Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases α and β. Interaction with rhodopsin—transducin complex and thermal stability

Abstract

To elucidate the physicochemical basis of differences between the isoforms of mammalian multifunctional nucleoside diphosphate kinase (NDP), we investigated the recombinant rat homohexameric NDP kinases α and β, consisting of highly homologous α or β subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase α^1–130β^131–152 and NDP kinase β^1–130α^131–152) and tagged derivatives (NDP kinase HA-α^1–130β^131–152, NDP kinase HA-β^1–130α^131–152, and NDP kinase HA-β). The thermal stability of these proteins and the ability of some of them to interact with the Rhodopsin—transducin (R*Gt) complex have been studied. It was found that NDP kinase α, NDP kinase α^1–130β^131–152, and NDP kinase HA-α^1–130β^131–152 were similar in their thermal stability (T _1/2=61−63°C). NDP kinase β, NDP kinase β^1–130α^131–152, NDP kinase HA-β^1–130α^131–152, and NDP kinase HA-β were inactivated at a lower temperature (T _1/2=51−54°C). NDP kinase HA-α^1–130β^131–152 interacted with the R*Gt complex in the same manner as NDP kinase α, whereas the interaction of NDP kinase HA-β^1–130α^131–152 and NDP kinase β with the photoreceptor membranes under the same conditions was very weak. It is suggested that the variability of the region V1 is a structural basis for the multifunctionality of NDP kinase hexamers in the cell.