The calmodulin-protein-kinase system of Mytilus edulis catch muscle

Abstract

1. 1. The Ca 2+-dependent modulator protein calmodulin (CaM) and a calmodulin-dependent protein kinase (CaM kinase) were isolated and characterized from catch musles of the bivalve Mytilus edulis L. 2. 2. CaM was purified to homogeneity from the heat-denatured supernatant of the muscles by DEAE-cellulose chromatography. The overall purification was estimated to be 30,000-fold. It was identified as calmodulin (CaM) on the basis of its ability to activate CaM-deficient PDE and CaM kinase in presence of Ca 2+, its inhibition by trifluoperazine and its apparent mol. wt of 15,900. 3. 3. The concentration of CaM in the muscle tissues of the mussel was about 10 times as high as in non-muscle tissues. 4. 4. Calmodulin kinase was purified from myofibrils of the catch muscles by a three step procedure involving chromatography on Affi-Gel Blue, Mono Q and Superose 6 columns. The overall purification was ca 5000-fold. Calmodulin kinase is a monomeric enzyme with an apparent mol. wt of 36,000. 5. 5. The activity of CaM kinase is absolutely dependent on Mg 2+. The Ca 2+-sensitivity of the Superose 6 preparation is absolutely dependent on external CaM, while the Mono Q preparation still contains CaM. The addition of CaM to the CaM-deficient kinase in a mole to mole ratio activates the enzyme 5-fold. 6. 6. The CaM kinase displays a narrow protein specificity. Its favorite substrate was molluscan paramyosin. It incorporates 4 moles 32P/mole paramyosin at 10 −5 moles Ca 2+/l. The K m for paramyosin is 2.3 μmoles/l. 7. 7. The CaM kinase itself seems to be the substrate of a cAMP kinase present in kinase preparation eluted from Mono Q columns.