Effects of Ionic Strength and Rapamycin on the Peptidylprolyl cis-trans Isomerase Activity of Immunoprecipitates Obtained From K562 Cell Membranes
| . | kobs (s−1) . |
|---|---|
| IP in presence of 150 mmol/L NaCl | |
| No additions | 0.3942 ± 5 × 10−4 |
| 20 nmol/L IP3 during determination, only | 0.0094 ± 5 × 10−5 |
| IP with 10.0 nmol/L rapamycin | 0.2973 ± 6 × 10−4 |
| IP with 10.0 nmol/L rapamycin, 20.0 nmol/L IP3 during determination | 0.0314 ± 8 × 10−4 |
| IP in presence of 50 mmol/L NaCl | |
| No additions | 0.3234 ± 4 × 10−4 |
| IP with 10.0 nmol/L rapamycin | 0.2022 ± 3 × 10−4 |
| IP with 200.0 nmol/L rapamycin | 0.0051 ± 5 × 10−5 |
| Uncatalyzed reaction | 0.0046 ± 2 × 10−5 |
| . | kobs (s−1) . |
|---|---|
| IP in presence of 150 mmol/L NaCl | |
| No additions | 0.3942 ± 5 × 10−4 |
| 20 nmol/L IP3 during determination, only | 0.0094 ± 5 × 10−5 |
| IP with 10.0 nmol/L rapamycin | 0.2973 ± 6 × 10−4 |
| IP with 10.0 nmol/L rapamycin, 20.0 nmol/L IP3 during determination | 0.0314 ± 8 × 10−4 |
| IP in presence of 50 mmol/L NaCl | |
| No additions | 0.3234 ± 4 × 10−4 |
| IP with 10.0 nmol/L rapamycin | 0.2022 ± 3 × 10−4 |
| IP with 200.0 nmol/L rapamycin | 0.0051 ± 5 × 10−5 |
| Uncatalyzed reaction | 0.0046 ± 2 × 10−5 |
Solubilized K562 membranes were incubated with biotin-conjugated, recombinant antiphosphotyrosine (RC20) under high (150 mmol/L NaCl) or low (50 mmol/L NaCl) ionic strength conditions, as indicated. Rapamycin and IP3 were present during the immunoprecipitation or added at the time of the isomerase determination, as indicated. Immune complexes were isolated using agarose-linked streptavidin. Aliquots of immobilized proteins (20 μL) were examined for peptidylprolylcis-trans isomerase 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). Reactions were observed for 7 to 9 half-lives. A sampling interval of 0.2 seconds was used with uninhibited reactions; it was 1.5 seconds when immunoprecipitation had been conducted in the presence of 200 nmol/L rapamycin or when the reaction was inhibited by 20 nmol/L IP3. The extent of the uncatalyzed reaction was assessed by conducting the determination using biotin-conjugated RC20 that had not been exposed to the K562 cell membrane preparation.
Abbreviation: IP, immunoprecipitation.