Regulation of fast skeletal muscle activity by SERCA1 vicinal-cysteines.
During prolonged skeletal muscle contractions free radicals are produced that may lead to fatigue. Vicinal cysteines, known as a Vicinal-thiol groups react preferentially among them depending on redox potential. Therefore, we examined the role of VT groups on the activity and conformational changes of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA1) from rabbit skeletal muscle isolated SR, by selective oxidation-reduction of VT-groups. After Ca(2+) is released from the SR to start contraction, SERCA1 pumps this cytosolic Ca(2+) back to the SR leading to muscle relaxation. Phenylarsine oxide (PAO) reacts selectively with VT-proteins forming dithioarsines, which are stable but exchanges rapidly with 2,3-dimercaptopropanol (BAL). When 0.1 mM PAO is added to isolated SR, 60 and 67% inhibition of SERCA1 hydrolytic and Ca(2+) uptake activities, respectively is observed. ATPase activity was fully reversible with 1 mM BAL. The SERCA1 thermal inactivation determined from isolated SR from muscle at rest showed a single transition for inactivation (T(i)) at 49 +/- 1.12 degrees C. In the presence of 0.1 mM PAO, SERCA1 shows two transitions at T(i) 34 +/-0.9 degrees C and at 27 +/-1.2 degrees C. The thermal denaturation profile of SERCA1 from muscle at rest, showed two transitions at T(m) = 51.5 +/-1.3 degrees C and 63 +/-1.02 degrees C related to nucleotide and Ca(2+) binding domains, respectively. Whereas isolated SR obtained after a protocol of tetanic stimulation to produce muscle fatigue, showed three transitions in the SERCA1 denaturation profile similar to the effect of PAO, addition of 1 mM BAL reverted the effect of fatigue on SERCA1 denaturation profile. These results indicate a mechanism relating VT group's oxidation to muscle fatigue.
Alvarez
Rocío
R
Department of Biochemistry, School of Medicine, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Vázquez
Pável
P
Pérez
Francisco
F
Jiménez
Aura
A
Tirado
Aldo
A
Irles
Claudine
C
González-Serratos
Hugo
H
Ortega
Alicia
A
D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009119:Muscle Contraction; D018763:Muscle Fatigue; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000502:physiology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015854:Up-Regulation
D001152:Arsenicals; D004791:Enzyme Inhibitors; C029341:oxophenylarsine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D003545:Cysteine; D002118:Calcium