Irradiation with Ultraviolet Light in the Presence of Vanadate Increases Ca<sup>2+</sup> Permeability of the Sarcoplasmic Reticulum Membrane <i>via</i> Ca<sup>2+</sup>-ATPase

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  • Hirose Takashi
    Department of Bilolgy, Faculty of Science, Osaka University
  • Yamasaki Kazuo
    Department of Bilolgy, Faculty of Science, Osaka University Department of Biochemistry, Asahikawa Medical College
  • Yamamoto Taibo
    Department of Bilolgy, Faculty of Science, Osaka University

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  • Irradiation with Ultraviolet Light in the Presence of Vanadate Increases Ca2+ Permeability of the Sarcoplasmic Reticulum Membrane via Ca2+-ATPase

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The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2mM EGTA, 10mM MgCl2, 20% DMSO, and 50mM PIPES (pH 6.5) at room temperature. In the presence of 100 μM vanadate, the Ca2+-uptake activity of SR rapidly decreased and was almost lost in 20min. The activity was inhibited as a function of vanadate concentration with an apparent Ki of about 20 μM. On the other hand, Ca2+-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 μM vanadate. The loss of the relationship between Ca2+-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 μM 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 Ca2+-ATPase fraction. Furthermore, the amount of fluoral-P incorporation into the Ca2+-ATPase increased with UV irradiation time in the presence of vanadate and reached the maximum level at 100min. About 7-8 nmol of fluoral-P was bound to 1mg of Ca2+-ATPase at the steady level. These results suggest that when SR is incubated with vanadate under UV irradiation, specific residues in Ca2+-ATPase are oxidized to form aldehyde, which causes an increase in the Ca2+ permeability of the SR membrane.

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