Purification of type 1 protein (serine/threonine) phosphatases by microcystin‐Sepharose affinity chromatography

Abstract

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the α,β-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PPI) with high capacity and purified human PP1γ in one step from E. coli extracts. It was also used to purify forms of PPI bound to myofibrils from skeletal muscle. Two of these comprised PP1 completed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.