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  Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop

Kiessling, P., Jahn, W., Maier, G., Polzar, B., & Mannherz, H. G. (1995). Purification and characterization of subtilisin cleaved actin lacking the segment of residues 43-47 in the DNase I binding loop. Biochemistry, 34(45), 14834-14842. doi:10.1021/bi00045a026.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-4C66-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-4C67-F
Genre: Journal Article

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Kiessling, Peter1, Author              
Jahn, Werner1, Author              
Maier, Gernot, Author
Polzar, Bernhard, Author
Mannherz, Hans Georg1, Author              
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1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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 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.

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Language(s): eng - English
 Dates: 1995-09-121995-05-261995-11-14
 Publication Status: Published in print
 Pages: 9
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 Rev. Type: Peer
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Title: Biochemistry
Source Genre: Journal
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Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 34 (45) Sequence Number: - Start / End Page: 14834 - 14842 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103