Disulfide formation within the regulatory light chain of skeletal muscle myosin.
Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)
Huber
P J
PJ
Institute of Pharmacology, University of Zürich, Switzerland.
Brunner
U T
UT
Schaub
M C
MC
D000494:Allosteric Regulation; D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology; D011817:Rabbits; D013438:Sulfhydryl Compounds
D004220:Disulfides; D015879:Myosin Subfragments; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; C011136:thionitrobenzoic acid; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide