Purification and properties of Bacillus subtilis aspartate transcarbamylase.

Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.