Synaptosomal proteins and axoplasmic flow: Fractionation by SDS polyacrylamide gel electrophoresis

Abstract

The neuronal cell body is known to supply the axon and the nerve terminal with various materials which migrate through the axon at several different velocities. A perikaryal origin of an important part of the syn- aptic proteins has been suggested and substantiated [1 ]. Using the visual system of the pigeon as a model, it was shown that proteins labelled in one retina reach the optic nerve terminals in the contralateral optic lobe in at least two waves. The first, corresponding to the fast axoplasmic flow, has a peak at 24 hr while the second, having the characteristics of slow axo- plasmic flow, has its maximum at 14 days [2-4], Previous attempts to fractionate the synaptosomal proteins [5-8] have not dealt with the source of origin of these proteins, i.e. the problem was not in- vestigated whether the proteins arrive with the fast or slow axonal flows or whether they are synthesized in the nerve endings. This report is concerned with the characterization, by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, of the synaptic proteins migrating from the perikaryon. 2. Materials and methods Each pigeon was injected intraocularly [2] with 50/aCi of aH-leucine (1-4,5-3H-leucine, specific activity 22 Ci[mM) or 50/aCi 14C-leucine (1-1- 14C- leucine, specific activity 62 mCi/rnM, both Radio- chemical Centre, Amersham, England) in 50 pl H20. Subcellular fractionation of the optic lobe's homo- genate was performed by the slightly modified [2] method of Gray and Whittaker [9]. The fraction enriched in synaptosomes, containing about 1.5 mg protein, was solubilized in 200/al of SUCK solution (1% SDS, 8 M urea, 0.01 M dithiotreitol and 0.05 M K2CO3) [10~ 11] by gentle agitation at room temper- ature for 30 min and used immediately for the electro- phoresis. An ORTEC SLAB electrophoresis unit was used with a pulsed constant-power source. 0.1% SDS acrylamide discontinuous gradient gel was prepared according to Grossfeld and Shooter [12]. In each cell, 7.5 ml of 12% acrylamide, 7.5 ml of 9.6%, 6 ml of 7.5% and 3 ml of 4.2% were layered on top of each other and polymerized simultaneously. The stacking gel, 8 ml of 2.5% acrylamide, was then polymerized with light. 50/al of synaptosomes solubilized in SUCK were applied on the stacking gel. Electrophoresis was performed in the discontinuous Tris glycine buffer system [12] using 300 pulses/see at 120 V, 10 mA and 0.1 /aF discharge capacitance. The proteins moved towards the anode at room temperature for 16 hr. The gels were fixed in concentrated acetic acid for 12 hr and stained with 1% amido black B in 7% (v/v) acetic acid for 3 hr. They were destained in 7% acetic acid and sectioned between successive bands, where mechanical separation was possible. The pieces of gel were solubilized [13] and counted in a Mark I Nuclear Chicago Scintillation Counter. The dpm were