The isolation and lipid composition of subcellular fractions derived from neuronal perikarya isolated in bulk from...

(1) Neuronal perikarya were isolated from rabbit cerebral cortex and were homogenized and separated into a number of subcellular membrane fractions using differential and discontinuous density gradient centrifugation. (2) The efficiency of homogenizing the nerve cell bodies could be greatly increased by the preliminary passage of the cell body fraction through a micropipet tip. (3) Of a number of density media, a discontinuous gradient of metrizamide gave the best resolution of the mitochondrial and lysosomal marker enzymes found in the crude mitochondrial fraction. This yielded a purified mitochondrial fraction and several lighter membrane fractions. (4) The lighter membrane fractions in metrizamide contained a degree of mitochondrial contamination which could be removed by a second gradient spin on 1.2 M sucrose, producing a second mitochondrial fraction and two light membrane fractions. (5) Based on marker analyses, fractions enriched in nuclei, mitochondria, microsomes and plasma membrane/lysosomes were produced. (6) The two mitochondrial fractions showed the highest, and the nuclear fraction the lowest specific phospholipid content. Cholesterol: phospholipid molar ratios showed a gradient of values from a low (0.2) for the mitochondria and nuclei to an intermediate value (0.4) for the microsomes to a high (0.6) for the light membrane fractions. (7) Phospholipid distributions indicated that for the nuclear and mitochondrial fractions phosphatidylinositol was more abundant than sphingomyelin or phosphatidylserine, while for the microsomes and the two light membrane fractions these three phospholipids were present in almost equal amounts.