Protein kinase C isoenzymes in airway smooth muscle

被引:19
|
作者
Webb, BLJ [1 ]
Lindsay, MA [1 ]
Barnes, PJ [1 ]
Giembycz, MA [1 ]
机构
[1] NATL HEART & LUNG INST,UNIV LONDON IMPERIAL COLL SCI TECHNOL & MED,LONDON SW3 6LY,ENGLAND
基金
英国惠康基金;
关键词
D O I
10.1042/bj3240167
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKC alpha, PKC beta(I), and PKC beta(II). In some experiments immunoreactive bands corresponding to PKC delta, PKC epsilon and PKC theta were also labelled, whereas the gamma, eta and zeta isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKC delta, PKC epsilon and PKC zeta, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using epsilon-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4 beta-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs alpha, beta(I), and beta(II). In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKC alpha', PKC beta(I)' and PKC beta(II)'. However, these novel enzymes were cofactor-independent and did not bind [H-3]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs alpha', beta(I)' and beta(II)' represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca(2+)and PDBu when epsilon-peptide is used as the substrate.
引用
收藏
页码:167 / 175
页数:9
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