title: Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop.
abstract: 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 and a small N-terminal peptide, which remains attached to the core fragment [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 actin core of 37 kDa and a nicked 4.4 kDa N-terminal peptide. Here we describe a procedure for purifying the actin core fragment and the attached N-terminal peptide from the linking pentapeptide comprising amino acid residues 43-47 under native conditions by anion exchange chromatography. After removal of the pentapeptide, the salt-induced polymerization 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 earlier reports proposing that cleavage in the DNase I binding loop is affecting the ion induced polymerization of actin [Higashi-Fujime, S., et al. (1992) J. Biochem. (Tokyo) 112, 568-572; and Khaitlina, S., et al. (1993) Eur. J. Biochem. 218, 911-920]. Monomeric and filamentous subactin exhibited reduced abilities to inhibit deoxyribonuclease I (DNase I) and to stimulate the myosin subfragment 1 ATPase activity. Direct binding of subactin to DNase I was verified by gel filtration and to myosin subfragment 1 by affinity chromatography, chemical cross-linking, and electron microscopy.
authors.affiliation: Institut für Klinische Cytobiologie and Cytopathologie, Philipps-Universität, Marburg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Kiessling
authors.forename: W
authors.initials: W
authors.lastname: Jahn
authors.forename: G
authors.initials: G
authors.lastname: Maier
authors.forename: B
authors.initials: B
authors.lastname: Polzar
authors.forename: H G
authors.initials: HG
authors.lastname: Mannherz
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010590:Phalloidine / Q000494:pharmacology; D011817:Rabbits; D013381:Subtilisins / Q000378:metabolism*
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010590:Phalloidine; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I; D013381:Subtilisins; D000251:Adenosine Triphosphatases