Crosslinking by Thiol Disulfide Interchange of 5,5′‐Dithiobis(2‐nitrobenzoic acid)‐Treated Light Chain and Heavy Chain of Rabbit Skeletal Myosin

Interchain disulfide crosslinks between the heavy‐chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs 2 ), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2‐nitro‐5‐thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs 2 ‐modified light chain 2 by 32–39%, if Ca 2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs 2 ‐modified light chain 2 attaches to the heavy chain in the region of the 21‐kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/sub‐fragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190 , 793–799]. Modification of thiol‐1 groups with iodoacetamide as well as crosslinking the thiol‐1 and thiol‐2 groups by the bifunctional reagent p‐N,N′ ‐phenylenedimaleimide prior to incubation with Nbs 2 ‐modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs 2 ‐modified light chain 2 to the heavy chain. An examination of K + , Ca 2+ , Mg 2+ and actin‐activated Mg 2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs 2 ‐modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs 2 ‐modified light chain 2 to light‐chain‐2‐deficient heavy meromyosin restored the original light‐chain‐2‐dependent Ca 2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light chain 2.