The activation of Cls with purified Clr

Abstract

The Cls activator obtained in neutral euglobulin precipitates (pH 7·5, RSC 0·04) was solubilized with 0·02 M EDTA, 0·005 M L-lysine buffer (pH 7·5, RSC 0·04) and subjected to DEAE and CM cellulose column chromatography. The final preparations showed a single major protein band on analytical acrylamide gel electrophoresis with a few minor bands near the cathode. They gave a single precipitation line on immunodiffusion against a potent horse anti-whole human serum. This line completely fused into a precipitation line made with anti-Clr and these fractions. Cls activator activity corresponded to the single major band on acrylamide gel and was associated with the hemolytic activity of Clr. The spontaneous activation in highly purified Cls (Sakai and Stroud, 1973) was minimized by treating with DFP. The stabilized Cls was still susceptible to activation by Clr. In other experiments using DFP- 32P binding to the smaller subunit of C ls was demonstrated, suggesting that this peptide chain contains the active site.