Abstract
The heavy chains of Acanthamoeba myosins. IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with UV light at 0 degrees C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.
Highlights
MATERIALS AND METHODSDivalent cationsb, y F-act,in,and by the stateof phospho- Acanthamoeba myosins IA and IB werehighlypurified by the rylation of either its heavy or light chains
Acantharnoeba myosin IB must contain the catalytic site, as well as the actin binding site, since it retains full myosin and actomyosin ATPase activity when the light chains have been completely removed [3]. It would be very useful if thecatalyticsite could bedirectlylabeled by a radioactive ligand through a stable covalent bond. Progress in this direction was made by Szilagyi et al [10] who found that an arylazide derivative of ATP photoaffinity labeled predominantlytheheavychain of rabbitskeletal muscle myosin
The myosins form a n unusual class of ATPases subject to we have utilized direct photoaffinitylabeling of myosins with multiple regulations.Generally,in theabsence of F-actin, radioactive nucleotide substrates as developed originally by their ATPase activities can be expressed in the presence of
Summary
Divalent cationsb, y F-act,in,and by the stateof phospho- Acanthamoeba myosins IA and IB werehighlypurified by the rylation of either its heavy or light chains. It is of proceduresofMaruta et al [13].Acanthamoeba myosin I heavy considerable interestto identify thecatalyticsitesonthe myosins and to study their interactions with the regulatory sites. Photoaffinity labeling was generally carried out with myosins at 2 mg/ml and 30 p~ radioactive nucleotide in 50 pl of 10 mM Tris/ chloride, pH 7.5, 2 mM MgC12, 1 mM dithiothreitol, and 10% sucrose.
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