UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification.
Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.
Grammer
J C
JC
Institute of Biological Chemistry, Washington State, Pullman 99164-4660.
Cremo
C R
CR
Yount
R G
RG
D000244:Adenosine Diphosphate; D000818:Animals; D001665:Binding Sites / Q000528:radiation effects; D000252:Calcium-Transporting ATPases / Q000528:radiation effects; D066298:In Vitro Techniques; D009132:Muscles / Q000032:analysis; D015879:Myosin Subfragments; D009218:Myosins / Q000528:radiation effects*; D010446:Peptide Fragments / Q000528:radiation effects*; D011817:Rabbits; D014466:Ultraviolet Rays; D014638:Vanadates
D015879:Myosin Subfragments; D010446:Peptide Fragments; D014638:Vanadates; D000244:Adenosine Diphosphate; D009218:Myosins; D000252:Calcium-Transporting ATPases