Purification of smooth muscle myosin phosphatase using a thiophosphorylated myosin light-chain-affinity resin.
Many protein kinases are able to catalyze the thiophosphorylation of protein substrates via a phospho-transfer reaction using adenosine-5'-o(3-thiotriphosphate) (ATPgammaS) (1), but, in general, thiophosphorylated proteins are very poor substrates for protein phosphatases (2,3). As a result, the protein substrate is essentially trapped in the phosphorylated state. This thiophosphorylation can be exploited in order to generate a strategy for the selective purification of protein phosphatases. Indeed, a number of thiophosphorylated protein substrates have been successfully used for the affinity purification of protein phosphatases (4-7). Here we describe the use of thiophosphorylated smooth muscle myosin regulatory light chains for the selective purification of the smooth muscle myosin phosphatase holoenzyme.
Borman
Meredith
M
Department of Biochemistry and Molecular Biology, Smooth Muscle Research Group, University of Calgary, AB, Canada.
MacDonald
Justin
J
D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D018994:Myosin Light Chains / Q000737:chemistry*; D043263:Myosin-Light-Chain Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D024745:Smooth Muscle Myosins / Q000378:metabolism*
D018994:Myosin Light Chains; D043263:Myosin-Light-Chain Phosphatase; D024745:Smooth Muscle Myosins