Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases alpha and beta. Interaction with rhodopsin-transducin complex and thermal stability.
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 alpha(1-130)beta(131-152) and NDP kinase beta(1-130)alpha(131-152)) and tagged derivatives (NDP kinase HA-alpha(1-130)beta(131-152), NDP kinase HA-beta(1-130)alpha(131-152), 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 alpha(1-130)beta(131-152), and NDP kinase HA-alpha(1-130)beta(131-152) were similar in their thermal stability (T(1/2) = 61-63 degrees C). NDP kinase beta, NDP kinase beta(1-130)alpha(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta were inactivated at a lower temperature (T(1/2) = 51-54 degrees C). NDP kinase HA-alpha(1-130)beta(131-152) interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta(1-130)alpha(131-152) 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.
Orlov
N Ia
NIa
Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, 173-0015, Japan. orlov@iteb.ru
Ishijima
Y
Y
Orlov
D N
DN
Orlova
T G
TG
Bursteĭn
E A
EA
Kimura
N
N
D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology; D004795:Enzyme Stability; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry* / Q000201:enzymology; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D007527:Isoenzymes; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D009701:Nucleoside-Diphosphate Kinase; D015293:Transducin