Ischemia-induced structural change in SR Ca-ATPase is associated with reduced enzyme activity in rat muscle

Tupling, R., H. Green, G. Senisterra, J. Lepock, and N. McKee. Ischemia-induced structural change in SR Ca21ATPase is associated with reduced enzyme activity in rat muscle. Am J Physiol Regulatory Integrative Comp Physiol 281: R1681–R1688, 2001.—In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca21-ATPase can explain the alterations in Ca21-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 6 6.7 g (mean 6 SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca21-ATPase activity (mmol zg protein21 zmin21) of C (15,802 6 1,246) and I (11,609 6 1,029) was 90 and 73% (P , 0.05) of CC (17,562 6 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca21 at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl-N9-(dimethylamino-a-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca21 binding sites of the Ca21-ATPase, respectively, indicated a 26% reduction (P , 0.05) in FITC binding capacity (absolute units) in I (0.22 6 0.01) compared with CC (0.29 6 0.02) and C (0.29 6 0.03). Our results suggest that the reduction in maximal SR Ca21-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.