A Ca2+ion-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle

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Abstract
Ca2+-activated Z-disk-removing activity in the P0-40 crude muscles extracts described by Busch et al. was purified from porcine skeletal muscle extracts by using 5 column chromatographic procedures in succession: 6% agarose; DEAE-cellulose; Sephadex G-200; DEAE-cellulose with a very shallow gradient; Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The 5 column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure produces a 17,800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 .mu.g of CAF/g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 .mu.g of purified CAF/g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HCl buffer but migrated as 2 bands with MW of 80,000 and 30,000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80,000- and 30,000-dalton subunits make up 85-90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.

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