Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

Studies on the Incorporation of Amino Acids by a Cell-Free System Obtained from Beef Retina

1972 ◽  
Vol 50 (5) ◽  
pp. 581-587 ◽  
Author(s):  
Y. Matuk
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Incubation Medium ◽  
Optimum Concentration ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The incorporation of 14C-leucine into proteins by a cell-free system from beef retina was studied. It was found that the optimum concentration of ATP depended on the concentration of ribosomes in the incubation medium. Very little incorporation of 14C-leucine was observed in the absence of K+. The optimum concentration of phosphocreatine required for incorporation of radioactive leucine depended on the concentration of Mg2+ in the incubation medium, and the optimum concentration of K+ appears to be independent of the concentrations of Mg2+ and phosphocreatine used.Retinol and retinal had no effect, but ethanol markedly inhibited protein synthesis at concentrations higher than 2%.Puromycin (10−4 M) inhibited incorporation of 14C-leucine by about 80%. The degree of inhibition by cycloheximide depended on the concentration of pH 5 fraction in the incubation medium.

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals Incorporation of 3H from δ-(l-α-amino (4,5-3H)adipyl)-l-cysteinyl-d-(4,4-3H)valine into isopenicillin N

1979 ◽  
Vol 184 (2) ◽  
pp. 421-426 ◽  
Author(s):  
J O'Sullivan ◽  
R C Bleaney ◽  
J A Huddleston ◽  
E P Abraham
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Adipic Acid ◽  
Experimental Error ◽  
Acid Oxidase ◽  
Cephalosporium Acremonium ◽  
Amino Acid Oxidase ◽  
Free System ◽  
A Cell ◽  
Isopenicillin N

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.

scholarly journals Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1969 ◽  
Vol 115 (4) ◽  
pp. 671-678 ◽  
Author(s):  
M. D. Herrington ◽  
A. O. Hawtrey
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Inhibitory Effect ◽  
Bovine Mammary Gland ◽  
Free System ◽  
Enzyme Fraction

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of 14C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of 14C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105° for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.

Peptidsynthese im zellfreien System unter dem Einfluß von Hefe-Ribonucleinsäure

1968 ◽  
Vol 23 (1) ◽  
pp. 50-58 ◽  
Author(s):  
W. Duntze ◽  
W. Atzpodien ◽  
B. Ulrich ◽  
H. Holzer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Gel Filtration ◽  
Amino Acid Incorporation ◽  
Free System ◽  
Total Rna ◽  
Acid Incorporation ◽  
Yeast Ribosomes ◽  
A Cell

Total RNA from yeast stimulates the incorporation of 14C-amino acids into material insoluble in hot trichloroacetic acid in a cell-free E. coli system. In a sucrose gradient the stimulating RNA fraction sediments together with the 28 S fraction of ribosomal RNA. RNA from isolated yeast ribosomes preincubated with RNase was active in amino acid incorporation as well.Preincubation of ribosomal RNA at 70° resulted in an increased incorporation activity of the RNA. However, attempts to separate an active messenger fraction from total RNA as well as from 28 S RNA by heating were unsuccessful. The presented data indicate that ribosomal RNA itself is active in cell-free amino acid incorporation.By hydrolysis of the incorporation products it could be shown that the 14C-amino acids used in the cell free system were incorporated into peptides. The bulk of the radioactive peptides had a molecular weight below 2000 as estimated by Sephadex gel filtration.

Thermolability of protein synthesis in a cell-free system from the obligately psychrophilic yeast Candida gelida

1969 ◽  
Vol 15 (4) ◽  
pp. 339-343 ◽  
Author(s):  
C. H. Nash ◽  
D. W. Grant ◽  
N. A. Sinclair
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Thermal Inactivation ◽  
Kinetic Studies ◽  
Temperature Sensitive ◽  
Free System ◽  
Psychrophilic Yeast ◽  
A Cell ◽  
Polypeptide Chains ◽  
Soluble Enzymes

A subcellular amino-acid-incorporating system from the obligately psychrophilic yeast, Candida gelida, was completely inhibited after incubation at 35 C for 30 minutes. The thermal inactivation of protein synthesis was due, in part, to the presence of unusually temperature-sensitive aminoacyl-sRNA synthetases in C. gelida extracts. Of the 13 specific synthetases examined, 7 retained less than 50% of their activity after being held at 35 C for 30 minutes. Kinetic studies of thermal inactivation of leucyl-sRNA synthetase demonstrated that this enzyme is 50% inactivated after only 7 minutes at 35 C. None of the 10 sRNA species tested was temperature sensitive. In addition to temperature-sensitive synthetases, C. gelida possesses thermolabile soluble enzymes involved in the formation of ribosomal-bound polypeptide chains.

Effect of Amino Acid Levels on a Cell-Free System for Protein Synthesis

1970 ◽  
Vol 135 (1) ◽  
pp. 180-183 ◽  
Author(s):  
R. W. Wannemacher ◽  
W. K. C. Cooper ◽  
K. Muramatsu
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Amino Acid Levels

scholarly journals Studies on the biosynthesis of gramicidin S in a cell-free system from Bacillus brevis. Further attempts to elucidate its mechanism of synthesis

1967 ◽  
Vol 105 (2) ◽  
pp. 451-453 ◽  
Author(s):  
K. J. Figenschou ◽  
L. O. Frøholm ◽  
S. G. Laland
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Free Amino Acid ◽  
Free Amino ◽  
Cell Free Extract ◽  
Bacillus Brevis ◽  
Free System ◽  
Cell Free System ◽  
Gramicidin S ◽  
A Cell

1. The pH optima for the incorporation of 14C-labelled amino acids into gramicidin S by an 11000g cell-free extract from Bacillus brevis have been determined. The pH optima for leucine, proline, phenylalanine, ornithine and valine were 7·5–7·7, 7·5–7·7, 7·7–7·9, 7·7–7·9 and 8·0–8·2 respectively. Hence the greatest difference in pH optima existed between leucine and valine, where it was 0·5pH unit. 2. The 11000g cell-free extract incorporated into gramicidin S only the l-isomers of valine, proline and ornithine. However, both isomers of leucine are utilized and the experiments indicate that a leucine racemase exists in the 11000g cell-free extract. With phenylalanine the l-isomer is utilized much more effectively than the d-isomer. This is noteworthy since it is the d-isomer that occurs in gramicidin S. The experiments indicate that conversion of the l-isomer into the d-form takes place at a stage beyond that of the free amino acid.

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