Table 4.
Relative Activities of Ca2+-Dependent TGase and Selected Enzymes in Homogenates, Mitochondria, and Mitoplasts Prepared from Mouse Livera
| enzyme/fraction | total units | recovery % | specific activity | specific activity relative to homogenate |
|---|---|---|---|---|
| GDH | ||||
| homogenate | 110 ± 7 | (100) | 33.0 ± 2.5 | (1) |
| mitochondria | 7.5 ± 2.5 | 6.8 | 172 ± 32 | 5.4 |
| mitoplasts | 3.0 ± 0.5 | 2.7 | 695 ± 188 | 21.0 |
| γ-cystathionase | ||||
| homogenate | 36.4 ± 3.1 | (100) | 10.6 ± 0.9 | (1) |
| mitochondria | <0.03 | <0.10 | <0.01 | <0.001 |
| mitoplasts | <0.03 | <0.10 | <0.01 | <0.001 |
| LDH | ||||
| homogenate | 333 ± 98 | (100) | 97 ± 25 | (1) |
| mitochondria | ∼10 | ∼3 | ∼3 | ∼0.03 |
| mitoplasts | <10 | <3 | <3 | <0.03 |
| mitAspAT | ||||
| homogenate | 226 ± 28 | (100) | 68.0 ± 9.8 | (1) |
| mitochondria | 7.1 ± 1.5 | 3.1 | 160 ± 27 | 2.4 |
| mitoplasts | 4.7 ± 1.0 | 2.1 | 1060 ± 240 | 15.6 |
| KGDHC | ||||
| homogenate | 11.1 ± 0.4 | (100) | 3.33 ± 0.32 | (1) |
| mitochondria | 0.57 ± 0.05 | 5.1 | 7.98 ± 0.23 | 2.4 |
| mitoplasts | 0.060 ± 0.015 | 0.56 | 3.36 ± 1.36 | 0.99b |
| TGase | ||||
| homogenate | 1336 ± 200 | (100) | 405 ± 74 | (1) |
| mitochondria | 0.070 ± 0.017 | 0.0052 | 9.7 ± 0.7 | 0.024 |
| mitoplasts | 0.047 ± 0.012 | 0.0035 | 10.0 ± 2.7 | 0.025 |
Except in the case of Ca2+-dependent TGase, total activity and specific activity are expressed as μmol/min and milliunits/mg of protein, respectively. In the case of TGase, total activity and specific activity are expressed as nmol/h and pmol h−1 (mg of protein)−1, respectively. The Ca2+-dependent activity was calculated using blank 2 (see the Experimental Procedures). A total of 18 6-month-old B6/CBA51/J mice were sacrificed in two lots of two on the same day. Thus, n = 4 separate preparations from the same group of mice. Each batch of mouse livers weighed ∼5 g. All enzyme activities, except TGase, were measured in triplicate. TGase activity was measured in duplicate. However, n = 4 because only the average value for each enzyme activity in each preparation was used in calculating the mean.
The large loss of KGDHC activity during the preparation of mitoplasts from liver mitochondria does not appear to be related to protease activity. Protease inhibitor cocktail was present during the preparation of the mitoplasts. Moreover, KGDHC activity in brain mitoplasts was not destroyed during preparation from mitochondria (Table 3). The unusual susceptibility of liver KGDHC to damage during mitoplast preparation requires further study.