Table 2.
Oligomeric state of full-length Lon and its chymotryptic fragments
| Protein (aa) | molecular mass, kDa (monomer) | Size-exlusion chromatography dataa; molecular mass, kDa (n) | Velocity sedimentation datae | Crystallo-graphic dataf (n) | |||
|---|---|---|---|---|---|---|---|
| Superdex 75 16/60 | Sephacryl S-300 26/60 | S20, w′s | molecular mass, kDa (n) | RQ% | |||
| N (1–207) | 23.2 | 27.5 (1) | ND | 1.93 | 23.6 (1) | 100 | - |
| Ab(235–584) | 39.4 | 42.3 (1) | 45 (1) | ND | - | - | - |
| AP-w.t. (235–784) | 60.8 | ND | 220 (4)C 125 (2)d |
3.75 8.80 21.50 |
64.0 (1) 230.0 (4) 878.4 (ass) |
50 20 30 |
- |
| AP-S679A (235–784) | 60.8 | ND | 220 (4)C 125 (2)d |
ND | - | - | - |
| α (491–584) | 10.8 | 10.0 (1) | ND | ND | - | - | (1) |
| P-w.t. or 5679A (585–784) | 21.4 | 23.8 (1) | ND | 1.75 | 20.4 (1) | 100 | (6) |
| Lon-w.t. or 5679A (1–784) | 87.4 | ND | ~ 1000 (?) | ~ 50–100 | ND | ||
(n) - Number of subunits; RQ - relative quantity; ND - not determined; ass - associates.
Chromatography was performed at 4°C in 50 mM Tris/HCl buffer, pH 7.5, containing 0.2 M NaCl. Protein concentration in all samples was similar and about 15 mg/ml. Molecular mass (Mr) was derived from the equation logXav= a - b logMr, were Kav is availability constant determined as (Ve-Vo)/(Vt-Vo); Ve - protein elution volume, Vo and Vt - column void and total volume, respectively; a and b are experimental constants estimated from the corresponding calibration plot.
identical results were observed in the presence of 0.5 mM ADP in the column buffer, irrespective saturation of protein sample with nucleotide before size-exclusion chromatography.
Protein was saturated with 1 mM ADP or AMPPNP on the concentrating step before chromatography performed in the absence of nucleotides.
Protein was concentrated without nucleotides before chromatography.
Data obtained at 25°C without additives of nucleotides.
Published in (Botos et al., 2004a; 2004b).