Reactivities of thiols in myosin rod: effect of magnesium and ionic strength.
There are six cysteines in each chain of myosin rod of rabbit skeletal muscle: three are in the S-2 portion, at residues 66, 108 and 410 (Lu, R.C. and Lehrer, S. (1984) Biochemistry 23, 5975-5981). The other three are in the light meromyosin portion, assigned at residues 572, 600 and 770 on the basis of homology between the amino acid sequence in the vicinity of these thiols and that of the rod of nematode myosin (McLachlan, A.D. and Karn, J. (1982) Nature 299, 226-231). Since the thiols are distributed in different regions of the rod, measuring their reactivities under various conditions may provide information on the conformations of these regions. Myosin rod was carboxymethylated with radioactive iodoacetic acid under various conditions. The cysteine-containing peptides were isolated using HPLC from the tryptic digests, and the radioactivity incorporated into each thiol was measured. In the denatured state all six thiols were equally reactive. In the native state, all thiols have low reactivity, the reactivity of Cys-108 or -410 is only 0.1% of that in the denatured state, Cys-600 exhibited the highest reactivity, about 20-times that of Cys-410; Cys-66, -572 and -770 had 2-4-times that of Cys-410. When the rods formed filaments, the reactivities of all cysteines further decreased: Cys-66, -108 and -770 were reduced to 50%, while Cys-410, -572 and -600, located in the middle of the rod, were reduced to 20-30% of their reactivities in the monomeric form. In the presence of Mg2+ the reactivity of Cys-108 increased by 20%, whereas Cys-572 decreased by 50%. The results are consistent with the view that metal ions affect the conformation of the rod. This may play a role in the mechanism of filament formation and the movement of crossbridges.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D046911:Macromolecular Substances; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D007461:Iodoacetates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014357:Trypsin; D009218:Myosins; D003545:Cysteine; D019807:Iodoacetic Acid