Table 1.
Specific binding and relative remaining activity in construct screening and T50 after preparative-scale expression.
| Mutant | Specific binding (dpm)a | RAb | T50c | |
|---|---|---|---|---|
| 25 °C | 40 °C | (%) | (°C) | |
| Ala56Asn2.40 | 9±32 | 20±4 | – | N.D. |
| His83Gly2.67 | 4933±88 | 3332±42 | 68 | N.D. |
| Lys88Gly3.21 | 5327±118 | 3993±71 | 75 | N.D. |
| Leu109Ser3.42 | 2179±274 | 889±92 | 41 | N.D. |
| Leu109Thr3.42 | 3193±232 | 1567±82 | 49 | N.D. |
| Ala56Asn2.40/Leu109Ser3.42 | 42±7 | −20±36 | – | N.D. |
| His83Gly2.67/Leu109Ser3.42 | 4398±207 | 3408±159 | 77 | N.D. |
| Lys88Gly3.21/Leu109Ser3.42 | 3783±148 | 2875±77 | 76 | N.D. |
| Ala56Asn2.40/Leu109Thr3.42 | 28±11 | 15±16 | – | N.D. |
| His83Gly2.67/Leu109Thr3.42 | 4758±207 | 3315±97 | 70 | N.D. |
| Lys88Gly3.21/Leu109Thr3.42 | 4836±278 | 3967±201 | 82 | N.D. |
| Ala56Asn2.40/His83Gly2.67 | 5±30 | 9±13 | – | N.D. |
| Ala56Asn2.40/Lys88Gly3.21 | 63±9 | 44±4 | – | N.D. |
| His83Gly2.67/Lys88Gly3.21 | 5097±482 | 3987±86 | 78 | 48.6±0.1 |
| Ala56Asn2.40/His83Gly2.67/Leu109Ser3.42 | −2±2 | 1±32 | – | N.D. |
| Ala56Asn2.40/Lys88Gly3.21/Leu109Ser3.42 | 21±30 | −6±37 | – | N.D. |
| His83Gly2.67/Lys88Gly3.21/Leu109Ser3.42 | 5473±133 | 5184±290 | 95 | 47.9±0.0 |
| Ala56Asn2.40/His83Gly2.67/Leu109Thr3.42 | 2±10 | 22±16 | – | N.D. |
| Ala56Asn2.40/Lys88Gly3.21/Leu109Thr3.42 | 4±32 | 18±36 | – | N.D. |
| His83Gly2.67/Lys88Gly3.21/Leu109Thr3.42 | 4856±114 | 4513±28 | 93 | 48.0±0.2 |
| Ala56Asn2.40/His83Gly2.67/Lys88Gly3.21 | 65±7 | 51±12 | – | N.D. |
| Ala56Asn2.40/His83Gly2.67/Lys88Gly3.21/ | ||||
| Leu109Ser3.42 | 12±7 | −9±8 | – | N.D. |
| Ala56Asn2.40/His83Gly2.67/Lys88Gly3.21/ | ||||
| Leu109Thr3.42 | 11±14 | 25±11 | – | N.D. |
| Original (dN15/Ser309Ala) | 2523±75 | 1136±81 | 45 | 43.5±0.5 |
The value is the average specific binding with the standard deviation. The average specific binding was calculated as the difference between the total binding (n=3) and the nonspecific binding (n=2). Total and nonspecific binding were set as the binding activity for 0.5 nM [3H]-LTB4 for each membrane fraction without or with 0.5 μM LTB4 treatment, respectively. The membrane fractions from the small-scale expression experiment were reacted using the same amount (10 μg) of total protein.
The remaining activity (RA, %) is the ratio of the specific binding at 25 °C to that at 40 °C.
T50 is the half remaining binding activity temperature measured using the membrane fraction gpBLT1 (0.4 μg protein) from preparative-scale expression. A representative result is shown in Fig. 2A. N.D.: not determined.