Modification of thiols of gizzard myosin alters ATPase activity, stability of myosin filaments, and the 6-10 S conformational transition.
The pattern of incorporation of [14C]N-ethylmaleimide (MalNEt) into gizzard myosin indicates the presence of two classes of thiols: rapidly and slowly modified. The first class contains two thiol residues, SH-A and SH-B, located in the myosin rod and the 17-kDa light chain, respectively, while the second contains at least two thiols located in the myosin heavy chain. Changes in ATPase activities upon modification occur rapidly or slowly, paralleling reaction of either the first or second class of thiols. Rapid changes include increases in the Ca2+- and Mg2+-activated activities of myosin alone, measured at ionic strengths below 0.3 M, and an increase and a decrease in the actin-activated activity of dephosphorylated and phosphorylated myosin, respectively. Modification of SH-A and SH-B with MalNEt is accompanied by stabilization of myosin filaments, seen as an increase in light-scattering intensity, and by destabilization of the folded, 10 S conformation of the myosin monomer. In the presence of 0.175 M NaCl and 1 mM MgATP, unmodified and MalNEt-modified myosin sediment in the ultracentrifuge as single components at 10.0 S and 6.0 S, respectively. The MalNEt-induced increase in the Ca2+- or Mg2+-activated ATPase activity, measured in the absence of actin, can be attributed either to stabilization of filaments or to destabilization of the 10 S conformation, depending on the ionic strength of the assay. Modification of the second class of thiols is accompanied by a decrease in K+-EDTA-activated activity and an increase in Ca2+-activated activity measured above 0.3 M NaCl, where myosin neither forms filaments nor assumes the 10 S conformation. These slow changes are characteristic of blocking the SH-1 thiols of skeletal-muscle myosin, but in gizzard myosin are attributable to modification of a less reactive thiol, SH-C.
Chandra
T S
TS
Nath
N
N
Suzuki
H
H
Seidel
J C
JC
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002250:Carbon Radioisotopes; D002645:Chickens; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding
D000199:Actins; D002250:Carbon Radioisotopes; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D005033:Ethylmaleimide