IT WAS recently reported that the non-ionic detergent, Triton X-100, will solubilize over half of the proteins of the mitochondrial fraction obtained from rat whole brain honiogenates (BRUNNGRABER and AGUILAR, 1962). This extract contained several glycolytic as well as tricarboxylic acid cycle enzymes. It is well established that the usual preparation of rat brain mitochondria is a heterogeneous mixture of cell particulates ( HEBB and WHITTAKBK, 1958; WHITTAKER, 1959; DE ROBERTIS et nl., 1962) and that this preparation is capable of glycolysis (GALLAGHER et d., 1956; BKUNNGRAREK and ABOOD, 1960). In view of the effort in this laboratory directed toward fractionation of "insoluble" proteins derived from the particulate mitochondrial fraction, it was considered desirable to ascertain the origin of enzymes solubilized by Triton X-100. By using two methods of subfractionation, it was found that isocitrate dehydrogenase, fumarate hydratase, malate dehydrogenase, aspartate and alanine transaminases, and glutamate dehydrogenase in the Triton extract of the whole mitochondrial fraction originate from the "tan" particles (mitochondria), whereas the glycolytic enzymes originate mainly from the lightcr "white" particles (myelin fragments, nerve endings, synaptic vesicles and synaptic membranes). M E T H O D SSiih/iactionation of the mitochondriaI ,fraction: The mitochondrial fraction was prepared as previously described by BRUNNGRABER and AGUILAR (1962). Subfractionation was accomplished by two methods. Method 1 utilized the density gradient ultracentrifugation procedure of WmTrAKER (1959) with the No. 40 head of the Spinco Model L ultracentrifuge. Whittaker's fractions A, B, and C were obtained.An alternative procedure was also used (Method 2). The mitochondrial fraction was rcsuspended in 0.25 M-sucrose containing 5 x M-ethylenediamine-tetraacetic acid, pH 7.0, and centrifuged at 100,OOO g for 20 rnin. The presence of a white upper layer and a more densely packed tan lower layer was apparent. The upper white material (subfraction A) was teased off with a spatula carefully avoiding disturbance of the tan material. A subfraction B was obtained by removing next all of the remaining white, including tan, so that only pure tan (subfraction C) remained at the bottom of the centrifuge tube. Fractions obtained either by Method 1 or 2 were taken up in 0.25 Msucrose and recentrifuged at 100,000 g for 20 rnin. The appearance of the pellet gave some indication of the effectiveness of the separation in Method 2. A similar method based on separation of tan from white material was reported by DAHL et al. (1960). The subfractions A, B, and C obtained by Methods 1 and 2 and a portion of the original whole mitochondria1 fraction were homogenized by hand in 10 ml (final volume) of 0.5% Triton X-100 in 0,005 M-phosphate buffer, pH 7.6 and centrifuged at 100,000 g for 30 min. The clear supernatant was used for the enzyme assays. Enzyme assays: The following enzymes were assayed: Isocitrate dehydrogenase (Ociroa, 1955); fumarate hydratase...

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.