Table 1.

Enzymatic, HNF1-binding, and transcriptional activity of DCoH mutants compared to the wild type

DCoH mutants	Dehydratase activity, %	Binding to HNF1, %	Transcriptional activity, %
Wild type	100  ±  5	100  ±  6	100  ±  16
H62N	2.6  ±  0.2	99  ±  3	90  ±  20
H63L	0.49  ±  0.03	106  ±  8	91  ±  19
H80L	20  ±  1	82  ±  22	98  ±  11
C82R	49  ±  4	93  ±  13	97  ±  11
H63L/H80L	0.15  ±  0.01	83  ±  11	54  ±  25
H62N/H63L	0.012  ±  0.002	111  ±  10	14  ±  10
K72E	96  ±  3	101  ±  7	100  ±  12
R31Q	118  ±  5	108  ±  11	122  ±  25
R31Q/K72E	135  ±  8	101  ±  10	432  ±  53
E65A	45  ±  2	105  ±  13	156  ±  46
E65A/K72E	53  ±  3	96  ±  14	310  ±  37
F67A	72  ±  2	65  ±  7	185  ±  28

The dehydratase activities were determined as described in Materials and Methods. The results are the means ± SD of three measurements. The HNF1-binding assays were performed in MaxiSorp 96 well microtiter plates (Nunc) and are based on standard ELISA protocols (37). In each well 400 ng of HNF1 in 40 μl Hepes buffered saline (HBS, 20 mM Hepes pH 7.5/150 mM NaCl) were immobilized. The wells were washed and blocked with HBS/5% milk for 1 hr, then rinsed with HBS/1% BSA. Incubation with wild-type DCoH or its mutants occurred in 40 μl of HBS/1% BSA for 2–4 hr at 22°C. The final concentration of DCoH was 30 μg/ml. After three washes, antibody against DCoH (30), was added and incubation proceeded overnight. The primary antibody was washed away and secondary antibody, anti-rabbit IgG alkaline phosphatase conjugate (Promega), was added for 1 hr at a dilution of 1:4,000. The color reaction was performed with 5 mM p-nitrophenyl-phosphate. Optical densities (405 nm) were measured with a microplate reader (Bio-Tek EL340). The following controls were included in each experiment: minus HNF1 and minus DCoH (Fig. 3). Each value represents the mean ± SD of at least three independent experiments. The antigenicity of each mutant was assessed by immobilizing wild-type and mutant DCoH (0.8–3.2 ng) either on Multisorb plates or nitrocellulose (dot blots) and then probing with antibody. As a control, gel shift assays were done as described (16) with the mutants C82R, H63L, and wild-type DCoH, and gave the same results as the ELISA assay. The determination of the in vivo transcriptional activity is described in Materials and Methods. Transfections were performed with duplicate samples. The results are means ± SD of four independent experiments each and are corrected for transfection efficiency. All listed activities are percentages of the wild-type activity.