TECHNICAL CONSIDERATIONS FOR ASSESSING ALTERATIONS IN SKELETAL-MUSCLE SARCOPLASMIC-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 muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96 +/- 0.1 and 0.99 +/- 0.1 mg/g in WG and RG, respectively. The percent Ca++-ATPase 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 of 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 dye 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 freezing. 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 data suggest that alterations in maximal SR Ca++-uptake function and maximal Ca++-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage.