Tryptophanyl-tRNA synthetase was purified to homogeneity from human placenta with standard techniques. A neutral detergent, polyoxyethylene cetyl ether (Brij 58), stabilized the enzyme throughout the purification. The enzyme appeared to be homogeneous by standard disc gel polyacrylamide electrophoresis, but a minor contaminant was defined with multiple gels of differing porosity. Inclusion of Brij 58 in polyacrylamide gels allowed us to recover 100% of the enzyme activity and to demonstrate that the activity was associated with the major protein band. The mol wt of the erzyme is 100,000 by sucrose density gradient centrifugation, 125,000 by gel filtration chromatography, and 118,000 by electrophoretic mobility on polyacrylamide gels of differing porosity. Sodium dodecyl sulfate-polyacrylamide electrophoresis demonstrated a single band of mol wt 58,000. Polyacrylamide electrophoresis in the presence of 8 M urea revealed a single major band. Therefore, the enzyme appears to be a dimer composed of subunits of identical mass and charge. We have modified the technique of isoelectric focusing in two ways with the result that up to 100% of our enzyme activity is recoverable after focusing. The isoelectric point of human tryptophanyl-tRNA synthetase is 5.2.
|Number of pages||6|
|State||Published - Dec 1 1974|
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