Peptidylprolyl cis-trans Isomerase Activity of the Inositolphosphate-Binding Immunophilin From K562 Cells
| . | kobs (s−1) . |
|---|---|
| Membrane preparation | |
| No additions | 0.3924 ± 1.6 × 10−3 |
| 2.5 nmol/L IP4 | 0.2382 ± 8 × 10−4 |
| 10 nmol/L rapamycin | 0.2282 ± 1.0 × 10−3 |
| 100 nmol/L wortmannin | 0.3285 ± 9 × 10−4 |
| 300 nmol/L wortmannin | 0.2709 ± 6 × 10−4 |
| 600 nmol/L wortmannin | 0.2391 ± 4 × 10−4 |
| Immobilized immunoprecipitate | |
| No additions | 0.3211 ± 1 × 10−5 |
| 20.0 nmol/L IP3 | 0.0064 ± 1 × 10−5 |
| 1.0 nmol/L IP4 | 0.0064 ± 1 × 10−5 |
| 20.0 nmol/L PI 4,5-bisP | 0.0060 ± 1 × 10−5 |
| 20.0 nmol/L PI 4-P | 0.0060 ± 2 × 10−5 |
| 200.0 nmol/L PI 4,5-bisP | 0.0059 ± 1 × 10−5 |
| 200.0 nmol/L PI 4-P | 0.0059 ± 1 × 10−5 |
| hrFKBP12 | 0.0453 ± 1 × 10−4 |
| 20.0 nmol/L IP3 | 0.0425 ± 1 × 10−4 |
| hrFKBP12 | 0.0405 ± 4 × 10−4 |
| 200.0 nmol/L PI 4-P | 0.0364 ± 1 × 10−4 |
| 2.0 mmol/L PI 4-P | 0.0359 ± 1 × 10−4 |
| Uncatalyzed reaction | 0.0052 ± 2 × 10−5 |
| . | kobs (s−1) . |
|---|---|
| Membrane preparation | |
| No additions | 0.3924 ± 1.6 × 10−3 |
| 2.5 nmol/L IP4 | 0.2382 ± 8 × 10−4 |
| 10 nmol/L rapamycin | 0.2282 ± 1.0 × 10−3 |
| 100 nmol/L wortmannin | 0.3285 ± 9 × 10−4 |
| 300 nmol/L wortmannin | 0.2709 ± 6 × 10−4 |
| 600 nmol/L wortmannin | 0.2391 ± 4 × 10−4 |
| Immobilized immunoprecipitate | |
| No additions | 0.3211 ± 1 × 10−5 |
| 20.0 nmol/L IP3 | 0.0064 ± 1 × 10−5 |
| 1.0 nmol/L IP4 | 0.0064 ± 1 × 10−5 |
| 20.0 nmol/L PI 4,5-bisP | 0.0060 ± 1 × 10−5 |
| 20.0 nmol/L PI 4-P | 0.0060 ± 2 × 10−5 |
| 200.0 nmol/L PI 4,5-bisP | 0.0059 ± 1 × 10−5 |
| 200.0 nmol/L PI 4-P | 0.0059 ± 1 × 10−5 |
| hrFKBP12 | 0.0453 ± 1 × 10−4 |
| 20.0 nmol/L IP3 | 0.0425 ± 1 × 10−4 |
| hrFKBP12 | 0.0405 ± 4 × 10−4 |
| 200.0 nmol/L PI 4-P | 0.0364 ± 1 × 10−4 |
| 2.0 mmol/L PI 4-P | 0.0359 ± 1 × 10−4 |
| Uncatalyzed reaction | 0.0052 ± 2 × 10−5 |
Where indicated (membrane preparation), aliquots (100 μL) of the solubilized K562 cell membrane preparation were used directly, without immunoprecipitation, and the effects of IP4, rapamycin, and wortmannin were determined. To prepare immunoprecipitates, solubilized K562 cell membranes were incubated with biotin-conjugated, recombinant antiphosphotyrosine (RC20) in the presence of buffers containing 50 mmol/L NaCl. Immunoreactive proteins were isolated using agarose-linked streptavidin. The phosphatidylinositol 4-phosphate (PI 4-P) and phosphatidylinositol 4,5-bisphosphate (PI 4,5-bisP) were sonicated under an atmosphere of nitrogen immediately before being added to reaction media. Aliquots (20 μL) of immunoprecipitated, immobilized proteins were examined for peptidylprolyl cis-transisomerase activity using the substrate Suc-Ala-Leu-Pro-Phe-p-nitroanilide as described.40 Values for kobs were calculated using the equation for a first order reaction with offset (EnzFitter; BIOSOFT, Cambridge, UK). Reactions were observed for 7 to 9 half-lives. A sampling interval of 0.2 seconds was used for uninhibited reactions and 1.5 seconds for strongly inhibited reactions. The extent of the uncatalyzed reaction was assessed by conducting the determination using biotin-conjugated RC20 that had not been exposed to the K562 membrane proteins.