Abstract
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.
Highlights
It is known that alkylation of SMM thiols can cause changes in the regulation of the ATPase activity of SMM (41, 48) similar to those observed here
800-fold 27-fold permeabilization and complete RLC thiophosphorylation, this suggests that H2O2 directly affects the force-generating complex
These data are consistent with a H2O2-induced loss of functional myosin heads
Summary
Protein Preparations—Protein preparations were essentially as described (25). HMM and S1 were prepared by digestion of gizzard SMM (26) with Staphylococcus aureus protease V8 (Sigma). Peroxide Treatment of HMM and S1—HMM samples were thiophosphorylated where indicated and spun through a 5-ml buffer exchange column (32) prepared with Sephadex G-50 – 80 resin (Sigma) in nonreducing buffer (10 mM MOPS, 50 mM NaCl (pH 7.0)). After flushing the cuvette three times, 50 l was shot from each syringe (at 45 s), and the data were collected with the anti-bleaching shutter engaged for upHMM (1000 –1400 s; 1024 points) or without the shutter for shots under 60 s for pHMM and S1 (1024 points). The samples were subjected to nanobore LC with on-line tandem mass spectrometry performed on a ThermoFinnigan LTQ-FT This instrument is a hybrid of a linear ion trap with a Fourier transform-infrared cyclotron resonance detector (34, 35). Tracheal Smooth Muscle Experiments—See the supplemental materials and the legend for Fig. 9 for information regarding the tracheal smooth muscle experiments
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