Protein kinase C in rat cerebral microvessels.
Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.
Hanson-Painton
O
O
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Morgenstern
K
K
Cooper
D R
DR
Moore
B
B
Botchlet
T
T
Grammas
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002196:Capillaries / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D010743:Phospholipids; D010750:Phosphoproteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D002118:Calcium