Quaternary structural analysis of nucleoside diphosphate kinases using capillary electrophoresis.
Capillary electrophoresis was used to monitor the quaternary structure and structural changes by denaturants of nucleoside diphosphate kinase (NDP kinase). NDP kinase from human erythrocyte consists of two kinds of polypeptide chains: A (Nm23-H1) and B (Nm23-H2), which are the products of nm23-H1 and -H2 genes, respectively. Structural characteristics of native NDP kinase, recombinant Nm23-H1 and -H2 were examined using capillary electrophoresis employing high pH running buffer in an uncoated fused-silica capillary as a separation column. The results were compared with those of the conventional sodium dodecyl sulfate and non-denaturing polyacrylamide gel electrophoresis. It shows that capillary electrophoresis is a possible tool to investigate protein structure, including the quaternary structure. This protocol was applied to the investigation of the denaturation process of each enzyme by denaturants (6 M urea or heating to 70 degrees C) and to the monitoring of the interaction between denatured Nm23-H1 and -H2. The structural information of NDP kinase obtained by analysis using capillary electrophoresis is valuable for understanding its suggested biological function as a tumor metastasis suppressor and c-myc transcription factor, etc.
Heo
Y J
YJ
College of Pharmacy, Ewha Womans University, Seoul, South Korea.
Kim
S Y
SY
Kim
E
E
Lee
K J
KJ
D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D009701:Nucleoside-Diphosphate Kinase