Abstract
Two soluble Ca 2+-dependent protein kinases (enzymes I and II) have been extensively purified from silver beet leaf tissue by means of a protocol involving batch-wise elution from DEAE-cellulose, Ca 2+-dependent binding to phenyl-Sepharose, gradient elution from DEAE-Sephacel, gel filtration and binding to Cibacron F3GA-Sepharose CL-6B. Protein kinases I and II are resolved on gradient elution from DEAE-Sephacel and are further distinguished by their different K m values for ATP and large differences in relative rates of phosphorylation of histone H1, casein and bovine serum albumin (the latter two proteins are relatively poor substrates for enzyme II but not enzyme I). Both enzymes have similar molecular weights as determined from gel filtration (56000 ± 2000 and 57000 ± 3000 for enzymes I and II, respectively). Both enzymes are absolutely dependent on free Ca 2+ for activity with maximal histone H1 kinase activity being obtained at 0.5 μM free Ca 2+. A millimolar concentration of Mg 2+ is required in addition to a micromolar concentration Ca 2+ for maximal activity. Both enzymes specifically phosphorylate serine residues of histone H1, are thiol activated and are inhibited by lanthanides and a range of calmodulin antagonists and inhibitors of protein kinase C.
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