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Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers
Author(s) -
Oláh Z.,
Novak R.,
Lengyel I.,
Dux E.,
Joó F.
Publication year - 1988
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1988.tb04834.x
Subject(s) - phosphorylation , second messenger system , kinetics , microbiology and biotechnology , protein phosphorylation , modulation (music) , chemistry , neuroscience , biophysics , biology , signal transduction , protein kinase a , physics , quantum mechanics , acoustics
The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the kinetics of 32 P incorporation into specific protein substrates were evaluated by computer‐aided x‐ray film densitometry. With the use of this method, Ca 2+ ‐calmodulin (CAM)‐, Ca 2+ /phospholipid (PK C)‐, cyclic GMP (cGMP)‐, and cyclic AMP (cAMP)‐dependent protein kinases were detected. CAM‐dependent protein ki‐nase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP‐dependent protein kinase was much higher than that of the cAMP‐dependent one. Autophosphoryla‐tion of both the α‐ and β‐subunits of CAM‐dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32 P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca 2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothe‐lial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood‐brain barrier.