期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2001
卷号：98
期号：12
页码：6635-6640
DOI：10.1073/pnas.111585098
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要："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 Ca2+-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 {approx}10-4 M free Ca2+ and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at {approx}10-8 M free Ca2+ in the presence of MgATP. At {approx}10-4 M free Ca2+, the thin filaments moved at {approx}4 {micro}m/s. Addition of cAMP and cAMP-dependent protein kinase at {approx}10-8 M free Ca2+ caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/{micro}m 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.
