Light-activated guanosinetriphosphatase in Musca eye membranes resembles the prolonged depolarizing afterpotential in photoreceptor cells.

Abstract

Measurement of light-dependent GTPase (EC 3.1.5.1) activity in a paradigm guided by electrophysiological experiments was used to examine the involvement of a guanine nucleotide binding protein in fly phototransduction. Cell-free membrane preparations of Musca eyes responded to blue light by a 10- to 20-fold increase in GTP-hydrolyzing activity. This light-dependent GTPase had a low Km for GTP (0.5 microM) and was effectively inhibited by guanosine (5'----O3)-1-thiotriphosphate and guanosine 5'-[beta-gamma-imino]triphosphate but not by adenosine 5'-[beta-gamma-imino]triphosphate and ATP. The action spectrum of GTPase activity measured with intense light resembled closely the photoequilibrium spectrum of metarhodopsin. After illumination with blue (less than 480 nm) light, which converted rhodopsin to metarhodopsin, the GTPase remained highly active for at least 60 min in the dark. Similarly, rhodopsin-to-metarhodopsin conversion in intact cells induced a prolonged excitation in the dark, known as the prolonged depolarizing afterpotential (PDA). The persistent GTPase activity (like the PDA) was suppressed to the low basal activity of the unilluminated membranes after conversion of metarhodopsin to rhodopsin with red light (greater than 570 nm), whereas during illumination with red light, some GTPase activity was maintained. The magnitude of the persistent GTPase activity in the dark, like the PDA, depended in a supralinear manner on the amount of pigment conversion. Thus, the dependence of GTPase activity of Musca membrane preparations on photopigment conversion resembles the induction and suppression of the PDA measured in intact photoreceptors of Musca. These findings indicate that a guanine nucleotide binding protein is part of the chain of events leading to both the generation of the receptor potential and the PDA.

Light‐modulated ADP‐ribosylation, protein phosphorylation and protein binding in isolated fly photoreceptor membranes

“…The association of light-dependent GTPase activities with crude Muscu eye membranes [27] and isolated blowfly rhabdoms [28] may indicate that a G-protein is also part of the chain of events leading to excitation of fly photoreceptors. Additional evidence for the presence of a G-protein in fly rhabdomeric membranes has been obtained by bacterial toxin-catalyzed ADP-ribosylation.…”

“…The light-modulated events which have so far been observed in fly photoreceptors are (a) the phosphorylation of proteins, in particular of rhodopsin [24-261, and (b) the activation of a GTPase in crude eye membranes [27] and in purified rhabdomeric membranes [28]. In the present study we have investigated, by toxin-catalyzed ADP-ribosylation, whether isolated photoreceptor membranes of the blowfly contain G-proteins.…”

Cloning of a Drosophila melanogaster guanine nucleotide regulatory protein beta-subunit gene and characterization of its expression during development.

“…Fig. 5 (18), and GTP analogs induce membrane depolarization in Musca photoreceptors (17). Lightdependent GTP binding and increased inositolphospholipid turnover are exhibited by fly (Musca and Drosophila) eye membranes (36).…”

“…The high degree of conservation of mammalian G proteins (16) and the biochemical evidence for the existence of G proteins in insects (17)(18)(19) and other invertebrates (20,21) prompted us to search for homologous genes in Drosophila melanogaster. We chose Drosophila, a metazoan organism amenable to genetic manipulation, to better understand the functions of these ubiquitous proteins.…”

Similarities between G‐proteins in visual cells ofSepiaand cattle

“…Activated membrane receptors stimulate the cr-subunit of G-proteins to bind GTP. In visual cells of invertebrates, photoactivated rhodopsin [1] catalyzes the exchange of GTP for bound GDP on a G-protein [2,3]. The active, GTP-bound form (G,~-GTP) activates a phospholipase C. This active PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2) to diacylglycerol (DAG) and inositol trisphosphate (IP3) [4][5][6][7].…”