Kinetics of smooth muscle heavy meromyosin with one thiophosphorylated head.
Actin-activated MgATPase of smooth muscle heavy meromyosin is activated by thiophosphorylation of two regulatory light chains, one on each head domain. To understand cooperativity between heads, we examined the kinetics of heavy meromyosin (HMM) with one thiophosphorylated head. Proteolytic gizzard heavy meromyosin regulatory light chains were partially exchanged with recombinant thiophosphorylated His-tagged light chains, and HMM with one thiophosphorylated head was isolated by nickel-affinity chromatography. In vitro motility was observed. By steady-state kinetic analysis, one-head thiophosphorylated heavy meromyosin had a similar K(m) value for actin but a V(max) value of approximately 50% of the fully thiophosphorylated molecule. However, single turnover analysis, which is not sensitive to small amounts of active heads, showed that one-head thiophosphorylated heavy meromyosin was 46-120 times more active than unphosphorylated HMM but only 7-19% as active as the fully thiophosphorylated molecule. Discrepancy between the single turnover and steady-state values could be explained by a small fraction of rigor heads. These rigor heads would have a large effect on the steady-state kinetics of one-head thiophosphorylated HMM. In summary, thiophosphorylation of one head leads to a molecule with unique intermediate kinetics suggesting that thiophosphorylation of one head cooperatively alters the kinetics of the partner head and vice versa.
Ellison
P A
PA
Department of Biochemistry, the University of Nevada, Reno, Nevada 89557, USA.
Sellers
J R
JR
Cremo
C R
CR
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002645:Chickens; D002846:Chromatography, Affinity; D005895:Gizzard, Avian; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase