Abstract
Membranes from the buds of Pisum sativum L. contain a protein kinase which is activated 5- to 15-fold by micromolar levels of calcium. Best calcium activations were found with light-membrane fractions, and on density gradients these band at a similar position to the plasma membrane. Other heavier membranes, however, also contain a calcium-dependent protein kinase. The activity of the calcium-dependent protein kinase is inhibited by added phospholipids and phospholipase, in contrast to protein-kinase C. Calcium-dependent protein-kinase activity can be inhibited by 40% by low concentrations of the calmodulin inhibitor, trifluoperazine, but inhibitions are detected only after prior incubation of the membranes for some hours in ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Substantial calcium-dependent protein-kinase activity remains uninhibited by trifluoperazine indicating that there may be calmodulin-dependent and calmodulin-independent, but calcium-activated, protein kinases in pea membranes. The calcium-activated protein kinase seems to be intrinsically bound to membranes and only slight or partial solubilisation is obtained by the detergents nonidet P-40, (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate or octyl glucose. Better solubilisation is obtained by acetone treatment. There is some retention of calcium activation after partial solubilisation. A calcium-independent protein kinase has also been detected in membrane preparations; it has a substrate specificity different from that the calcium-dependent enzyme. Our results indicate, therefore, that there may be at least three protein kinases attached to pea shoot membranes.
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Abbreviations
- EGTA:
-
ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- Hepes:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- TFP:
-
trifluoperazine
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Hetherington, A.M., Trewavas, A. Activation of a pea membrane protein kinase by calcium ions. Planta 161, 409–417 (1984). https://doi.org/10.1007/BF00394571
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DOI: https://doi.org/10.1007/BF00394571