Carbodiimide-catalyzed cross-linking sites in the heads of gizzard heavy meromyosin attached to F-actin.
In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(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*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000378:metabolism; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000302:isolation & purification
D000199:Actins; D000596:Amino Acids; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; C048876:1,5-AEDANS; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide