Isolation of the native form of chicken gizzard myosin light-chain kinase

Abstract

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin L-chain kinase (MWr 136,000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of MWr 141,000, which previously co-purified with the myosin L-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin L-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cAMP-dependent protein kinase. This MWr-141,000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit MWr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.