TABLE 1.
Competitive inhibition analysis with cold pheromones and inhibitors
| Pheromone or inhibitor | Sequence | Inhibition of [3H]cPD1 binding (IC50 [nM])a to:
|
cPD1 activitye (nM) | ||
|---|---|---|---|---|---|
| TraCb | Donor spheroplastc | GST-TraAd | |||
| cPD1 | H-Phe-Leu-Val-Met-Phe-Leu-Ser-Gly-OH | 34.2 | 1.49 | 0.50 | 0.1 |
| iPD1 | H-Ala-Leu-Ile-Leu-Thr-Leu-Val-Ser-OH | 78.5 | 0.45 | 0.33 | >350 |
| cAD1 | H-Leu-Phe-Ser-Leu-Val-Leu-Ala-Gly-OH | >1,000 | >1,000 | >1,000 | >350 |
| iAD1 | H-Leu-Phe-Val-Val-Thr-Leu-Val-Gly-OH | >1,000 | >1,000 | >1,000 | >350 |
| cCF10 | H-Leu-Val-Thr-Leu-Val-Phe-Val-OH | 126 | >1,000 | >1,000 | >1,000 |
| iCF10 | H-Ala-Ile-Thr-Leu-Ile-Phe-Ile-OH | 100 | >1,000 | >1,000 | >350 |
| cAM373 | H-Ala-Ile-Phe-Ile-Leu-Ala-Ser-OH | >1,000 | >1,000 | >1,000 | >350 |
| iAM373 | H-Ser-Ile-Phe-Thr-Leu-Val-Ala-OH | >1,000 | >1,000 | >1,000 | >350 |
| cOB1 | H-Val-Ala-Val-Leu-Val-Leu-Gly-Ala-OH | >1,000 | >1,000 | >1,000 | >350 |
| iOB1 | H-Ser-Leu-Thr-Leu-Ile-Leu-Ser-Ala-OH | 16.5 | >1,000 | >1,000 | >350 |
Concentrations of cold peptides that produce 50% inhibition of the specific binding (IC50) were estimated from the plots of [3H]cPD1 binding. The specific binding was calculated by subtraction of the nonspecific binding determined in the presence of 1 μM cold cPD1 from the maximum binding.
Binding to TraC was assayed with TraC-expressing cells (intact cells of OG1X carrying pDLES23) and 0.5 nM [3H]cPD1.
Binding to donor spheroplasts was assayed with spheroplasts of OG1X carrying pAM351 and 0.5 nM [3H]cPD1.
Binding to GST-TraA was assayed with 20 nM GST-TraA and 0.2 nM [3H]cPD1.
cPD1 activity represents the minimum concentration inducing cell clumping of a pPD1 donor strain (39-5Sα) in a microtiter bioassay (40).