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ABSTRACT

The accuracy and precision with which Ca++-dependent proteinase (CAF) activity can be assayed in skeletal muscle tissue was determined by using two experimental approaches: (1) repeated sampling of a homogeneous batch of minced rat skeletal muscle to estimate variation among CAF assays done on fractions made from the same muscle tissue and to ascertain the effect of sample size on assays of CAF activity; and (2) comparison of CAF assays done on muscle samples of similar weights obtained from different animals that had been treated alike. Muscle CAF activity can easily be detected in 0.5g muscle samples, but the measured activity is not accurate and increases with increasing sample size. The decreased CAF activity assayed in small muscle samples seemed to originate from failure to extract all the CAF in these samples, possibly because of the different homogenizer that must be used to homogenize small samples. If a Waring Blendor is used at 8000 rpm, muscle samples must be 19g or larger to obtain accurate assays of CAF activity. The coefficient of variation for duplicate assays of CAF activity on the same P045 crude CAF fraction was 5.85% (assay variation); for assays of CAF activity on different samples of the same muscle tissue, 7.18% (sampling variation and variation in procedure for preparing crude CAF fractions); and for assays of CAF activity on muscle tissue obtained from the different groups of animals that had been treated alike, 10.30% (animal variation). Hence, CAF activity can be measured with acceptable precision in skeletal muscle tissue, but treatments designed to alter muscle CAF activity must cause changes of at least 20% to be detectable against the natural variation of muscle CAF activity in different animals.