Effect of cAMP-PK CAT on (1,4,5)IP3 and (1,3,4,5)IP4 induced Ca2+ flux in highly purified plasma membranes
| Incubation conditions . | 45Ca2+ Uptake . | % Release . |
|---|---|---|
| Experiment 1 | ||
| 1. Control uptake (+CAT, no ATP) (15-min incubation) | 59 500 ± 1600 | 0 |
| 2. Same as 1 + (1,3,4,5)IP4 | 43 900 ± 300* | 27 |
| 3. Same as 1 + ATP, + (1,3,4,5)IP4 | 41 600 ± 5000* | 30 |
| 4. Same as 1 + (1,4,5)IP3 | 5900 ± 1400* | 40 |
| 5. Same as 1 + ATP + (1,4,5)IP3 | 2300 ± 700 | 12 |
| Experiment 2 | ||
| 6. Control same as 1 (+CAT, no ATP) | 22 300 ± 1990 | 0 |
| 7. Same as 6 + (1,4,5)IP3 | 16 700 ± 527* | 25 |
| 8. Same as 6 + ATP + (1,4,5)IP3 | 21 600 ± 996 | 3 |
| 9. Same as 6 + ATP + inh. pep. + (1,4,5)IP3 | 18 800 ± 1920 | 16 |
| Incubation conditions . | 45Ca2+ Uptake . | % Release . |
|---|---|---|
| Experiment 1 | ||
| 1. Control uptake (+CAT, no ATP) (15-min incubation) | 59 500 ± 1600 | 0 |
| 2. Same as 1 + (1,3,4,5)IP4 | 43 900 ± 300* | 27 |
| 3. Same as 1 + ATP, + (1,3,4,5)IP4 | 41 600 ± 5000* | 30 |
| 4. Same as 1 + (1,4,5)IP3 | 5900 ± 1400* | 40 |
| 5. Same as 1 + ATP + (1,4,5)IP3 | 2300 ± 700 | 12 |
| Experiment 2 | ||
| 6. Control same as 1 (+CAT, no ATP) | 22 300 ± 1990 | 0 |
| 7. Same as 6 + (1,4,5)IP3 | 16 700 ± 527* | 25 |
| 8. Same as 6 + ATP + (1,4,5)IP3 | 21 600 ± 996 | 3 |
| 9. Same as 6 + ATP + inh. pep. + (1,4,5)IP3 | 18 800 ± 1920 | 16 |
Incubations as in Figure 5. Control incubations contained no ATP (which is required for both phosphorylation and inhibition of Ca2+ effect). cAMP-PK catalytic subunit (CAT) was added to all experiments at 50 U/incubation; ATP, where used, 100 μmol/L final concentration, inhibitor peptide (inh. pep.) at 10 μmol/L. Composition of inh. pep. is H-Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile-His- Asp-OH. 50 μmol/L (1,4,5)IP3 or (1,3,4,5)IP4was added to the mixtures after 10-minute incubation (equilibrium), and reactions were stopped after a further 5 minutes by rapid filtration through 0.45-μm filters. Values shown are means ± SEM of triplicate observations, with similar data obtained in 3 other membrane preparations.
P < .05 compared with respective controls using Mann-WhitneyU Confidence Interval and Test using the Minitab Data Analysis Software (Coventry, UK).