2Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: (496) 733-0553; E-mail: firstname.lastname@example.org
* To whom correspondence should be addressed.
Received February 5, 2007; Revision received April 19, 2007
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 alpha and beta, consisting of highly homologous alpha or beta subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase alpha1-130beta131-152 and NDP kinase beta1-130alpha131-152) and tagged derivatives (NDP kinase HA-alpha1-130beta131-152, NDP kinase HA-beta1-130alpha131-152, and NDP kinase HA-beta). 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 alpha, NDP kinase alpha1-130beta131-152, and NDP kinase HA-alpha1-130beta131-152 were similar in their thermal stability (T1/2 = 61-63°C). NDP kinase beta, NDP kinase beta1-130alpha131-152, NDP kinase HA-beta1-130alpha131-152, and NDP kinase HA-beta were inactivated at a lower temperature (T1/2 = 51-54°C). NDP kinase HA-alpha1-130beta131-152 interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta1-130alpha131-152 and NDP kinase beta 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.
KEY WORDS: nucleoside diphosphate kinase, G protein transducin, thermal stability of proteins