Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox.
Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.
Strosova
Miriam
M
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia.
Karlovska
Jana
J
Spickett
Corinne M
CM
Grune
Tilman
T
Orszagova
Zuzana
Z
Horakova
Lubica
L
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002839:Chromans / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006997:Hypochlorous Acid / Q000633:toxicity*; D007700:Kinetics; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D016877:Oxidants / Q000633:toxicity*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D050050:Protein Carbonylation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology
D000975:Antioxidants; D002839:Chromans; D016877:Oxidants; D006997:Hypochlorous Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D002118:Calcium