The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNAVal from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNAVal to valyl-tRNA synthetase from B. stearothermophilus were determined between 5° and 60°C.
