Fractionation of brain macromolecules. Chromatography on hydroxyapatite of soluble proteins and nucleic acids from whole rat brain and the effect of autolysis.
1. A procedure for the chromatographic fractionation of soluble brain proteins on calcium hydroxyapatite is described. Chromatograms obtained are reproducible; approximately 11 protein and at least three nucleic acid components can be identified. The effects of column dimensions and flow rate on the chromatograms obtained are described. 2. 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 0.25m-sucrose at 105400g for 90min. causes the sedimentation of ribosomes as well as the disappearance of the component corresponding to ribosomal RNA. Extraction of brain tissue, or particulate 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. 3. 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 90min. 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. 4. 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.
D000818:Animals; D001923:Brain Chemistry; D002623:Chemistry Techniques, Analytical; D002845:Chromatography; D006358:Hot Temperature; D006882:Hydroxyapatites; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins; D009696:Nucleic Acids; D012313:RNA; D051381:Rats; D012270:Ribosomes; D013347:Subcellular Fractions
D006882:Hydroxyapatites; D009419:Nerve Tissue Proteins; D009696:Nucleic Acids; D012313:RNA