EFFECT OF SPERMINE ON MEMBRANE-ASSOCIATED AND MEMBRANE-INSERTED FORMSOF PROTEIN-KINASE-C

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane -associated form) and an irreversible one which is chelator stable (me mbrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane -inserted forms of protein kinase C were investigated using a reconsti tuted system consisting of partially purified protein kinase C from ra t brain and phospholipid vesicles of defined composition. The active m embrane-bound complex was conveniently determined by its ability to bi nd radioactive phorbol ester with an exact 1:1 stoichiometry. Our expe rimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by th e presence of spermine whereas the PDBu binding affinity was not signi ficantly affected. The addition of the divalent cation increased the a ffinity of phorbol ester for the active complex but had no effect on N (max); spermine added in this experimental conditions was no longer ab le to decrease the total number of enzyme molecules bound to liposomes . Moreover gel filtration experiments of the protein kinase C-phosphol ipids complex formed in the presence of calcium, indicated that polyam ine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipi d concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to mem brane binding sites both in the absence and in the presence of Ca++, c ould promote binding conditions that oppose to the formation of the in serted form of the enzyme. As a consequence the distribution between t he reversible and the irreversible membrane-bound forms of protein kin ase C is affected.