An in vitro assay reveals essential protein components for the "catch" state of invertebrate smooth muscle.
"Catch," a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the "latch" state of vertebrate smooth muscles. Its induction and release involve Ca(2+)-dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with approximately 10(-4) M free Ca(2+) and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at approximately 10(-8) M free Ca(2+) in the presence of MgATP. At approximately 10(-4) M free Ca(2+), the thin filaments moved at approximately 4 microm/s. Addition of cAMP and cAMP-dependent protein kinase at approximately 10(-8) M free Ca(2+) caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/microm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo. Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation.
Yamada
A
A
Kansai Advanced Research Center, Communications Research Laboratory, Iwaoka 588-2, Nishi-ku, Kobe 651-2492, Japan. c2v@crl.go.jp
Yoshio
M
M
Kojima
H
H
Oiwa
K
K
D000818:Animals; D049872:Bivalvia; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D009119:Muscle Contraction; D009124:Muscle Proteins / Q000032:analysis*; D009130:Muscle, Smooth / Q000737:chemistry / Q000502:physiology*; D009218:Myosins / Q000502:physiology
D009124:Muscle Proteins; D000242:Cyclic AMP; D009218:Myosins; D002118:Calcium