Simple purification and functional reconstitution of octopus photoreceptor Gq, which couples rhodopsin to phospholipase C.
In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.
Kikkawa
S
S
Department of Life Science, Himeji Institute of Technology, Hyogo, Japan.
Tominaga
K
K
Nakagawa
M
M
Iwasa
T
T
Tsuda
M
M
D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008027:Light; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D049831:Octopodiformes; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012243:Rhodopsin / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D010738:Type C Phospholipases / Q000378:metabolism*
D017607:Aluminum Compounds; D008081:Liposomes; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride