Results: 5

1.
FIGURE 3.

FIGURE 3. From: Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice.

Transgenic expression of a H46R/H48Q SOD1 results in decreased copper metallation of the endogenous mouse SOD1. The metallation states of mouse SOD1 from a non-transgenic and transgenic mouse are shown. The metallation state of mouse SOD1 was measured from the brain, spinal cord, and liver of a nontransgenic and a H46R/H48Q transgenic mouse. Because the H46R/H48Q SOD1 mutant protein eluted differently from the mouse SOD1 off the HPLC column and does not bind copper, it was possible to measure the effect of overexpression separated from effects of insufficient copper. Overexpression of this hSOD1 leads to decreased copper metallation for the mouse SOD1 as inferred when comparing mSOD1 in NTG to mSOD1 in H46R/H48Q (*) for each tissues examined. *, p < 0.05.

Herman L. Lelie, et al. J Biol Chem. 2011 January 28;286(4):2795-2806.
2.
FIGURE 1.

FIGURE 1. From: Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice.

Concentration of copper and zinc in whole spinal cord, brain, and liver reflect tight copper regulation. Sections of spinal cord, brain, and liver from a NTG and transgenic mice overexpressing human WT, G37R, G93A, or H46R/H48Q SOD1 were weighed, completely digested in nitric acid, then subjected to metal quantitation by ICP-MS. The H46R/H48Q mouse brain and liver zinc concentrations were consistently lower than all other mice (arrow, p < 0.005). Far right panel, a box plot graph incorporating all measurements from the same tissue demonstrates that copper concentrations remain relatively constant in the brain and spinal cord, whereas zinc was lower in spinal cord, somewhat higher in brain (p value 0.019), and highest in liver (*, p < 0.0001 relative to spinal cord). The bar graph results are reported in mean μg of metal/g of tissue (wet weight), and error bars represent ±S.D. of three or more independent mice per genotype.

Herman L. Lelie, et al. J Biol Chem. 2011 January 28;286(4):2795-2806.
3.
FIGURE 4.

FIGURE 4. From: Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice.

Copper and zinc concentrations from detergent-insoluble and -soluble species suggest minimal SOD1 metallation in detergent-extracted SOD1. A, the detergent-insoluble species were prepared from nontransgenic and G93A, G37R, H46R/H48, and hWT SOD1 transgenic mice. SOD1 concentration was measured by densitometry from a Western blot, and the copper and zinc concentrations were measured by ICP-MS. Despite the variable levels of SOD1 from each detergent insoluble pellet preparation, the levels of copper and zinc did not fluctuate across all the mice types. B, detergent-insoluble and detergent-soluble species were analyzed for activity and SOD1 concentration. The relative activity was divided by the SOD1 concentration to gain a rough idea of the level of copper bound to the SOD1. The axis on the left is in arbitrary units. The soluble hWT, H46R/H48Q, and G37R SOD1s were measured as 2123, 172, and 1682, respectively, which was significantly higher activity per dimer than their detergent-insoluble counterparts, which measured as 143, 9, and 18, respectively. Note that very little WT hSOD1 was found in the detergent-resistant pellets. The box plots represent median values and 25th and 75th percentile ranges, and bar graphs represent mean ± S.D. of three or more independent mice per genotype.

Herman L. Lelie, et al. J Biol Chem. 2011 January 28;286(4):2795-2806.
4.
FIGURE 5.

FIGURE 5. From: Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice.

Infrared and x-ray fluorescence microscopy images data analysis of spinal cord cross-sections from NTG mice or mice expressing WT, G93A, G37R, or H46R/H48Q SOD1. A, image maps prepared from infrared and x-ray fluorescence data collection are shown. Top row, principal component analysis of the infrared images clearly differentiates between the white and gray matter based on differences in lipid and protein composition. The copper (middle row) and zinc (bottom row) XFM images illustrate changes in metal concentration and distribution as a function of SOD1 expression and mutation state. XFM images are presented in micromolar units as determined by the NIST standard reference. The scale bar represents 0.1 mm. At least three cross-sections were analyzed from four mice of each genotype, and representative images are shown. B, median copper and zinc concentrations (mm) in the same cross-sections of spinal cord, as measured by XMF are shown. All spinal cords from mice expressing mutant hSOD1 showed elevated zinc in the white matter as compared with NTG. In the gray matter, copper was elevated over NTG in WT, G37R, and G93A but not in the double mutant H46R/H48Q, which does not bind copper (*, significantly different from NTG; **, significantly different from WT by Kruskal-Wallis and Mann-Whitney U test). At least three cross-sections were analyzed from four mice of each genotype.

Herman L. Lelie, et al. J Biol Chem. 2011 January 28;286(4):2795-2806.
5.
FIGURE 2.

FIGURE 2. From: Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice.

SOD1 metallation in various tissues from transgenic mice. Copper and zinc in SOD1 was measured by HPLC-ICP-MS as described under “Experimental Procedures.” The metallation of WT, G37R, G93A, and H46R/H48Q hSOD1 from transgenic mice and the endogenous mSOD1 from a nontransgenic mouse are shown. A, spinal cord is shown. B, brain is shown. C, liver is shown. Note that in spinal cord and brain the total metal site occupancy does not differ significantly for the G37R, G93A, or hWT SOD1 or the mouse SOD1. However, the copper and zinc ratios are different, with less copper and more zinc being found in the overexpressed human SOD1s as compared with mouse SOD1 in the non-transgenic animals. Human WT-SOD1 had a slightly lower copper content and a higher zinc content compared with the mutant SOD1s G93A and G37R (*). The mutations in H46R/H48Q SOD1 destroy the copper binding site, and as such no detectable copper levels were found. Zinc metallation is also significantly lower in the H46R/H48Q mutant compared with the other transgenic SOD1s (*). In liver, SOD1 metallation is starkly different; in particular, G93A and hWT had dramatically reduced zinc occupancy levels relative to nervous tissue (#). D, the coppers bound to the SOD1 are active. A plot of the copper concentrations measured by the HPLC-ICP method compared with the copper measured by SOD activity shows a strong correlation, which indicates that the majority of copper bound to these SOD1 species are active. The measurements are from all the endogenous and exogenously expressed SOD1s from the mice analyzed in this study. E, right, shown is a stacked bar graph representation of metallation to illustrate total metallation. The dashed line at four metals/dimer delineates a fully occupied metal binding site. With the exception of the H46R/H48Q mutant, most other SOD1s are highly metallated. Left, shown is a box plot graph of combined metallation states for each tissue demonstrating tissue variability. Despite the vastly different trends among mutant metallation states, it is interesting to note that median copper and zinc values remain consistent across all tissues examined; these are also similar to as-isolated SOD1s from various host organisms. All bars represent the mean ± S.D. of three or more independent mice per genotype. Box plots represent median values and 25th to 75th percentile ranges. * p < 0.001; # p < 0.01.

Herman L. Lelie, et al. J Biol Chem. 2011 January 28;286(4):2795-2806.

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