IDENTIFICATION AND PROPERTIES OF PHOSPHATASES IN SKELETAL-MUSCLE WITH ACTIVITY TOWARDS INHIBITORY SUBUNIT OF TROPONIN, AND THEIR RELATIONSHIP TO OTHER PHOSPHOPROTEIN PHOSPHATASES

Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysin-rich histone (fraction F1) were fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behavior during chromatography. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 .mu.M) was significantly lower than that previously reported. Phosphatase activities towards troponin I and phosphorylase a did not show a requirement for divalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. Discontinuous polyacrylamide-gel electrophoresis studies indicated that several of the fractions contained more than 1 phosphatase activity, and showed that several of the activities could exist in different aggregation states. On the basis of these studies at least 2 phosphatases with activity towards troponin I only were identified. Phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all 3 substrates were found. A fraction with MW of 150,000 could be activated by freezing with 2-mercaptoethanol or by heating to 55.degree. C. This activation was accompanied by a decrease in MW to 25,000. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10 s.