title: The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA.
abstract: The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.
authors.affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
authors.forename: H
authors.initials: H
authors.lastname: Brzeska
authors.forename: T J
authors.initials: TJ
authors.lastname: Lynch
authors.forename: E D
authors.initials: ED
authors.lastname: Korn
mesh_terms: D000048:Acanthamoeba / Q000201:enzymology*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins