PURIFICATION AND CHARACTERIZATION OF SUBTILISIN CLEAVED ACTIN LACKINGTHE SEGMENT OF RESIDUES-43-47 IN THE DNASE-I BINDING LOOP

The protease subtilisin has been reported to cleave skeletal muscle G- actin between Met 47 and Gly 48 generating a core fragment of 33 kDa a nd a small N-terminal peptide, which remains attached to the core frag ment [Schwyter, D., Phillips, M., & Reisler, E. (1989) Biochemistry 28 , 5889-5895]. However, amino acid sequencing and mass spectroscopy of subtilisin cleaved-actin revealed two cleavage sites, one between Met 47 and Gly 48 and a second between Gly 42 and Val 43, generating an ac tin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we de scribe a procedure for purifying the actin core fragment and the attac hed N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromato graphy. after removal of the pentapeptide, the salt-induced polymeriza tion of actin was abolished. However, the purified fragments could be polymerized by addition of salt plus myosin subfragment 1 or salt plus phalloidin as shown by sedimentation and fluorescence increase using N-(1-pyrenyl)iodoacetamide labeled actin. These results confirm earlie r reports proposing that cleavage in the DNase I binding loop is affec ting the ion induced polymerization of actin [Higashi-Fujime, S., et a l. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subac tin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct b inding of subactin to DNase I was verified by gel filtration and to my osin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.