tumour. The experiments reported here support the applicability of this type of study to various human tissues. Poly U was found to stimulate the incorporation of phenylalanine into ribonucleoprotein (RNP) particles of human liver, ovary, lung and prostate. Rat liver RNP preparations are similarly stimulated.

INHIBITION OF PHENYLALANINE INCORPORATION BY POLYURIDYLIC ACID.

ADDITION of polyuridylic acid (poly U) to cell-free systems prepared from a number of tissues results in enhanced incorporation of phenylalanine into polypeptide material1–4. Arnstein et al.2 noted that, when poly U was added to a reticulocyte ribosome system, the incorporation of other amino-acids was not inhibited and they concluded that natural messenger RNA (m-RNA) was not displaced from the ribosomes by the poly U. They questioned whether the poly U effect on phenylalanine incorporation had any physiological significance. Weinstein et al.5 later showed that incorporation of some amino-acids was inhibited when poly U was added to reticulocyte ribosomes. The present report concerns an instance when poly U, added to rat liver polysomes prepared in the absence of magnesium, actually inhibited phenylalanine incorporation. It is argued that this anomalous occurrence, paradoxically, strengthens the concept that poly U stimulation of phenylalanine incorporation is a reflexion of physiological events.

Stimulation of phenylalanine incorporation by polyuridylic acid in homogenates of sea urchin eggs

Stimulation of phenylalanine incorporation by polyuridylic acid in homogenates of sea urchin eggs

Investigations on the polyuridylic acid-dependent stimulation of phenylalanine incorporation in Escherichia coli cell-free systems

Investigations on the polyuridylic acid-dependent stimulation of phenylalanine incorporation in Escherichia coli cell-free systems