Abstract
In vitro selection technology has been used to purify RNA aptamers from a random sequence pool that can bind to, and specifically inhibit, protein kinase C beta II. Two of the selected RNA aptamers bind to this isozyme of protein kinase C with nanomolar affinities and inhibit activation with unprecedented selectivity; the highly related, alternatively spliced beta I isozyme, which differs by 23 residues, is inhibited with 1 order of magnitude lower potency; the next most similar isozyme, alpha, shows no detectable inhibition. The production of isozyme-specific inhibitors of protein kinase C opens the possibilities for dissecting the roles of specific protein kinase Cs in the myriad of intracellular signalling pathways.
Highlights
Materials-Bovine brain La-phosphatidylserine (PS)' and sn-l,a-dibRiNInnAdvtaoipt,rtaoanmsdeelsrepsceftricooifnmictaealclyrhainnnohdlioobgmiyt,spherqaousteebnienceeknpinouaossleedthatoCt cpPauInIr.ifyoLhchebippataitsdaiennsd,eodIyfnrlcopfr.mhooHmsSEpihPPgaEimteiSrdacy.beluT,cfhrfaioetnolrdinnaen[XdY(-P1s-Cn0~0,~)1wP,(21Ie-0Arde%iT(o3loPwe0bo0/vty0aCliganiqleyudcemeforrmuoolsmo(l"sDA)oGlvua)wtniwaotisner)PefpowroulaamrsrT w o of the selectedRNA aptamers bind to this isozyme DuPont NEN
A protein kinase C-selectivepeptide ( FKFKL - N H, of protein kinase C with nanomolar affinities and in- (Chakravarthy et al, 1991))was synthesized by the IndianaUniversity hibitactivationwithunprecedentedselectivity;the
Protein kinase C isozymes were highly related, alternatively splicedPI isozyme, which purified from the baculovirus expression system, as described (Orr et differs by 23 residues, is inhibited with1 order of mag- al., 19921,and stored at -20 "C in 10 mM Tris buffer, pH 7.5 (4 "C), 0.5 nitude lower potency; the most similar isozyma,e, showsnodetectableinhibitionT. heproduction of isozyme-specific inhibitors of protein kinase C opens thepossibilitiesfordissectingtheroles of specific mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, and 50% glycerol
Summary
Materials-Bovine brain La-phosphatidylserine (PS)' and sn-l,a-dibRiNInnAdvtaoipt,rtaoanmsdeelsrepsceftricooifnmictaealclyrhainnnohdlioobgmiyt,spherqaousteebnienceeknpinouaossleedthatoCt cpPauInIr.ifyoLhchebippataitsdaiennsd,eodIyfnrlcopfr.mhooHmsSEpihPPgaEimteiSrdacy.beluT,cfhrfaioetnolrdinnaen[XdY(-P1s-Cn0~0-,~)1wP,(21Ie-0Arde%iT(o3loPwe0bo0/vty0aCliganiqleyudcemeforrmuoolsmo(l"sDA)oGlvua)wtniwaotisner)PefpowroulaamrsrT w o of the selectedRNA aptamers bind to this isozyme DuPont NEN. Heproduction of isozyme-specific inhibitors of protein kinase C opens thepossibilitiesfordissectingtheroles of specific mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, and 50% glycerol.
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