Do adult rat ventricular myocytes express protein kinase C-alpha?
Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.
Rybin
V
V
Department of Medicine, Columbia University, New York, New York 10032, USA.
Steinberg
S F
SF
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D006352:Heart Ventricles / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar
D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha