Mode of Activation and Kinetic Properties of Three Distinct Forms of Protein Kinase C from Rat Brain1

Abstract

Three types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones gamma, beta, and alpha, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by beta I and beta II sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1,2-didecanoylglycerol and 1,2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase D activation in fibroblast membranes by the α and β isoforms of protein kinase C

“…The rat brain PKC that was found to be capable of activating PLD consisted of a mixture of the 01, p and y isofoirns [ 161. In order to determine which isoforms were active, they were resolved by hydroxylapatite chromatography [21]. Fig.…”

“…PKCa, /I and y were purified from rat brain as described previously [21]. After resolution by hydroxylapatite chromatography, the identity of the isoforms was confirmed by immunoblot analysis using specific antisera.…”

“…Homogenates prepared from rat brain [21] and CCL39 cells [16] were centrifuged at 100,000 x g for 30 min. Samples of the soluble fraction (4 pug protein) were subjected to 10% SDS-PAGE.…”

Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C

“…> Na'). Because Ba2'-can activate some Ca?+-dependent processes (Knight et al, 1988;Sekiguchi et al, 1988;Przywara et al, 1993), the enhanced ethanol inhibition of Ca2+/Ba'+ flux may be due to activation of a secondary Ca"-dependent mechanism. Injection of EGTA could reduce the ethanol inhibition of Ca'+ responses if this involved an intracellular Ca2+-dependent mechanism; however, EGTA is a slow Ca'+ chelator and was only present in micromolar concentrations.…”

Direct activation of a protein kinase activity from rat lacrimal gland by PMA in a phospholipid‐free system

“…The activation process involves the formation of a quaternary complex between kinase C, calcium, phospholipid and phorbol ester. In some studies, it has been shown that calcium is not absolutely necessary for the action of PMA, whilst the effect of phorbol esters is dependent upon the presence of phospholipids [7,19]. However, in the course of our study, as a control, we tested the effect of PMA on the kinase activity of the fractions eluting from the DE 52 column in the absence of both calcium and phosphatidylserine.…”