Bacterial protease ECP32 specifically hydrolyzing actin and its effect on cytoskeleton in vivo

A procedure for isolation of bacterial protease ECP32 yielding 100 mug of t he enzyme from 10 liters of the Escherichia coil strain A2 liquid culture h as been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation containe d about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-spe cific antibodies have been produced, and a two-stage procedure for the isol ation of protease ECP32 involving affinity chromatography has been elaborat ed. Microinjection of the purified ECP32 into Amoeba proteus cells caused r eversible distortions in amoeba locomotion. The effect was not observed upo n inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysi s of actin functions in vivo.