Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca2+ and K+ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163-202). The Ca2+-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and bindng to negatively-charged groups on the membrane surface. The K+-induced reaction includes only dimerization and is shown to be due to screening interactions alone. The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca2+-induced reaction has a 'bimolecular' rate constant of 4.77 . 10(8) M-1 . s-1. These values are close to the limit of diffusion control (8.03 . 10(9) M-1 . s-1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca2+-induced aggregation showed a break at 5 degrees C. Above this temperature, the activation energy is low (+0.65 kcal/mol), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing theenergetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of 'recognition sites' on the membrane surface. The results were analyzed in terms of an encounter complex model in which vesicles with separations of 26-126 A are considered capable of transformation into a stable complex. The rate constant of the transformation step is 1.4 . 10(3) s-1 for Ca2+ and approx. 1.6 . 10(5) s-1 for K+. The values are compared with previous results for chromaffin granule membranes and for phospholipid vesicles derived from chromaffin granule lipids and from acidic phospholipids. The half-time for Ca2+-induced transformation of the encounter complex into the stable complex is 435 microseconds. It is concluded that the recognition sites are almost as optimally deployed as the vesicle plasma membrane recognition sites involved in exocytotic release.