Sub‐second turnover of transducin GTPase in bovine rod outer segments A light scattering study

Abstract

A fast, regenerative light scattering signal from bovine ROS, the PA-signal, reflects the light-induced, transient activation of transducin.Its rate of recovery depends on the number of photolysed rhodopsin molecules, indicating that rhodopsin deactivation and not GTPase activity is rate limiting in our in vitro system. When rhodopsin deactivation is accelerated (in the presence of NH,OH), PA-signal recovery is also accelerated. A GTPase turnover number of more than 2 s-l (at 37°C) can bc derived from these experiments. This is more than one order of magnitude faster than the GTPase rates so far described in the literature and is rapid enough for a physiological shut-off mechanism. The fast GTPase is attributed to a highly intact disk stack, which never releases transducin into the free aqueous space.

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction

“…We have previously shown that the time course of the recovery of PA reflects rhodopsin disactivation due to phosphorylation, and this effect, rather than GTPase activity, is rate limiting under our usual measuring conditions [12]. The calcium effects reported in this communication would therefore suggest a calcium dependence of kinase activity.…”

“…under conditions where kinase activity is suppressed, an artificial quencher of rhodopsin activity was required for a speedy signal recovery. Hydroxylamine is such a quencher, and in its presence rhodopsin disactivation occurs so fast that the GTPase activity of the activated G-protein becomes (at least partially) rate limiting [12]. Under such conditions the rate of recovery became independent of the calcium concentration ( fig.3b).…”

G Proteins and Phototransduction

“…The early studies of transducin GTPase reaction revealed a paradox: The rate of GTP hydrolysis measured with purified transducin was ∼100 times slower than the apparent time constant with which rods recover from flashes (compare, e.g., 92 and 93 with 4 and 5). The resolution of this paradox began when experimental measurements of transducin's GTPase activity were made under the more physiological conditions of either concentrated suspensions of rod outer segments (94,95) or of rod outer segments with highly preserved disc stack structure (96). These studies found a GTPase rate much higher than had been observed in cell-free systems reconstituted with purified proteins.…”

Model Systems For The Study Of Seven-Transmembrane-Segment Receptors

“…By monitoring activation and inactivation of transducin in rod outer segment membranes, a soluble protein extract from rod cells was observed to greatly accelerate the arrestin dependent inactivation of rhodopsin signaling (135,136). Moreover, in a reconstituted system consisting of purified transducin and rhodopsin, no diminution of rhodopsin-mediated activation of transducin was detected in the presence of arrestin, regardless of the extent of phosphorylation of rhodopsin (137), suggesting that the present model of competition between arrestin and transducin for binding to rhodopsin might be inadequate.…”