Localization, Solubilization and Characterization of Plant Membrane-Associated Calcium-Dependent Protein Kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca(2+) -dependent protein kinase. The Ca(2+) -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as beta-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca(2+) -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca(2+) -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca(2+) -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca(2+) -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K(m) values for ATP, and in Ca(2+) -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca(2+) -dependent protein kinases present in the soluble phase.