TECHNICAL CONSIDERATIONS FOR ASSESSING ALTERATIONS IN SKELETAL-MUSCLESARCOPLASMIC-RETICULUM CA-SEQUESTRATION FUNCTION IN-VITRO(+)

A multiple measurement system for assessing sarcoplasmic reticulum (SR ) Ca++-ATPase activity and Ca++-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle. In vitro measurements were performed on whole m uscle homogenates (HOM) and crude microsomal fractions (CM) enriched i n SR vesicles before and after quick freezing in liquid nitrogen. Isol ation of the CM fraction resulted in protein yields of 0.96 +/- 0.1 an d 0.99 +/- 0.1 mg/g in WG and RG, respectively. The percent Ca++-ATPas e recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca ++-activated Ca''-ATPase activity was not affected by quick freezing o f HOM or CM, but basal ATPase was reduced (P < 0.05) in frozen HOM (5. 12 +/- 0.18-3.98 +/- 0.20 mole/g tissue/min in WG and from 5.39 +/- 0. 20-4.48 +/- 0.24 mu mole/g tissue/min in RG). Ca++-uptake was measured at a range of physiological free [Ca++] using the Ca++ fluorescent dy e Indo-1. Maximum Ca++-uptake rates when corrected for initial [Ca++]( f) were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca++ was reduced (P < 0.05) in quick frozen HOM (1. 30 +/- 0.1-0.66 +/- 0.1 mu mole/g tissue/min in WG and 1.04 +/- 0.2-0. 60 +/- 0.1 mu mole/g tissue/min in RG). Linear correlations between Ca ++-uptake and Ca++-ATPase activity measured in the presence of the Ca+ ionophore A23187 were r = +0.25, (P < 0.05) and r = +0.74 (P < 0.05) in HOM and CM preparations, respectively, and were not altered by fre ezing. The linear relationships between HOM and CM maximum Ca++-uptake (r = +0.44, P < 0.05) and between HOM and CM Ca++-ATPase activity (r = +0.34, P < 0.05) were also not altered by tissue freezing. These dat a suggest that alterations in maximal SR Ca++-uptake function and maxi mal Ca++-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage.