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Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. the interaction of cholesteryl 14-methylhexadecanoate.

ABSTRACT: 1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11-26% and the simultaneous binding of GTP to 32-43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA-GTP-transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.

SUBMITTER: Hradec J

PROVIDER: S-EPMC1178546 | biostudies-other | 1972 Mar

REPOSITORIES: biostudies-other

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Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. Formation and properties of an aminoacyl-transfer ribonucleic acid-transferase I complex.

Project description:1. Transferase I of rat liver binds aminoacyl-tRNA to form a relatively stable complex, which is retained on cellulose nitrate filters. This reaction proceeds at both 0 degrees C and 37 degrees C and is inhibited by GTP. The resulting product is stabilized by GTP and Mg(2+). 2. Only very low quantities of deacylated tRNA are bound by transferase I. 3. Methods are described for the preparative isolation of the transferase I-aminoacyl-tRNA complex from incubation mixtures by using ion-exchange procedures. 4. The transferase I-aminoacyl-tRNA complex becomes readily bound to ribosomes. The presence of Mg(2+) is essential for the binding. GTP stimulates this reaction but is not absolutely required. 5. It is concluded that the formation of the transferase I-aminoacyl-tRNA complex may be the primary reaction in the binding of aminoacyl-tRNA to mammalian ribosomes and that, unlike in bacterial systems, GTP is not absolutely required for this step.

| S-EPMC1178500 | biostudies-other

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The role of guanosine triphosphate.

Project description:1. Transferase I from rat liver binds relatively low quantities of GTP when incubated with this nucleotide in the absence of aminoacyl-tRNA. 2. Transferase I reacts with both aminoacyl-tRNA and GTP to form a relatively stable complex that is retained on cellulose nitrate filters. The ternary complex transferase I-aminoacyl-tRNA-GTP is also formed when the transferase I-aminoacyl-tRNA complex is incubated with GTP or during the incubation of the transferase I-GTP complex with aminoacyl-tRNA. Synthesis of this complex does not require the presence of Mg(2+). 3. In the presence of Mg(2+) the ternary complex becomes readily bound to ribosomes without requirements for any other cofactors. 4. An extensive cleavage of GTP takes place when aminoacyl-tRNA becomes bound to ribosomes. 5. The low interdependence of reactions leading to the formation of transferase I complexes with aminoacyl-tRNA and GTP indicates that the mechanisms of the binding reaction in mammalian systems may be different from those in bacterial cells.

| S-EPMC1178501 | biostudies-other

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions.

Project description:1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.

| S-EPMC1177096 | biostudies-other

Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues.

Project description:1. l-Tyrosine-, l-alanine-, l-tryptophan- and l-threonine-tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70-100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid-tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.

| S-EPMC1185228 | biostudies-other

Effect of lipids, in particular cholesteryl 14-methylhexadecanoate, on the incorporation of labelled amino acids into transfer ribonucleic acid in vitro.

Project description:1. Rat liver pH5 enzymes and cell sap extracted with various organic solvents showed a variable decreased incorporation of labelled amino acids into s-RNA (;soluble' or transfer RNA) in vitro. 2. The original enzymic activity could be fully restored, though at different rates, by the addition of lipid extracts in quantities corresponding to those originally present. 3. Of the main lipid groups separated from the extract, only free cholesterol and cholesteryl esters were able to reactivate the extracted pH5 enzymes in the same way as the whole lipid extract. 4. Addition of pure cholesteryl 14-methylhexadecanoate also fully restored the enzymic activity. 5. There was no energy-dependent incorporation of labelled amino acids into ribosomal protein in the presence of extracted cell sap. Addition of cholesteryl 14-methylhexadecanoate fully restored the activity of the cell sap to incorporate labelled leucine and lysine into ribosomal protein and enhanced the incorporation of labelled protein hydrolysate and phenylalanine over the level found with the corresponding non-extracted preparations. 6. It is concluded that lipids play an important role in the synthesis of aminoacyl-s-RNA complexes and that cholesteryl 14-methylhexadecanoate may be the active lipid in this respect.

| S-EPMC1187102 | biostudies-other

Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate.

Project description:1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.

| S-EPMC1174086 | biostudies-other

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation.

Project description:1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.

| S-EPMC1166189 | biostudies-other

Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity.

Project description:The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of (14)C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[(14)C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.

| S-EPMC1178388 | biostudies-other

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

Project description:The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

| S-EPMC1183673 | biostudies-other

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

Project description:The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.

| S-EPMC1183672 | biostudies-other

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