Basic Study
Copyright ©The Author(s) 2015.
World J Biol Chem. Nov 26, 2015; 6(4): 366-378
Published online Nov 26, 2015. doi: 10.4331/wjbc.v6.i4.366
Table 1 Mutations introduced to glyceraldehyde 3-phosphate dehydrogenase cDNA ORF
GAPDH variantMutagenic primers1Nucleotide changeCodon change
Y94ACGATGCTGGCGCTGAGGCCGTCGTGGAGTCCACTGG280-281UAC- > GCC
CCAGTGGACTCCACGACGGCCTCAGCGCCAGCATCG
S98AGAGTACGTCGTGGAGGCCACTGGCGTCTTCACC290UCC- > GCC
GGTGAAGACGCCAGTGGCCTCCACGACGTACTC
T99AAGTACGTCGTGGAGTCCGCTGGCGTCTTCACCACC295ACU- > GCU
GGTGGTGAAGACGCCAGCGGACTCCACGACGTACT
T99IGAGTACGTCGTGGAGTCCATTGGCGTCTTCA296ACU- > AUU
TGAAGACGCCAATGGACTCCACGACGTACTC
Table 2 Kinetic parameters of wild type and mutated glyceraldehyde 3-phosphate dehydrogenase variants
GAPDH variantKm (G3P) (μmol/L)Km (NAD+) (μmol/L)
Wild type77 ± 25.957 ± 11.1
Y94A87 ± 33.197.4 ± 11
S98A100 ± 36.7101 ± 7.2
T99A112 ± 44.7105 ± 17.7
T99I106 ± 38.3741 ± 257
Table 3 Fluorescence recovery after photobleaching analysis of dynamic properties of wild type and mutated enhanced green fluorescent protein-glyceraldehyde 3-phosphate dehydrogenase
ProteinCompartment(1-Mf)t1/2, sD,μm2/s
EGFP-GAPDH-WTcyto0.18 ± 0.0380.19 ± 0.05527.17 ± 1.632
EGFP-GAPDH-Y94Acyto0.17 ± 0.1290.09 ± 0.01443.76 ± 7.158
EGFP-GAPDH-S98Acyto0.20 ± 0.0090.12 ± 0.01629.15 ± 3.885
EGFP-GAPDH-T99Acyto0.11 ± 0.0620.11 ± 0.02335.52 ± 8.133
EGFP-GAPDH-T99Icyto0.17 ± 0.060.22 ± 0.04117.46 ± 1.733
EGFP-GAPDH-WTnuclei0.62 ± 0.0481.41 ± 0.0332.91 ± 0.331
EGFP-GAPDH-Y94Anuclei0.23 ± 0.1270.09 ± 0.03439.05 ± 6.291
EGFP-GAPDH-S98Anuclei0.35 ± 0.1360.13 ± 0.01826.57 ± 3.747
EGFP-GAPDH-T99Anuclei0.38 ± 0.1200.11 ± 0.02733.01 ± 9.209
EGFP-GAPDH-T99Inuclei0.29 ± 0.0280.21 ± 0.05914.97 ± 1.855
Table 4 Average potential energy, kinetic energy, total energy and temperature calculated from dynamic simulations
Polypeptide variantAverage potential (kcal)Average kinetic energy (kcal)Average total energy (kcal)Average temperature (°C)
T99-5021.897.1-4924.7293.4
A99-5007.393.7-4913.6293.7
I99-4997.8101.4-4896.4293.3