Title 
       Antibodies specific for proteolyzed forms of protein
       kinase C alpha. 
Author 
       Kikuchi H; Imajoh-Ohmi S 
Address 
       Institute of Medical Science, University of Tokyo, Japan. 
Source 
       Biochim Biophys Acta, 1995 Nov 30, 1269:3, 253-9 
Abstract 
       The activation of protein kinase C (PKC) is irreversibly
       regulated by limited proteolysis catalyzed by a
       calcium-activated neutral cysteine protease, calpain.
       Calpain cleaves PKC alpha at specific sites in the hinge
       region between the catalytic and the regulatory domains of
       this kinase. Here we show a novel method for production of
       antibodies that bind specifically to the catalytic fragment
       of PKC alpha but not to the unproteolyzed protein. To
       detect proteolyzed PKC alpha 'cleavage site-directed
       antibodies,' which recognize amino-terminal regions in the
       nascent catalytic fragments and do not cross-react with
       the unproteolyzed enzymes, were raised using synthetic
       peptides corresponding to the amino-terminal sequences.
       The synthetic peptides used in this study were the
       sequences of human PKC alpha at the cleavage sites by m-
       and mu-types of calpains (LGPAGNKV and
       VISPSEDRKQPSNNLDRVKLT, respectively) and they are
       designated as CF alpha 2, CF alpha 4, in this order. Each
       synthetic peptide was injected into rabbit after
       conjugation with a carrier protein. The antibodies thus
       obtained (anti-CF alpha 2 or -CF alpha 4) specifically
       reacted with either the 46- or 45-kDa catalytic fragment
       of PKC alpha, respectively, whereas they did not
       cross-react with other fragments. Furthermore, the
       antibodies did not bind to the unproteolyzed enzyme nor
       fragments of PKC alpha obtained by treatment with other
       proteinases unless the fragment carried the same
       amino-terminal sequence. When human platelets were
       treated with calcium ionophore, the catalytic fragments of
       PKC alpha (45- and 46-kDa) were detected in the cytosol
       by immunoblotting with the antibodies. However, these
       antibodies did not bind unproteolyzed 80-kDa PKC alpha,
       although this form was dominant in the cytosol of the
       calcium ionophore-treated human platelets. In addition,
       the 45-kDa catalytic fragment of PKC alpha was detected
       in an apoptotic human fibroblast TIG-3 cells cultured in
       serum-free medium. Our method is applicable for analysis
       of proteolysis in various cellular states.