Effect of Bisimidate Cross-Linking Reagents on Synaptosomal Plasma Membrane

The bifunctional amino reagents dimethyl malonimidate, dimethyl adipimidate, and dimethyl suberimidate were tested for their ability to link covalently components of purified synaptosomal plasma membrane. Dimethyl malonimidate, the shortest of the three molecules, had no observable effect on the membrane at concentrations up to 50 mM. Dimethyl adipimidate, at concentrations of 10-50 mM, resulted in the disappearance from NaDodSO4 gels of three polypeptides with molecular weights of approximately 25 000, 65 000, and 75 000, and in the appearance of three new species with molecular weights of about 45 000, 125 000, and 200 000. Dimethyl suberimidate, at concentrations of 10-50 mM, resulted in the partial or complete disappearance of nearly all the polypeptide species; most of the new species formed had molecular weights greater than 106. The effects of dimethyl suberimidate were maximal at pHs greater than 9, showed a slight time dependence over a period of several hours at 25 °C, and were reltively unaffected by temperature in the range 0-45 °C. The results of two types of control experiments suggested that the occurrence of cross-linking between membranes was insignificant except under extreme conditions. Treatment of the membrane with 50 mM dimethyl suberimidate had no effect on the activities of any enzyme tested, except (Na+, K+)-ATPase, which was drastically inhibited by either dimethyl suberimidate or by the monofunctional reagent ethyl acetimidate. These observations suggest that many of the polypeptides of synaptosomal plasma membrane exist in the native membrane as monomers, and that individual molecules of most species come into close proximity with several neighbors. The large size of the complexes formed during extensive cross-linking is consistent with either stabilization of previously existing ordered arrays, or with random collisions of protein molecules following lateral diffusion in the lipid bilayer. Our results suggest that both mechanisms are involved to some extent.