Optimal concentration of iodonitrotetrazolium for the isolation of junctional fractions from rat brain.

Abstract

The yield and purity of synaptic plasma membranes (SPM) and synaptic junctions (SJ) from rat brain has been examined as a function of the concentration of rho-iodonitrotetrazolium (INT)--succinate used during their preparation. An INT concentration of 1 mg/g brain tissue (wet weight) was sufficient to obtain SPM and SJ of purity comparable to that obtained using 4--6 times that concentration of dye (1--3). At this lower level of INT the yield of SPM increased by about 100%, whereas mitochondrial contamination remained at 10--13% of the total SPM protein. At concentrations of INT below 0.5 mg/g brain tissue (wet weight) the contamination of SPM by mitochondria increased rapidly. At very low concentrations of INT (0.13 mg/g tissue) the contaminating protein of mitochondrial origin was 40--50% of the total protein in the SPM fraction. Examination by gel electrophoresis of SPM, SJ, and mitochondrial fractions with different degrees of cross-contamination allowed the assignment of marker polypeptides for mitochondrial, junctional, and nonjunctional plasma membranes. Under the conditions used to prepare SJ, a variable amount of particulate material floated over 1.0 M sucrose. It consisted of SJ and many membrane vesicles and had a protein composition similar to that of SJ contaminated by extrajunctional membrane proteins. An analogous fraction arose during in the preparation of postsynaptic densities.