| ironjustice@aol.com 2006-03-28, 10:29 am |
| ..
Inhibition of skeletal muscle s1-Myosin ATPase by peroxynitrite.
Tiago T, Sim=E3o S, Aureliano M, Mart=EDn-Romero FJ, Guti=E9rrez-Merino C
Biochemistry. 2006 Mar 21; 45(11): 3794-804
Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine
(SIN-1) produced a time-dependent inhibition of the F-actin-stimulated
S1 Mg(2+)-ATPase activity, reaching 50% inhibition with 46.7 +/- 8.3
muM SIN-1 for 8.7 muM S1, that is, at a SIN-1/S1 molar ratio of
approximately 5.5. The inhibition was due to the peroxynitrite produced
by SIN-1 decomposition because (1) decomposed SIN-1 was found to have
no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence
of superoxide dismutase and catalase fully prevented inhibition by
SIN-1, and (3) micromolar pulses of chemically synthesized
peroxynitrite produced inhibition of F-actin-stimulated S1
Mg(2+)-ATPase activity. In parallel, SIN-1 produced the inhibition of
the nonphysiological Ca(2+)-dependent and K(+)/EDTA-dependent S1 ATPase
activity of S1 and, therefore, suggested that the inhibition of
F-actin-stimulated S1 Mg(2+)-ATPase activity is produced by the
oxidation of highly reactive cysteines of S1 (Cys(707) and Cys(697)),
located close to the catalytic center. This point was further confirmed
by the titration of S1 cysteines with 5,5'-dithiobis(2-nitrobenzoic
acid) and by the parallel decrease of Cys(707) labeling by
5-(iodoacetamido)fluorescein, and it was reinforced by the fact that
other common protein modifications produced by peroxynitrite, for
example, protein carbonyl and nitrotyrosine formation, were barely
detected at the concentrations of SIN-1 that produced more than 50%
inhibition of the F-actin-stimulated S1 Mg(2+)-ATPase activity.
Differential scanning calorimetry of S1 (untreated and treated with
different SIN-1 concentrations) pointed out that SIN-1, at
concentrations that generate micromolar peroxynitrite fluxes, impaired
the ability of ADP.V(1) to induce the intermediate catalytic transition
state and also produced the partial unfolding of S1 that leads to an
enhanced susceptibility of S1 to trypsin digestion, which can be fully
protected by 2 mM GSH.
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