H(2)O(2)-induced kinetic and chemical modifications of smooth muscle myosin: correlation to effects of H(2)O(2) on airway smooth muscle.
The effect of H(2)O(2) 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. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) 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 H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-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, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.
Penheiter
Alan R
AR
Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota 55905, and the.
Bogoger
Michelle
M
Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557.
Ellison
Patricia A
PA
Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557.
Oswald
Barbara
B
Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota 55905, and the.
Perkins
William J
WJ
Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota 55905, and the.
Jones
Keith A
KA
Department of Anesthesiology, University of Alabama-Birmingham, Birmingham, Alabama 35249-6810.
Cremo
Christine R
CR
Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557. Electronic address: cremo@unr.edu.
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D024745:Smooth Muscle Myosins / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014132:Trachea / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D000251:Adenosine Triphosphatases; D024745:Smooth Muscle Myosins