Article Figures & Data
Figures
- Figure 1.
Distribution of hSOD1 and organelles in density gradient ultracentrifugations. Tissue homogenization, preparation of gradients, ultracentrifugation, and analysis of fractions were performed as described in Materials and Methods. A, Comparison of fractionation of mitochondria from brain, spinal cord, and liver. B, C, Location of markers for organelles in separations of spinal cord homogenates. Synaptotagmin is a marker for synaptic vesicles and plasma membrane. D–K, Relative distributions of hSODs and the mitochondrial marker SDH in spinal cord extracts from different transgenic models in presymptomatic and terminal stages of disease, as indicated in the framed insets. Note that the relative amounts of hSODs in the gradients have been multiplied by 10 to make the data easier to visualize. L, M, Ca2+ and succinate were added to a filtrate of a brain homogenate from a D90A mouse to cause permeability transition pore opening and mitochondrial swelling. L and M show density gradient ultracentrifugations of aliquots of the filtrate either untreated or treated with Ca2+ plus succinate. AP, Acid phosphatase; β-COP, Golgi β-coatomer protein; Mito (SDH), the mitochondrial marker SDH; PMP70, 70 kDa peroxisomal membrane protein.
- Figure 2.
Examples of Western immunoblots for localization of hSOD1 in tapped gradient fractions. To allow visualization in the same blot, the fractions were diluted or concentrated as indicated. The arrows indicate the location of the peak of the mitochondrial marker SDH (Mito). A, Density gradient centrifugation of a spinal cord extract from a presymptomatic G93A mouse; see also Figure 1D. Density gradient centrifugations of spinal cords from three different presymptomatic (B) and one terminally ill (C) G127X mice; see also Figure 1, J and K. The blots of the 10-fold concentrated fractions from the presymptomatic mice were overexposed to visualize the very faint bands.
- Figure 3.
Quantification of amounts and proportions of SOD1 associated with mitochondria in spinal cord (black columns) and brain (gray columns). The amount of hSOD1 in the three fractions in the gradient with the highest levels of the mitochondrial marker SDH (mostly fractions 9–11) was determined. The ratio of hSOD1/SDH in these was then multiplied by the SDH activities in all 18 fractions to estimate the total amount of hSOD1 associated with mitochondria. Both absolute and relative (percentage) amounts of hSOD1 associated with mitochondria per gram wet weight of tissue are presented. A, Amounts of hSOD1 associated with mitochondria in the different transgenic mouse strains. B, Corresponding results for mSOD1 in the transgenic strains and in nontransgenic control C57BL/6 mice. The error bars represent SDs. wt, Wild type; Sp.c., spinal cord.
- Figure 4.
Detection of hSOD1 aggregates in density gradient separations of spinal cord homogenates from presymptomatic and terminally ill mice, as indicated by the framed insets and in the figures. In each figure, the largest amount of aggregates found in a fraction was set to 100, and the results for all other fractions have been presented relative to that. A–D, Filter trap assay for hSOD1 aggregates. E, F, Filtrates of spinal cord extracts were treated with 0.5% of the detergent NP-40 and sonicated before density gradient ultracentrifugation. SDH and hSOD1 were analyzed in the tapped fractions. Mito (SDH), The mitochondrial marker SDH.
- Figure 5.
Gel chromatography of spinal cord homogenates from G85R and G127X mice. The hSOD1 in the eluted fractions was analyzed by Western immunoblot, and the resulting relative distributions (percentage) are shown in the figures. The actual immunoblots are shown under the figures. For comparison, the SOD activity of the dimeric mSOD1 is also shown. A, Results for a G85R spinal cord. B, G127X spinal cord. In the immunoblot, both the 17 kDa monomer and a 33 kDa hSOD1 complex of unknown composition are shown.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplementary Figure 1 Density gradient separation of homogenates of brains from a presymptomatic and a terminally ill G127X transgenic mouse by density gradient ultracentrifugation. Tissue homogenization, preparation of gradients, ultracentrifugation and analysis of fractions were carried out as described in Materials and Methods. A, a presymptomatic mouse and B, a terminally ill mouse.
- supplemental material - Supplementary Figure 2 Density gradient separation of a homogenate of a kidney from a presymptomatic G85R hSOD1 transgenic mouse. Tissue homogenization, preparation of gradients, ultracentrifugation and analysis of fractions were carried out as described in Materials and Methods. The mitochondrial marker SDH and the human and murine SOD1s were analyzed in the tapped fractions.
