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J. Biol. Chem., Vol. 282, Issue 7, 4336-4344, February 16, 2007

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H₂O₂-induced Kinetic and Chemical Modifications of Smooth Muscle Myosin

CORRELATION TO EFFECTS OF H₂O₂ ON AIRWAY SMOOTH MUSCLE^*

Alan R. Penheiter, Michelle Bogoger, Patricia A. Ellison, Barbara Oswald, William J. Perkins, Keith A. Jones^¶, and Christine R. Cremo¹

From the Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557, the Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota 55905, and the ^¶Department of Anesthesiology, University of Alabama-Birmingham, Birmingham, Alabama 35249-6810

The effect of H₂O₂ on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P_i release were measured by single-turnover kinetics. H₂O₂ treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, 50% of the molecules lost the ability to bind to ATP. H₂O₂ treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H₂O₂-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H₂O₂-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H₂O₂ caused a 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H₂O₂ directly affects the force generating complex. Dithiothreitol treatment reversed the H₂O₂ inhibition of the maximal force by 50%. These data, when compared with the in vitro kinetic data, are consistent with a H₂O₂-induced loss of functional myosin heads in the muscle.

Received for publication, October 10, 2006

^* This work was funded by NIAMS, National Institutes of Health Grant AR 40917 (to C. R. C.), National Institutes of Health Grant HL 54757 (to K. A. J.), and National Institutes of Health Grant 1P20RR018751 (to the COBRE Smooth Muscle Plasticity, Cell to Proteomics Interface Core facility). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and supplemental methods.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology/330, 1664 N. Virginia St., University of Nevada School of Medicine, Reno, NV 89557. Tel.: 775-784-7033; Fax: 775-784-1419; E-mail: cremo{at}unr.edu.

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