Irradiation with ultraviolet light in the presence of vanadate increases Ca2+ permeability of the sarcoplasmic reticulum membrane via Ca(2+)-ATPase.
The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2 mM EGTA, 10 mM MgCl2, 20% DMSO, and 50 mM PIPES (pH 6.5) at room temperature. In the presence of 100 microM vanadate, the Ca(2+)-uptake activity of SR rapidly decreased and was almost lost in 20 min. The activity was inhibited as a function of vanadate concentration with an apparent Ki of about 20 microM. On the other hand, Ca(2+)-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20 min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 microM vanadate. The loss of the relationship between Ca(2+)-uptake and ATPase reaction was found to be mainly caused by an increase in the Ca2+ permeability of the SR membrane, which was raised by increasing the vanadate concentration or UV irradiation time in a manner similar to that observed for the Ca2+ uptake. No rise in Ca2+ permeability occurred in liposomes reconstituted from SR lipid when they were irradiated with UV in the presence of 100 microM vanadate. When the vanadate-UV-treated SR was allowed to react with fluoral-P (4-amino-3-penten-2-one), an indicator of aldehyde, and the membrane proteins were separated by HPLC in the presence of SDS, the fluorescent probe was found to be closely associated with the Ca(2+)-ATPase fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Hirose
T
T
Department of Biology, Faculty of Science, Osaka University.
Yamasaki
K
K
Yamamoto
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism / Q000494:pharmacology*
D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium