Reduction of specific activity of CD45 PTP in CD45-AP-null thymocytes
Experiment no. and cell type . | Before IP . | After IP . | Amount removed by IP . | Specific activity (relative specific activity, %) . |
|---|---|---|---|---|
| 1 | ||||
| Wild type | ||||
| PTP activity | 12 783 | 2494 | 10 289 | 23.4 (100) |
| CD45 amount | 520.4 | 81.0 | 439.4 | |
| CD45-AP-null | ||||
| PTP activity | 5649 | 991 | 4658 | 17.2 (73.5) |
| CD45 amount | 338.6 | 68.3 | 270.3 | |
| 2 | ||||
| Wild type | ||||
| PTP activity | 3465 | 959 | 2506 | 17.87 (100) |
| CD45 amount | 140.2 | 0 | 140.2 | |
| CD45-AP-null | ||||
| PTP activity | 1766 | 456 | 1 310 | 14.92 (83.5) |
| CD45 amount | 87.8 | 0 | 87.8 | |
| 3 | ||||
| Wild type | ||||
| PTP activity | 15 758 | 4708 | 11 050 | 74.8 (100) |
| CD45 amount | 190.8 | 43.1 | 147.7 | |
| CD45-AP-null | ||||
| PTP activity | 15 300 | 3693 | 11 607 | 53.5 (71.5) |
| CD45 amount | 249.0 | 32.1 | 216.9 |
Experiment no. and cell type . | Before IP . | After IP . | Amount removed by IP . | Specific activity (relative specific activity, %) . |
|---|---|---|---|---|
| 1 | ||||
| Wild type | ||||
| PTP activity | 12 783 | 2494 | 10 289 | 23.4 (100) |
| CD45 amount | 520.4 | 81.0 | 439.4 | |
| CD45-AP-null | ||||
| PTP activity | 5649 | 991 | 4658 | 17.2 (73.5) |
| CD45 amount | 338.6 | 68.3 | 270.3 | |
| 2 | ||||
| Wild type | ||||
| PTP activity | 3465 | 959 | 2506 | 17.87 (100) |
| CD45 amount | 140.2 | 0 | 140.2 | |
| CD45-AP-null | ||||
| PTP activity | 1766 | 456 | 1 310 | 14.92 (83.5) |
| CD45 amount | 87.8 | 0 | 87.8 | |
| 3 | ||||
| Wild type | ||||
| PTP activity | 15 758 | 4708 | 11 050 | 74.8 (100) |
| CD45 amount | 190.8 | 43.1 | 147.7 | |
| CD45-AP-null | ||||
| PTP activity | 15 300 | 3693 | 11 607 | 53.5 (71.5) |
| CD45 amount | 249.0 | 32.1 | 216.9 |
The PTP activity present in microsomal fractions of wild-type and CD45-AP-null thymocytes was determined with the use of Raytide as a substrate, before and after removal of CD45 by immunoprecipitation. The PTP activity value shown for each experiment is an average of duplicate or triplicate assays. PTP activity removed by immunoprecipitation was obtained by subtracting the activity present after immunoprecipitation from the activity present before immunoprecipitation; this represents PTP activity attributable to CD45. Similarly, the amount of CD45 before and after immunoprecipitation was obtained by scanning densitometry of immunoblots; the difference between the 2 amounts represents the amount of CD45 removed by immunoprecipitation. The specific activity of CD45 PTP was then calculated by dividing the CD45 PTP activity removed by immunoprecipitation by the amount of CD45 removed by immunoprecipitation. The relative specific activity of CD45-AP-null thymocytes is expressed as a percentage of the specific activity of wild-type counterparts. The arithmetic mean of relative specific activities ± SD of the 3 experiments is 76.2 ± 6.4. The difference between wild-type and CD45-AP- null thymocytes was statistically significant as determined by Student t test (P < .005).
IP indicates immunoprecipitation.