A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.

The key metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), induces rapid cellular responses that constitute a so-called "non-genomic" response. This effect is distinguished from its "classic" genomic role in calcium homeostasis involving the nuclear 1,25-D3 receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D3 at physiological concentrations (EC50 = 16 +/- 1 nM). The effect was demonstrable with single PKC-alpha, -gamma, and -epsilon isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC-epsilon was also activated by 1,25-D3 shows that the calcium binding C2 domain is not required. The level of 1,25-D3-induced activation, paired with either diacylglycerol or 4 beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4 beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D3-induced activity decreased, consistent with 1,25-D3-activated PKC possessing a distinct conformation. The results suggest that PKC is a "membrane-bound receptor" for 1,25-D3 and that it could be important in the control of non-genomic cellular responses to the hormone.

Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKC␣ results in dramatically different effects on membraneassociated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKC␣ differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKC␣ 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 PKC␣ activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the nalkanols 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.

Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol esteractivated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160 -17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC␣. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation doseresponse curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4␤-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKC␣ contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively. Protein kinase C (PKC)1 comprises a family of isozymes that are pivotal in intracellular signal transduction (1-4). Activation of PKC requires association with the membrane and is induced by a number of activators and cofactors, the requirements for which differ for each isoform (2). Thus, with the exception of the so called atypical PKC isoforms, the activity of membrane-associated PKC is potentiated by the second messenger, diacylglycerol, a product of hormone receptor-phospholipase C-catalyzed phospholipid hydrolysis.In addition to the natural activators, including diacylglycerol, the enzyme is activated with high specificity by the tumorpromoting phorbol esters (5). For this reason, phorbol esters are often used in the study of the mechanism of PKC activation, based on the assumption that they compete directly with diacylglycerols for a common binding site on the enzyme (6, 7). Also it is commonplace in studies of the regulation of cellular processes to infer PKC involvement from a response elicited by phorbol esters (5). However, a number of studies have provided evidence indicating that the activated conformational forms of PKC induced by diacylglycerol compared with phorbol esters may not be equivalent based on observations of marked differences in the biological (8, 9) and enzymatic properties (10 -1...

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