ABSTRACT
The relationship between the tetrameric organization of the ryanodine receptor (RyR) and its activity in binding of ryanodine was approached through cross-linking studies using several bifunctional reagents, differing in their linear dimensions and flexibility, as well as in the reactivity of the active groups. Cross-linking with: 1,5-difluoro-2,4-dinitrobenzene (DFDNB); di(fluoro-3-nitrophenyl)sulfone (DFNPS), 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC); dimethyl suberimidate (DMS); ethylene glycol bis(succinimidylsuccinate) (EGS); and glutaraldehyde resulted in the disappearance of the, 470 kDa, RyR monomer protein band with concomitant appearance of additional bands of molecular masses higher than the monomer. At the relatively low concentrations of the reagents and the conditions used, RyR is the only cross-linked protein of SR membranes. The 'new' protein bands cross-react with antibodies against the RyR and correspond to dimers and tetramers of the RyR subunits while trimers were not detectable. DFDNB and DFNPS produced also a 560 kDa protein band which probably represents an intramolecular cross-linked monomer. The SDS-electrophoretic patterns of the cross-linked purified RyR resemble those of the membrane-bound receptor. Ryanodine binding to the high-affinity site was inhibited by modification of SR membranes with DFDNB and DFNPS, but not with DMS, EDC, EGS and glutaraldehyde, although RyR was completely cross-linked. The inhibition by DFDNB and DFNPS is due to modification of a specific lysyl residue which is also involved in the control of Ca2+ release. On the other hand, cross linking of the RyR with glutaraldehyde or EGS resulted in inhibition of ryanodine binding to the low-affinity, but not to the high-affinity binding sites. Thus, the cross-linking of two or more sites in each monomer (which lead to fixation of dimers or tetramers) did not prevent the conformational changes involved in the binding and occlusion of ryanodine at the high-affinity site, but inhibited its binding to the low-affinity sites.
