Abstract
Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Sodium oleate is able to activatesoluble protein ki- tioned into platelet membranes with the majority of nase C
Chem. 265, 16437-16443B)e.cause physiologic interactions of fatty acids with protein kinasCe occur in thepresence of membranes, the following studies were conducted to evaluate the effects of surfaces on the activation of proposed that physiologic activation of protein kinase C mayoccur intwocompartments:solubleprotein kinase C would bea primary target for cis-unsaturated fatty acids, especially arachidonate, while membraneassociated protein kinase C is a target for activation by diacylglycerols
The observation that sodium oleate is unable to activate protein kinase C in the presence of Triton X-lOO/PS mixed micelles [9] raises important questions as to the proposed physiologic relevanceof activation of protein kinase C by cisunsaturated fattyacids [7,8]:does the presence of physiologic membranes negate the effects of fatty acids on protein kinase C? The following study was undertaken to clarify the role of Triton X-100, 11%oleate was eluted in the void volume
Summary
From the Departmentsof Medicine and Cell Biology,Duke University Medical Center, Durham, North Carolina 27710. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleaitne the presence of different concentrationsof Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Protein kinase C is activated in vitro by arachidonic, Activation of protein kinase C by oleate in a physi- oleic, and other cis-unsaturated fatty acids [7,8,9] This actiologic setting employing platelet substrates eannddog- vation does not require phospholipids [8, 9] but is dependent enousplateletproteinkinase. When corrected for studies suggest that fatty acids do not interact with protein free oleate concentration, activation of protein kinase kinase C at the phorbol ester/DAG-binding site.
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