PHOSPHORYLATION OF A HIGH-MOLECULAR-WEIGHT (SIMILAR-TO-600 KDA) PROTEIN REGULATES CATCH IN INVERTEBRATE SMOOTH-MUSCLE

A unique property of smooth muscle is its ability to maintain force wi th a very low expenditure of energy. This characteristic is highly exp ressed in molluscan smooth muscles, such as the anterior byssus retrac tor muscle (ABRM) of Mytilus edulis, during a contractile state called `catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+](i), high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxatio n (release of catch) is initiated by serotonin, and mediated by an inc rease in cAMP and activation of protein kinase A. We sought to determi ne which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, havin g a molecular weight of similar to 600 kDa and a molar concentration a bout 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical con ditions studied (rest, activation, catch, and release of catch), chang es in phosphorylation of all other proteins, including myosin light ch ains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the similar to 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorpo rated per mole of similar to 600 kDa protein. Inhibition of A kinase b locked both the cAMP-induced increase in phosphorylation of the protei n and the release of catch. In addition, irreversible thiophosphorylat ion of the protein prevented the development of catch. In intact muscl e, the degree of phosphorylation of the protein increases significantl y when catch is released with serotonin. In muscles pre-treated with s erotonin, a net dephosphorylation of the protein occurs when the muscl e is subsequently put into catch. We conclude that the phosphorylation state of the similar to 600 kDa protein regulates catch.