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Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls

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Abstract

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg2+, Na+ and K+ on phosphorylation, measured as 32P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca2+. One peptide (relative molecular weight: 180,000) exhibits strong inhibition of 32P incorporation at physiological Ca2+ concentrations between 0.1 and 1 μM. Phosphorylation of about 10 other proteins was enhanced by Ca2+, being maximal in most cases at a concentration of about 3 μM free Ca2+. Five out of these 10 peptides show increased phosphorylation in the presence of 1 μM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.

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Abbreviations

EDTA:

ethylenediaminetetraacetic acid

EGTA:

ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

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  1. Institut für Biologie III der Universität, Schänzlestrasse 1, D-7800, Freiburg i. Br., Germany

    Bharathi P. Salimath & Dieter Marmé

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  1. Bharathi P. Salimath
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  2. Dieter Marmé
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Salimath, B.P., Marmé, D. Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls. Planta 158, 560–568 (1983). https://doi.org/10.1007/BF00397247

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  • DOI: https://doi.org/10.1007/BF00397247

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