Phosphorylation properties of twitchin from Yesso scallop catch and striated muscles

Abstract

Molluscan smooth muscle exhibits a unique muscle contraction, called catch, where the tension is maintained for long periods with little energy consumption. Catch contraction is regulated by twitchin, also called mini-titin, a member of the titin/connectin family. Twitchin is also found in molluscan non-catch striated muscle. We isolated twitchins from catch and striated muscles in the Yesso scallop Mizuhopecten yessoensis. Both twitchins are ~500 kDa in size. Western blotting using the anti-twitchin kinase domain antibody revealed that both twitchins have a kinase domain. Both twitchins were phosphorylated by the catalytic subunit of cAMP-dependent protein kinase in vitro. Stoichiometric analysis revealed that 0.6 and 1.7 mol Pi were incorporated into catch and striated muscle twitchins, respectively. Phosphopeptide mapping revealed that catch muscle twitchin has one phosphorylation site, whereas striated muscle twitchin has two. cDNA cloning gave partial sequences of two Yesso scallop twitchin isoforms. One had two putative phosphorylation sites and the other had one.