Chemical Modification of the Ca2+-dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle. I. Binding of N-Ethylmaleimide to Sarcoplasmic Reticulum: Evidence for Sulfhydryl Groups in the Active Site of ATPase and for Conformational Changes Induced by Adenosine Tri- and Diphosphate1

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20–100 μM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.

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Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum

Journal of Applied Physiology ◽

10.1152/jappl.1998.84.2.425 ◽

1998 ◽

Vol 84 (2) ◽

pp. 425-430 ◽

Cited By ~ 59

Author(s):

Terence G. Favero ◽

David Colter ◽

Paul F. Hooper ◽

Jonathan J. Abramson

Keyword(s):

Reactive Oxygen Species ◽

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Atpase Activity ◽

Structural Damage ◽

Hypochlorous Acid ◽

Reactive Oxygen ◽

Sulfhydryl Groups ◽

Dependent Manner ◽

Oxygen Species

Favero, Terence G., David Colter, Paul F. Hooper, and Jonathan J. Abramson. Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 84(2): 425–430, 1998.—Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 μM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (ATPase), inhibiting transport function. HOCl inhibits sarcoplasmic reticulum Ca2+-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 μM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca2+-handling kinetics in muscle cells.

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