alpha-chymotrypsin and dithiobis(succinimidylpropionate)

When chymotryptic myosin subfragment 1 (S1) of fast skeletal muscle myosin is treated with dithiobis(succinimidylpropionate) (DSP), the alkali light chains A1 and A2 become intramolecularly crosslinked to the N-terminal 27 kDa fragment of the S1 heavy chain (Labbé et al. (1981) Biochem. Biophys. Res. Commun. 102, 466-475). The results presented here show that in the presence of MgATP the efficiency of the crosslinking is markedly reduced. The results may indicate a nucleotide-induced structural rearrangement within the myosin head. It was also observed that crosslinking depressed the nucleotide-promoted tryptic conversion of the 27 kDa fragment to its 22 kDa derivative, suggesting that the crosslinks are in the vicinity of the additional tryptic cleavage site in the 27 kDa fragment or that the crosslinking prevents nucleotide-induced conformational changes in this region of the S1 heavy chain.

Topics: Adenosine Triphosphate; Animals; Chymotrypsin; Cross-Linking Reagents; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Molecular Weight; Muscles; Myosin Subfragments; Protein Conformation; Rabbits; Succinimides

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.

Topics: 1-Carboxyglutamic Acid; Animals; Calcium; Cattle; Chemical Phenomena; Chemistry; Chymotrypsin; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Glutamine; Macromolecular Substances; Peptide Fragments; Prothrombin; Sodium; Structure-Activity Relationship; Succinimides

Topics: Adenosine Triphosphatases; Animals; Chymotrypsin; Cross-Linking Reagents; Dimethyl Suberimidate; Disulfides; Macromolecular Substances; Molecular Weight; Muscles; Myosins; Peptide Fragments; Rabbits; Succinimides