ESSENTIAL LIGHT-CHAIN EXCHANGE IN SMOOTH-MUSCLE MYOSIN

The exchange of 17-kDa essential light chain (LC17) in smooth muscle m yosin was carried out by incubating myosin with a 10-fold molar excess of exogenously added LC17 over the corresponding endogenous light cha in in the presence of trifluoperazine and 4.5 M ammonium chloride. Por cine aorta myosin contains two kinds of LC17 isoform, LC17nm and LC17g i, while chicken gizzard myosin contains only one kind of LC17 isoform . As LC17gi can be separated from LC17nm and gizzard LC17 by urea-gel electrophoresis, LC17nm in aorta myosin and LC17 in gizzard myosin wer e exchanged with LC17gi and LC17gi in aorta myosin was exchanged with LC17nm, and the degree of exchange was estimated by urea-gel electroph oresis. Under the optimal conditions (6 and 10 degrees C for aorta and gizzard myosin, respectively), nearly 90% of exchange, which is close to the theoretical value, was achieved for the former combinations, a nd a slightly lower exchange was obtained for the latter. The LC17-exc hanged myosins contained stoichiometric amounts of the heavy and light chains and retained the original nature in the phosphorylation-depend ent actin-activated ATPase activity, 6S-10S conformational transition, and filament assembly. (C) 1997 Academic Press.