Detection of phosphorylation by enzymatic techniques.
Reversible protein phosphorylation is an important mechanism for regulating physiological processes in both plant and animal cells. There are a number of techniques to demonstrate the presence of covalently bound phosphate in proteins. The general strategy of the protocols in this unit is to first examine the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Protein phosphatases that selectively hydrolyze phosphoserine and phosphothreonine or phosphotyrosine residues can then be used to identify a functionally important covalent modification. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as (32)Pi based on its ability to form a complex with ammonium molybdate.
Shenolikar
S
S
Duke University Medical Center, Durham, North Carolina, USA.
D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation
D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases