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  • Title: Optimal concentration of iodonitrotetrazolium for the isolation of junctional fractions from rat brain.
    Author: Nieto-Sampedro M, Bussineau CM, Cotman CW.
    Journal: Neurochem Res; 1981 Mar; 6(3):307-20. PubMed ID: 7279108.
    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.
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