Abstract
The generation of the physiological response of a retinal rod cell to an incident photon involves activation of a cGMP phosphodiesterase (PDE) by a GTP-binding protein, transducin (T). This activation has been shown to occur by formation of a membrane-bound T alpha GTP-PDE complex (Clerc, A., and Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627; Catty, P., Pfister, C., Bruckert, F., and Deterre, P. (1992) J. Biol. Chem 267, 19489-19493). The recovery of the response involves turning-off of T by its intrinsic GTPase activity. We show here that the formation of the membrane-bound T alpha GTP-PDE complex correlates with an enhanced rate of GTP hydrolysis. In vivo, this would provide an appropriate mechanism for fast turn-off of cGMP hydrolysis.
Highlights
Rod outer segment (ROS)suspensions have given widelyvarying rates, ranging from 0.5-1 to 30-60 s/Pi.T. (Kuhn, 1981; Fung, 1983; Yamanaka et al, 1985; Sitaramayya et al, 1988; Phillips and Cerione, 1988; Archavsky et al, 1989,1991; Vuong and Chabre, 1990,1991; Chabre et al, 1990; Guyet al., 1990; Tingand Ho, 1991; Archavsky and Bownds, 1992), mostly too slow to account for the rapid turn-off of the electrophysiological signal of the rod (200-400 ms, see Baylor et al (1984))
Bovine rod outer segment (ROS) suspensions were supplemented with PDE, and we observed an acceleration of GTP hydrolysis selectively due to T.Parallel experiments on aliquots allowed us to directly correlate this acceleration with an increase in the amount of T u bound to the membranes via interaction with PDE
ROS pellets were stored under argon, in the dark, at -80 “C. Washed membraneswere obtained from ROS pellets by two successive washes in buffers I and H at about 0.5 mgof rhodopsin/ml
Summary
In the rods of vertebrate retina, transducin, T,’ conveys buffer (H) consisted of 5 mM HEPES, pH 7.5.All the buffers were extemporaneously degassed and complemented with0.15 mM phenylinformation from the light sensor, rhodopsin, to the cellular methylsulfonyl fluoride and 2 mM dithiothreitol. Spontaneous hydrolysis of GTP in Ta deactivates T, with subsequent deactivation of PDE It has been a matter of debate why the rates of GTP hydrolysis measured in vitro in Transducin and PDEwere extracted as described by Kuhn (1981) and Deterre et al (1986) by successive centrifugations of a bleached ROS suspension (2.5 mg of rhodopsin/ml) in buffer H (“crude PDE,” 80% pure PDE), buffer H supplemented with 50 p~ GTP-yS and 50 ~ L MMgClz (“crude T,” 90% pure T). The abbreviations used are: T, transducin; ROS, rod outer seg- 2.5 mg of rhodopsin/ml supplemented with 1mM GTP, by successive ment(s); Ta, Toy,a and P-y subunits of T T ~ G D ~ , TTc~uGloaTdPed, extractions inbuffers I (supernatant SI)and H (supernatant SZ),each with GDP or GTP; PDE, cGMP phosphodiesterase; PDEa, PDEP, extraction carried out twice.
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