FIG. 8.

Digestion of wild-type and mutant PCKs with trypsin. (A) SDS-gradient PAGE (8 to 18% polyacrylamide) of purified wild-type and partially purified mutant PCKs treated with trypsin for 30 min as described in Materials and Methods. The arrows (▸, ◂) indicate the position of soya bean trypsin inhibitor. Lanes: 1, 7 μg of wild-type PCK (WT); 2, WT + 0.6 μg of trypsin; 3, WT + 0.2 μg of trypsin; 4, WT + 0.06 μg of trypsin; 5, WT + 0.02 μg of trypsin; 6, size standards; 7, Arg2Ser mutant; 8, Arg2Ser + 0.6 μg of trypsin; 9, Arg2Ser + 0.2 μg of trypsin; 10, Arg2Ser + 0.06 μg of trypsin; 11, Arg2Ser + 0.02 μg of trypsin; 12, Arg396Ser mutant; 13, Arg396Ser + 0.6 μg of trypsin; 14, Arg396Ser + 0.2 μg of trypsin; 15, Arg396Ser + 0.06 μg of trypsin; 16, Arg396Ser + 0.02 μg of trypsin; 17, Arg2Ser Arg396Ser double mutant; 18, Arg2Ser Arg396Ser + 0.6 μg of trypsin; 19, Arg2Ser Arg396Ser + 0.2 μg of trypsin; and 20, Arg2Ser Arg396Ser + 0.06 μg of trypsin. (B) Model of trypsin hydrolysis of wild-type PCK. I represents uncut PCK, with N terminus at the left; II, cut at Arg2; III, cut at Arg396; IV, larger product of cutting at Arg2 and at Arg396; and V, small C-terminal fragment. Size standards for lane 7: bovine serum albumin, 66 kDa; ovine serum albumin, 45 kDa; pepsin, 34.7 kDa; α-lactoglobulin, 18.4 kDa; and lysozyme, 14.3 kDa. Activity of PCK after digestion with different amounts of trypsin as described in Materials and Methods. (C) Wild-type PCK. (D) Arg2Ser PCK.(E) Arg396Ser PCK. (F) Arg2Ser Arg396Ser double mutant. All assays had 1 mM MgATP and 2.4 mM Mg²⁺. In panels C to F, • represents activity with 400 μM Ca²⁺ and ▪ represents activity with no Ca²⁺.