TABLE 1.
GSTZ1 Expression per Gram Livera |
GSTZ1 Expression per Milligram Proteina |
GSTZ1 Activityb |
||||
---|---|---|---|---|---|---|
Cytosolc | Mitochondriac | Cytosol | Mitochondria | Cytosol | Mitochondria | |
nmol/min/mg | ||||||
Human (n = 4) | 40.1 ± 11.8 | 6.78 ± 1.11 | 0.54 ± 0.10 | 0.23 ± 0.04 | 0.49 ± 0.08 | 0.20 ± 0.07 |
Control rat (n = 4) | 0.92 ± 0.06 | 0.14 ± 0.01 | 0.94 ± 0.07 | 0.24 ± 0.02 | 1.47 ± 0.09 | 0.48 ± 0.02 |
DCA rat (n = 4) | 0.11 ± 0.03 | 0.012 ± 0.003 | 0.10 ± 0.02 | 0.021 ± 0.006 | 0.11 ± 0.06d | N.D. |
N.D., not detectable.
GSTZ1 expression was analyzed by using 5 μg of human cytosol, 5 μg of mitochondria, 40 μg of rat cytosol, and 100 μg of rat mitochondria on Western blots probed with hGSTZ1 polyclonal antibody. Band intensity was quantitated by ImageJ software. For human samples, the amounts of GSTZ1 were quantitated by comparing the immuno-intensity to a standard curve constructed with pure hGSTZ1. Data are shown as μg hGSTZ1/g liver or μg hGSTZ1/mg protein. For rat samples, individual GSTZ1 expression was normalized as a fraction of one control cytosol that had highest level of GSTZ1 expression. Data are shown as relative GSTZ1 expression/g liver or relative GSTZ1 expression/mg protein.
Samples were dialyzed in 1.15% KCl, 0.05 M potassium phosphate buffer, pH 7.4 before being assayed with 0.2 mM 14C-DCA as substrate.
Cytosolic yield was calculated based on the total cytosolic protein recovered experimentally. Mitochondrial yield was standardized to 30 mg of mitochondrial protein per gram liver to correct for loss of mitochondria during differential centrifugation (Fleischer et al., 1979).
Of the cytosol of four DCA rats, only two showed detectable activities. The limit of detection was 0.067 nmol/min/mg.