Chemical and physical studies on the structure of the histidyl transfer ribonucleic acid synthetase from Salmonella typhimurium.

The histidyl-tRNA synthetase from Salmonella typhimurium was purified to homogeneity by methods described previously. The molecular weight of the native enzyme was determined as 78,000 by high speed equilibrium centrifugation and as 92,000 by gel filtration; a subunit molecular weight of about 40,000 was found by centrifugation in 6 M guanidine HCl, and by sodium dodecyl sulfate gel electrophoresis. The isoelectric point was found to be 5.9 by focusing in polyacrylamide gels; the extinction coefficient at 280 nm was determined to be 80,000 M-I cm-l. The amino acid composition of the enzyme was determined. In particular, 8 cysteine, 34 lysine, 53 arginine, and 13 histidine residues were found for each molecule, calculated on the basis of an 80,000 molecular weight. Thirteen tryptophan residues were found by absorption spectroscopy in 6 M guanidine HCl; a tyrosine content of 20 residues per 80,000 was estimated. Titration with 5,5’-dithiobis(2nitrobenzoic acid) reagent showed that 2 cysteine residues were free ; titration with 5,5’-dithiobis(2-nitrobenzoic acid) reagent in 8 M urea demonstrated 2 additional cysteine residues. A peptide map of a tryptic digest showed the presence of about 33 ninhydrin staining spots; no NH2-terminal amino acid could be detected. It is inferred that the histidyltRNA synthetase in its native state is a dimer, probably composed of identical subunits. The enzyme is stable to storage in 50% glycerol at -20’; at 4’ in dilute solution the enzyme slowly loses activity. Gel chromatography shows the appearance of a high molecular weight, inactive form during storage at 4’. This material can be reactivated by incubation with reducing agents.