title: Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1.
abstract: Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
authors.forename: D
authors.initials: D
authors.lastname: Schwyter
authors.forename: M
authors.initials: M
authors.lastname: Phillips
authors.forename: E
authors.initials: E
authors.lastname: Reisler
mesh_terms: D000199:Actins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D003850:Deoxyribonuclease I / Q000378:metabolism; D066298:In Vitro Techniques; D008274:Magnesium; D015636:Magnesium Chloride; D008854:Microscopy, Electron; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011108:Polymers; D013381:Subtilisins
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011108:Polymers; D015636:Magnesium Chloride; D003850:Deoxyribonuclease I; D013381:Subtilisins; D009218:Myosins; D008274:Magnesium