Myosin subfragment-1 is fully equipped with factors essential for motor function.
The sliding velocity of actin filaments propelled by chicken skeletal myosin subfragment-1 (S1) was measured when the tail end of S1 was specifically bound to the glass surface. To achieve the specific binding, a regulatory light chain was replaced by a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC) and chicken gizzard smooth muscle regulatory light chain (cgmRLC). The BDTC-cgmRLC of S1 was then attached to the glass surface using a biotin-avidin system. The velocity of actin filaments caused by S1 bound to the surface in this manner was 6.8 +/- 0.6 microm/sec at 29 degrees C, which was 3.5-fold greater than that (1.9 +/- 0.3 microm/sec) when bound directly to the surface as in previous studies, but similar to that caused by native chicken skeletal myosin (6.5 +/- 0.6 microm/sec). The actin-activated Mg-ATPase activity was similar to that of S1 before the RLC of S1 was exchanged for BDTC-cgmRLC. The results indicate that S1 can produce a normal fast movement of actin filaments as well as hydrolyse ATP and generate force.
Iwane
A H
AH
Faculty of Engineering Science, Osaka University, Toyonaka, Japan.
Kitamura
K
K
Tokunaga
M
M
Yanagida
T
T
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005895:Gizzard, Avian; D005898:Glass; D018482:Muscle, Skeletal / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*
D017931:DNA Primers; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins