The use of permeabilized cells to assay protein phosphorylation and catecholamine release.
A number of approaches can be used to determine the protein kinases and protein phosphatases acting on particular phosphoproteins in vivo. Cell permeabilization represents one such approach. In this overview we discuss the different permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on the extent of digitonin-permeabilization, protein phosphorylation and catecholamine release are also discussed. The factors include the permeabilization medium, the ions such as calcium, and the second messengers, such as cAMP, IP3, cADPR and calmodulin. The effect of specific peptide inhibitors of protein kinases on tyrosine hydroxylase phosphorylation is illustrated. Advantages and disadvantages of cell permeabilization procedures are discussed throughout the text.
Gonçalves
C A
CA
Dept de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Brazil.
Gottfried
C
C
Dunkley
P R
PR
D000818:Animals; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability; D003470:Culture Media; D008565:Membrane Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D010766:Phosphorylation; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D002395:Catecholamines; D003470:Culture Media; D008565:Membrane Proteins; D014446:Tyrosine 3-Monooxygenase