Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin.
The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Onishi
H
H
Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka, Japan.
Maita
T
T
Matsuda
G
G
Fujiwara
K
K
D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002645:Chickens; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates
D000199:Actins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; C048876:1,5-AEDANS; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide