A native electrophoretic technique to study oligomerization and activity of cytosolic 5%26apos;-nucleotidase II

作者：Filoni Daniela Nicole; Pesi Rossana^*; Allegrini Simone; Camici Marcella; Tozzi Maria Grazia

来源：Analytical and Bioanalytical Chemistry, 2013, 405(27): 8951-8954.

DOI：10.1007/s00216-013-7313-3

• Native electrophoresis
• 5 %26apos
• -Nucleotidase
• Oligomeric state
• Activity staining

The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5%26apos;-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

• 出版日期2013-11