Tropomyosin is in a reduced state in rabbit psoas muscle

Abstract

Tropomyosin (Tm) purified from skeletal and cardiac muscle often contains disulfide bonds due to oxidation of cysteine groups that are in close proximity in the coiled-coil structure. Are these disulfide crosslinks present in the muscle or produced by oxidation during preparation? To answer this question we reacted one part of freshly dissected rabbit psoas muscle fibers, which was permeabilized with Triton X-100, with N-ethyl maleimide (NEM) to block cysteine groups and another part with 5,5'-dithiobis(2-nitro benzoate) (DTNB) to facilitate disulfide bond formation by interchain sulfhydryl-disulfide exchange. We found, by high resolution gradient SDS polyacrylamide gels, that the NEM-treated muscle was only composed of uncrosslinked Tm and the DTNB treated muscle was composed of disulfide-crosslinked Tm. This work indicates that Tm exists in a reduced state in rabbit psoas muscle.

Fig. 1

A SDS gradient gel of psoas muscle fibers treated with NEM or DTNB. A rabbit skeletal actin control, Tm rabbit skeletal Tm control; TnT rabbit skeletal troponin T control, Muscle 2 loadings of NEM- or DTNB-treated muscle fibers. The crosslinked Tm region (upper) and the uncrosslinked region (lower) are magnified in B. B Magnified upper and lower regions of the gradient gel of A. Note: (1) presence of α- and β-Tm and absence of crosslinked α-α and α-β Tm for NEM treatment; (2) presence of crosslinked α-α and α-β Tm and absence of uncrosslined α- and β-Tm for DTNB treatment

Fig. 2

SDS gradient gel of supernatants after boiling the NEM and DTNB samples to purify the Tm. Note: (1) presence of α-Tm and absence of crosslinked Tm species for NEM treatment; (2) absence of α-Tm and presence of crosslinked Tm for DTNB treatment; (3) loss of myosin heavy chain, actin and some low molecular weight species