Abstract:
:Yeast actin mutants with acidic residues at the N terminus either neutralized (DNEQ) or deleted (delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an approximately 3-fold decrease in the binding constant for DNEQ and delta-DSE actins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (strong binding). DNEQ and delta-DSE actins protected S1 from tryptic digestion as well as the wild type and rabbit actins. The activation of S1 ATPase by DNEQ and delta-DSE actins (up to 50 microM) was very low but increased greatly after cross-linking these mutant actins to S1 by dimethyl suberimidate. Thus, the increased dissociation of mutant actins from S1 in the presence of ATP is the main cause for the low acto-S1 ATPase activities. At low-ionic strength conditions and in the presence of methylcellulose, the DNEQ and delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 microns/s). Yet, the sliding velocity of the N-terminal and D24A/D25A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indicates a lower relative force generation with the mutant actins. In contrast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than with wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved. These results indicate the importance of electrostatic actomyosin interactions under physiological salt conditions and show functionally distinct roles for the different myosin binding sites on actin.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Miller CJ,Wong WW,Bobkova E,Rubenstein PA,Reisler Edoi
10.1021/bi962388+subject
Has Abstractpub_date
1996-12-24 00:00:00pages
16557-65issue
51eissn
0006-2960issn
1520-4995pii
bi962388+journal_volume
35pub_type
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