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The tyrosyl-tRNA synthetase from Escherichia coli binds only 1 mol of tRNA, tyrosine, and tyrosyl adenylate per mol of enzyme dimer. However, like the enzyme from Bacillus stearothermophilus, once one active site is occupied by tyrosyl adenylate the other becomes accessible to bind a further molecule each of tyrosine and ATP. Both bacterial enzymes show biphasic kinetics with respect to tyrosine in the aminoacylation of tRNA. Equilibrium dialysis experiments show that this is due to 2 mol of tyrosine binding in the presence of ATP and tRNA. A method is given for a correction for the effects of hydrolysis of the charged tRNA on the aminoacylation kinetics.

A simple, rapid, and economical procedure is described for the determination of the number of catalytically competent active sites on aminoacyl-tRNA synthetases based on the stoichiometry of aminoacyl adenylate formation. On mixing tRNA synthetase, cognate amino acid, (gamma-32P)ATP, and inorganic pyrophosphatase under suitable conditions there is an initial rapid stoichiometric "burst" (rate constant k1) of depletion of ATP as enzyme bound aminoacyl adenylate is formed. There is then an initially linear decrease in ATP concentration as the complex hydrolyzes (with rate constant k2) releasing enzyme to form further adenylate. Provided k2 less than k1 the initial burst gives the stoichiometry of aminoacyl adenylate formation. Complexes which are too unstable to be isolated by the usual gel or nitrocellulose disk filtration procedure may be assayed in this way. This technique has been applied to five highly purified aminoacyl-tRNA synthetases. The tyrosyl-tRNA synthetase from Bacillus stearothermophilus is shown to bind only one aminoacyl adenylate per dimer.

A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus. This has been used to measure the initial rate of acylation of tRNATyr by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme, and also the rate constants of the partial reactions of amino acid activation and transfer to the tRNA. It is shown that at saturating conenctration of tRNA the reaction proceeds by a ternary complex mechanism. The rate-determining step is either the aminoacyltion process or a step preceding it. At low concentrations of tRNA the reaction proceeds by the stepwise process of formation of tyrosyl adenylate followed by acylation of the tRNA. The rate constants for these partial reactions are faster than that for the ternary complex reaction. But the prior binding of tRNA greatly decreases the rate of tyrosyl adenylate formation. Both pathways are probably important at physiological concentrations. 88% of the tyrosine from the tyrosyl adenylate complex is transferred to tRNA. The presence of added tyrosine and ATP reduces this to 78%. However, the addition of aliquots of ATP to a mixture of enzyme, tyrosine, and a saturating concentration of tRNA (i.e., ternary complex conditions) leads to at least 0.97 mol of tRNA being acylated/mol of ATP hydrolyzed. Trapping experiments show that the 12% of adenylated that is not transferred to tRNA is hydrolyzed on the enzyme rather than expelled into solution.

A procedure is presented for the purification of 3 g of homogeneous methionyl-tRNA synthetase and 1 g of homogeneous tyrosyl-tRNA synthetase from 50 kg batches of Escherichia coli. The procedure permits the isolation of many enzymes simultaneously and the elution positions of seven other aminoacyl-tRNA synthetases, catalase, rhodanese, phosphofructokinase, elongation factor Tu and cytochrome b-562 are indicated. The problems of extraction work on this scale are discussed.

Alamethicin, an antibiotic that can transport cations and induce action potentials in synthetic membranes, is shown to be a cyclic peptide with 18 residues including 7-alpha-aminoisobutyric acid residues, two glutamine residues and one free carboxyl group. The composition indicates microheterogeneity. Alamethicin itself and many peptides derived from it are immune to enzymic digestion, but specific partial acid cleavages have allowed determination of the complete sequence. Diborane reduction has shown that the alpha-carboxyl group of glutamine-18 is free, but the ring is formed by a peptide bond between the imino group of proline-1 and the gamma-carboxyl group of glutamic acid-17. The structure is contrasted with that of other cation-transporting antibiotics. Model building allows a structure that could stack to form a tunnel with a lipophilic exterior and hydrophilic interior and flexible internal arms formed by the pendant C-terminal glutamine residue.