Abstract
The key signal transduction enzyme protein kinase C (PKC) contains a hydrophobic binding site for alcohols and anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKCalpha results in dramatically different effects on membrane-associated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKCalpha differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKCalpha contains two distinct phorbol ester binding regions of low and high affinity for the activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). Short chain n-alkanols competed for low affinity phorbol ester binding to the enzyme, resulting in reduced enzyme activity, whereas high affinity phorbol ester binding was unaffected. Long chain n-alkanols not only competed for low affinity phorbol ester binding but also enhanced high affinity phorbol ester binding. Furthermore, long chain n-alkanols enhanced phorbol ester induced PKCalpha activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the n-alkanols alone were weak activators of the enzyme. The cellular effects of n-alkanols and general anesthetics on PKC-mediated processes will therefore depend in a complex manner on the locality of the enzyme (e.g. cytoskeletal or membrane-associated) and activator type, apart from any isoform-specific differences. Furthermore, effects mediated by interaction with the region on the enzyme possessing low affinity for phorbol esters represent a novel mechanism for the regulation of PKC activity.
Highlights
Driven by the need to improve anesthetic efficacy and to remove deleterious side effects, much effort has been expended in determining the site and mechanism of anesthetic action, yet both have remained elusive [1, 2]
Strong support for this came from a series of important studies that showed that the soluble enzyme firefly luciferase contains a hydrophobic binding site for alcohols and anesthetics, which mediated effects on enzyme activity that obeyed the Meyer-Overton rule [5, 6]
The challenge now is to find a site for anesthetics on a central nervous system protein that could be relevant to the mechanism of anesthesia
Summary
The fluorescence intensities at the emission maxima of PKC tryptophans and SAPD (333 and 425 nm, respectively), obtained on excitation of the tryptophan fluorophore at 290 nm, were determined using a PTI Alphascan fluorimeter (Photon Technology International, Princeton, NJ). The direct excitation of the SAPD fluorophore was corrected for by measuring the fluorescence intensity corresponding to each SAPD concentration in the presence of all assay components (including alcohols or anesthetics where added) except PKC␣. This signal was subtracted from the corresponding fluorescence intensities observed in the presence of the enzyme, isolating the contribution of resonance energy transfer to the observed signal. The probe was used to assess the effect of SAPD on head group organization by measuring the fluorescence anisotropy [38]
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