Synaptic plasma membranes from rat brain synaptosomes: Isolation and partial characterization

Abstract

We have isolated a highly purified plasma membrane fraction directly from synaptosomes prepared by centrifugation on a Ficoll-sucrose gradient. A critical feature of the preparation method is use of alkaline conditions to achieve effective osmotic shock so that membranes effectively resolve from mitochondria. If osmotic shock is carried out at alkaline pH, 70 % of the (Na +-K +)-activated-ouabain-sensitive ATPase (EC 3.6.1.4) of the synaptosomes can be recovered with only 7 % of total cytochrome oxidase (EC 1.9.3.1) in synaptosomal membrane fractions. In contrast, if osmotic shock is carried out at neutral pH, 50 % of the mitochondria overlays 85 % of the membranes. A fraction enriched in synaptic plasma membranes (SPM fraction) can be prepared on a simple discontinuous gradient in relatively good yield. The SPM fraction bands between 25–32.5 % sucrose; the yield is about 1.5 mg of protein per g tissue wet wt. An alkaline phosphatase (EC 3.1.3.1) is found in the SPM fraction. The distribution pattern of alkaline phosphatase parallels that of Na +-K +)-ATPase . 5′-Nucleotidase (EC 3.1.3.5) and acetylcholinesterase (EC 3.1.1.7) sediment at lighter densities than (Na +-K +)-ATPase or alkaline phosphatase although there is considerable overlap. The SPM fraction contains β-N- acetylglucosaminidase . This enzyme is not completely washed out of the SPM fraction by salt treatment but is released by low concentrations of Triton X-100.