mid-s]ice was immediately frozen with liquid Np and the remaining sliees were stained with TTi and giant mount slides for light microseopy were prepared.A method of NADH-F was based on the evidence that NA)+ + NADH decreases in the neerotic tissue though it remains well
Abstract Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.
The phorbol ester TPA may be intercalated into the membrane phospholipid bilayer and selectively binds to every molecule of C-kinase in exhibiting its full enzymatic activity. Available evidence suggests that C-kinase is the receptive protein of this tumor promoter, and the results presented seem to provide clues for clarifying the mechanism of controlling cell growth and differentiation.
Protein kinase C distributed widely in tissues and organs has the potential to play a crucial role in signal transduction for a variety of biologically active substances including growth factors, which elicit activation of cellular functions and proliferation. When cells are stimulated, this protein kinase is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumor-promoting phorbol esters are intercalated into the membrane, substitute for diacylglycerol, and permanently activate protein kinase C irrespective of the feedback control by cyclic AMP. Under normal conditions in many tissues this cyclic nucleotide blocks the signal-induced inositol phospholipid breakdown and thereby prevents the activation of this protein kinase. Available evidence suggests that protein kinase C is most likely a receptor protein of tumor promoters, and exploration of the roles of this enzyme may provide clues for understanding better the biochemical basis of cell growth and differentiation.
Journal Article Rapid Assay of Binding of Tumor-Promoting Phorbol Esters to Protein Kinase C Get access Yusushi TANAKA, Yusushi TANAKA Department of Biochemistry, Kobe University School of MedicineChuo-ku, Kobe, Hyogo 650 Search for other works by this author on: Oxford Academic PubMed Google Scholar Ryohei MIYAKE, Ryohei MIYAKE Department of Biochemistry, Kobe University School of MedicineChuo-ku, Kobe, Hyogo 650 Search for other works by this author on: Oxford Academic PubMed Google Scholar Ushio KIKKAWA, Ushio KIKKAWA 4 Department of Biochemistry, Kobe University School of MedicineChuo-ku, Kobe, Hyogo 650 4To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Yasutomi NISHIZUKA Yasutomi NISHIZUKA Department of Biochemistry, Kobe University School of MedicineChuo-ku, Kobe, Hyogo 650 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 99, Issue 1, January 1986, Pages 257–261, https://doi.org/10.1093/oxfordjournals.jbchem.a135467 Published: 01 January 1986 Article history Received: 12 August 1985 Published: 01 January 1986
