The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf

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Abstract
Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3-5-fold when Na thioglycollate (30 m[image]) and Mg chloride (16 m[image]) were included in the extraction medium. Omitting sucrose (0.45 [image]) from the extraction medium did not alter the activity. Activity was a linear function of enzyme concentration up to 1 disk (30 mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1 m[image]) was added to the reaction mixture. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity Km [Michaelis constant] (ATP) was 0.65 m[image] and Km (L-amino acids) was 70 (i[image]. Activity above the control value was found with all amino acids andlunides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.