Bacterial Protease ECP32 Specifically Hydrolyzing Actin and Its Effect on Cytoskeleton in vivo

A. V. Morozova, I. N. Skovorodkin, S. Yu. Khaitlina, and A. Yu. Malinin^*

Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064 Russia; fax: (812) 247-0341; E-mail: malinin@mail.cytspb.rssi.ru

^* To whom correspondence should be addressed.

Received April 19, 2000; Revision received June 28, 2000

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A procedure for isolation of bacterial protease ECP32 yielding 100 µg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.

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KEY WORDS: protease ECP32, protein purification, limited proteolysis of actin, actin, antibodies, microinjection, amoeboid locomotion

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