Partial purification and properties of a protein kinase C type enzyme from plants

Abstract

Partial purification of a protein kinase with a dependence on micromolar concentrations of free calcium has been achieved from seedlings of Amaranthus tricolor. The enzyme has a M r of 77 600 as determined by gel filtration and 84 500 by SDS-PAGE analysis. Interaction of the enzyme with membranes (inside-out erythrocyte vesicles) is regulated by calcium, a characteristic of animal protein kinase C. Phospholipid and diolein activation of the enzyme is markedly dependent on the phospholipid used and on both calcium and phospholipid concentration. K m values for Ca 2+ in the absence of phospholipid was 20–40 μM and in the presence of phosphatidylserine 5–10 μM. Diolein plus phosphatidylserine lowered the K m to < 1.5 μM. The best activation was achieved at 1OOμM calcium with 40μg/ml phosphatidylserine and 8μg/ml diolein. These properties indicate a protein kinase C type enzyme. The plant enzyme reacted with antiserum directed against the regulatory domain of bovine brain protein kinase C in an immunoblot experiment.