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A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66 000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.

Horse liver alcohol dehydrogenase is irreversibly inactivated by Procion blue MX-R, a dichlorotriazinyl structural analogue of Cibacron blue F3G-A, with over 90% loss of activity within 30 min at pH 8.5 and 37 degrees C at a reactive dye concentration of 1 mM and enzyme subunit concentration of 5 microM. Methoxylated Procion blue MX-R does not inactivate the enzyme. The inactivation of horse liver alcohol dehydrogenase by Procion blue MX-R is competitively inhibited by the pyridine nucleotides NAD+ and NADH. Quantitatively inhibited horse liver alcohol dehydrogenase contains 1 mol dye/mol subunit of Mr 40 000. Chymotryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a single blue peptide which on sequencing and analysis yields an amino acid sequence of: (formula; see text) with the affinity label, Procion blue MX-R, unambiguously identified as being attached to the thiol side chain of Cys-174 in the catalytic domain of the enzyme. The specific active-site-directed reaction of Procion blue MX-R with horse liver alcohol dehydrogenase is interpreted in terms of the known crystallographic structure of the enzyme.

A procedure is described for the purification of cysteinyl-tRNA synthetase as a side product of a multi-enzyme isolation from Bacillus stearothermophilus. The native and denatured enzyme are both shown to have a molecular weight of 54000 by gel filtration and sodium dodecyl sulphate/polyacrylamide gel electrophoresis respectively. Fingerprinting and peptide counting indicate that the polypeptide chain has a nonrepeating primary structure. The enzyme has only one binding site for each of its substrates (cysteine, ATP and tRNACys) as judged by equilibrium dialysis, active-site titration and fluorescence quenching. No evidence for the dimerisation of the enzyme in the presence of these substrates could be found. We conclude that cysteinyl-tRNA synthetase, which is the smallest aminoacyl-tRNA synthetase yet described, is both structurally and functionally monomeric.

Brown MX-5BR specifically and irreversibly inactivates tryptophanyl-tRNA synthetase from Bacillus stearothermophilus at pH 8.5. The enzyme is protected from inactivation by the substrates tryptophan and ATP and to lesser extents by ADP, AMP, the product inorganic pyrophosphate and other nucleotides such as GTP. The Kd of the pure reactive dye for the enzyme was measured to be 6.7 X 10(-6) M. The Km values of the two substrates tryptophan and MgATP were found to be 1 x 10(-5) M and 5 x 10(-5) M respectively. The aminated dye is a competitive inhibitor of tryptophanyl-tRNA synthetase with respect to both tryptophan and MgATP with Ki values of 2 x 10(-4) M against both substrates. The use of this dye as an active-site-directed affinity label is discussed.