Fractionation of brain macromolecules. Chromatography on hydroxyapatite of soluble proteins and nucleic acids from whole rat brain and the effect of autolysis

Abstract

A procedure for the chromatographic fractionation of soluble brain proteins on calcium hydroxyapatite is described. Chromatograms obtained are repro- clucible; approximately 11 protein and at least three nucleic acid components can be identified. The effect of column dimensions and flow rate on the chromatograms obtained are described. One of the nucleic acid components appears to correspond to soluble RNA and another to ribosomal RNA. Treatment of homogenates by procedures that cause the removal of ribosomes from soluble extracts also cause the disappearance of the component corresponding to ribosomal RNA. Centrifugation of rat-brain homogenates prepared in 0.25 M-sucrose at 105400g for 90 min. causes the sedimentation of ribosomes as well as the disappearance of the component corresponding to ribosomal RNA. Extraction of brain tissue, or particu-late fractions prepared from brain, with dilute buffers causes the solubilization of part of the ribosomal RNA that then can subsequently be identified in the chromatogram. Autolysis under aerobic conditions has been shown to cause an increase in the nucleic acid component in the chromatogram that corresponds to the ribosomal RNA. Aerobic autolysis causes part of the ribosomal RNA to be solubilized, as evidenced by its failure to be sedimented by centrifugation at 105400g for 90 min. and by its appearance in the fraction corresponding to ribosomal RNA in the chromatogram. These changes were not observed when autolysis was carried out under anaerobic conditions. Aerobic autolysis also caused changes in those proteins that are strongly adsorbed on the gel. In general, the proteins of the cell sap appeared to be fairly stable to autolysis. Marked differences in the chromatograms are observed when soluble proteins from the particle fraction were autolysed and chromatographed.