Binding between maleimidobenzoyl-G-actin and myosin subfragment 1.
It is well known that the structural interactions between S-1 moieties of myosin molecules ("cross bridges") and actin molecules in polymerized ("F") form are thought to underlie muscle contraction. It is surmised that such interactions are unitary (actin:S-1 = 1:1), but actual demonstration thereof is handicapped by intrinsic properties of the proteins. Recently, it has been reported that chemically modified [with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)] actin maintains its monomeric ("G") form and makes a stable unitary complex with S-1 but does not activate the S-1 ATPase [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. However, we recently showed that when MBS-G-actin and S-1 are covalently cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), ATPase activity is restored [Hozumi, T. (1991) Biochem. Int. 23, 835-843]. Here we investigated the interface between MBS-G-actin and S-1 using the techniques of tryptic digestion and EDC-cross-linking. MBS-G-actin specifically protected the N-terminal region of S-1 heavy chain against tryptic cleavage at the 25 kDa/50 kDa junction, which is different from the effect that a protomer within F-actin has on the protection of the 25 kDa/50 kDa junction. In addition, the cross-linking pattern between MBS-G-actin and S-1 was different from that between F-actin and S-1. When MBS-G actin was cross-linked to trypsin-treated S-1, no cross-linked product was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Hozumi
T
T
Department of Physiology, Nagoya City University Medical School, Japan.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D005456:Fluorescent Dyes; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C066969:maleimidobenzoyl-G-actin; D014357:Trypsin; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide