SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.
To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.
Kojima
S
S
Department of Structural Analysis, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan.
Fujiwara
K
K
Onishi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C006422:1,8-I-AEDANS; D009218:Myosins; D003545:Cysteine