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J Biol Chem, Vol. 275, Issue 20, 15142-15151, May 19, 2000

Kinetics of Smooth Muscle Heavy Meromyosin with One Thiophosphorylated Head*

Patricia A. EllisonDagger , James R. Sellers§, and Christine R. CremoDagger

From the Dagger  Department of Biochemistry, the University of Nevada, Reno, Nevada 89557 and § Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762

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 Km value for actin but a Vmax value of ~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.

* This work was supported in part by NIAMS Grant AR40917 from the National Institutes of Health (to C. R. C).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an American Heart Association Research Program Support award. To whom correspondence should be addressed: University of Nevada Central Receiving, Biochem/330/Cremo, 1664 N. Virginia St., Reno, NV 89503. Tel. 775-784-7033; Fax: 775-784-1419; E-mail: Cremo@unr.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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