Purification of T beta gamma subunit of transducin.
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Animals↗
Biomedical subjects
M Chabre
Publications and source records attributed to M Chabre.
Myristoylation is not required for GTP-dependent binding of ADP-ribosylation factor ARF1 to phospholipids.
Membrane binding of ADP-ribosylation factors (ARFs) is GTP-dependent and seems to require amino-terminal myristoylation. Recently it has been proposed that myristoylation is needed not for the activation of ARF by GTP but for its subsequent association to membranes. Here we show that unmyristoylated bovine ARF1, expressed in bacteria, can be efficiently loaded with GTP gamma S (guanosine 5'-O-(thio)triphosphate) at 1 microM free Mg2+, in the presence of phospholipids. Unmyristoylated ARFGTP gamma S cosediments with phospholipid vesicles and totally binds to phospholipid-cholate micelles, as seen by gel filtration chromatography. We therefore propose that, in vivo, myristoylation is required for the interaction of ARFGDP with its membrane-bound exchange factor rather than for the association of ARFGTP with lipid membranes. Phospholipid-bound ARFGTP gamma S can also stably interact with and activate the catalytic subunit of cholera toxin, suggesting that ARFGTP provides a membrane anchor for cholera toxin and thereby facilitates its access to membrane-bound substrates.
ADP-Ribosylation Factor 1↗
Interaction between the retinal cyclic GMP phosphodiesterase inhibitor and transducin. Kinetics and affinity studies.
In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
3',5'-Cyclic-GMP Phosphodiesterases↗
GTP hydrolysis by purified alpha-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor.
The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
3',5'-Cyclic-GMP Phosphodiesterases↗
ADP-ribosylation of Gs by cholera toxin is potentiated by agonist activation of beta-adrenergic receptors in the absence of GTP.
Purified Gs is a substrate for ADP-ribosylation catalyzed by cholera toxin (CTx). In S49 cyc- membranes complemented with in vitro translated Gs alpha, the beta-adrenergic agonist isoproterenol enhanced the ADP-ribosylation rate. This effect was maximal if all guanyl nucleotides were suppressed but was blocked by the beta-adrenergic antagonist alprenolol. Enhancement was partially diminished if addition of GDP followed that of isoproterenol. When added in the absence of agonist, the GTP analogues guanosine 5'-O-(gamma-thiotriphosphate) and guanosine 5'-(beta, gamma-imido)triphosphate potentiated CTx-catalyzed ADP-ribosylation of Gs alpha consistent with their activating ADP-ribosylation factors. However, this effect was lessened when the same nucleotides were tested in the presence of agonist. Taken altogether, these results indicate that like Gt and Gi, Gs is an optimal substrate for CTx when coupled to an agonist-activated receptor and depleted of nucleotide. Therefore, coupling to the receptor and subsequent departure of the GDP turn out to be the common features underlying the sensitivity of all GTP-binding proteins to CTx-catalyzed ADP-ribosylation.
Adenosine Diphosphate Ribose↗
The mechanism of aluminum-independent G-protein activation by fluoride and magnesium. 31P NMR spectroscopy and fluorescence kinetic studies.
With magnesium present, fluoride and aluminum ions activate heterotrimeric G-proteins by forming AlFx complexes that mimic the gamma phosphate of a GTP. We report compelling evidence for a newly proposed process of G-protein activation by fluoride and magnesium, without Al3+. With millimolar Mg2+ and F-, Gs and Gt activate adenylylcyclase and cGMP-phosphodiesterase, respectively. In 31P NMR, addition of magnesium to Gi1 alpha GDP or Gt alpha GDP solutions containing fluoride, but no Al3+, modifies the chemical shift of the GDP beta phosphorus, suggesting that magnesium interacts with the beta phosphate. Titration of this effect indicates that two Mg2+ are bound per G alpha. Biphasic activation kinetics, monitored by G alpha tryptophan fluorescence, suggests the rapid binding of one Mg2+ to G alpha GDP and the slow association of another Mg2+, in correlation with fluoride binding and G alpha activation. The deactivation rate upon fluoride dilution shows a second order dependence with respect to the residual F- concentration, suggesting the sequential release of at least three F-/G alpha. Thus, in millimolar Mg2+ and F-, and without Al3+, two Mg2+ and three F- bind sequentially to G alpha GDP and induce the switch to an active G alpha (GDP-MgF3)Mg state, which is structurally analogous to G alpha (GDP-AlFx)Mg and to G alpha (GTP)Mg.
3',5'-Cyclic-GMP Phosphodiesterases↗
The G protein cascade of visual transduction: kinetics and regulation.
In retinal rods photoexcited rhodopsin (R*) catalyses the activation of transducin (T) by GTP, which in turn activates the cGMP phosphodiesterase (PDE). The ensuing decrease in cGMP concentration reduces the cell membrane's channel conductance. To account for the kinetics of the response to light, all underlying biochemical reactions must reach maximum speed and be turned off within a second. Kinetic analysis of transducin activation suggests that because of the fast lateral diffusion of T, the rate-limiting step is not the collision between R* and T but the entry of GTP after the release fo GDP from the R*-bound T alpha. T alpha-GTP dissociates from both R* and T beta gamma and diffuses through the cytoplasm to activate PDE. In suspensions of bovine rod outer segments, time-resolved microcalorimetry yields rates of approximately 1-2 s-1 for the GTPase of T alpha and the correlated deactivation of PDE. But for isolated T alpha-GTP the single turnover GTPase rate measured by a stopped-flow technique is only 0.05 s-1. To activate PDE, T alpha-GTP binds tightly to the PDE gamma subunit. In vitro the soluble T alpha-GTP.PDE gamma complex dissociates from activated PDE alpha beta. Thus PDE gamma might be the GTPase activator of T alpha, but no GTPase acceleration was observed in isolated T alpha-GTP.PDE gamma. The GTPase activation must depend on the interaction of T alpha-GTP.PDE gamma with membrane-bound PDE alpha beta.
3',5'-Cyclic-GMP Phosphodiesterases↗
Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.
We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.
3',5'-Cyclic-GMP Phosphodiesterases↗
Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.
Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)
3',5'-Cyclic-GMP Phosphodiesterases↗
Kinetics and energetics of the rhodopsin-transducin-cGMP phosphodiesterase cascade of visual transduction.
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3',5'-Cyclic-GMP Phosphodiesterases↗
Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.
Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.
Aluminum↗
Kinetic analysis of the activation of transducin by photoexcited rhodopsin. Influence of the lateral diffusion of transducin and competition of guanosine diphosphate and guanosine triphosphate for the nucleotide site.
The activation of transducin (T) by photoexcited rhodopsin (R*) is kinetically dissected within the framework of Michaelis-Menten enzymology, taking transducin as substrate of the enzyme R*. The light scattering "release" signal (Vuong, T.M., M. Chabre, and L. Stryer, 1984, Nature (Lond.). 311:659-661) was used to monitor the kinetics of transducin activation at 20 degrees C. In addition, the influence of nonuniform distributions of R* on these activation kinetics is also explored. Sinusoidal patterns of R* were created with interference fringes from two crossed laser beams. Two characteristic times were extracted from the Michaelis-Menten analysis: t(form), the diffusion-related time needed to form the enzyme-substrate R*-transducin is 0.25 +/- 0.1 ms, and T(cat), the time taken by R* to perform the chemistry of catalysis on transducin is 1.2 +/- 0.2 ms, in the absence of added guanosine diphosphate (GDP) and at saturating levels of guanosine triphosphate (GTP). With t(form) being but 20% of the total activation time t(form) + t(cat), transducin activation by R* is not limited by lateral diffusion. This is further borne out by the observation that uniform and sinusoidal patterns of R* elicited release signals of indistinguishable kinetics. When (GDP) = (GTP) = 500 microM, t(cat) is lengthened twofold. As the in vivo GDP and GTP levels are comparable, the exchange of nucleotides may well be the rate-limiting process.
Animals↗
[Transduction of the visual signal in retinal cells].
Transduction of a visual signal is a complex process. It involves photochemical, enzymatic and ionic reactions. An electrophysiological response is generated on absorption of a photon by a photoreceptor cell's pigment molecule, then propagates to the synapses. The first photochemical reaction, isomerization of retinal, in vertebrates occurs in the photoreceptor cells--rods and cones--of the retina, so changes conformation and activity of a pigment-bound protein, rhodopsin, in membranes of intracellular discs. Rhodopsin becomes enzymatically active and catalyses the activation by GTP of a great number of transducins, which in turn activate cGMP phosphodiesterase. This enzymatic chain propagates and greatly enhances hydrolysis of cytoplasmic cGMP. One photon incites hydrolysis of 10(5) cGMP molecules in 100 ms. Local cGMP decrease frees it from specific binding sites in cytoplasm occupied by proteinaceous canals in cell membrane around the activated disc. In darkness high cGMP concentration, hence binding, kept canals open, maintaining high cellular cation permeability, especially to Na+, and a strong cellular depolarization. Ca2+ influx, also allowed, balanced Na+ movement. Canal closure induces local hyperpolarization, the first electrophysiological response, which propagates through the cell to synaptic contacts. It also lowers intracellular Ca2+ concentration which initiates cGMP synthesis--from GTP by a guanylate cyclase controlled negatively by recoverin, a calcium-dependent protein--to restore cGMP towards at rest level. Although all macromolecules involved in this now fairly complete scheme have been isolated and characterized, cloned and sequenced, no three-dimensional structure has yet been established. The proteins are membrane-bound rather than in independent crystal form, which renders such structural studies difficult.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Deactivation kinetics of the transduction cascade of vision.
The response of the retinal rod cell to a dim flash lasts less than a second. This phototransduction is mediated by a guanine nucleotide-binding (G) protein cascade in which rhodopsin is the receptor, transducin is the G-protein, and the cGMP-specific phosphodiesterase (PDE) is the effector. Photoexcited rhodopsin activates transducin which in turn activates PDE. For this underlying biochemistry to be kinetically compatible with the photoresponse, both transducin and PDE must be deactivated in subsecond times. We report here direct measurements of their deactivation kinetics. The rate of heat release when transducin and PDE hydrolyze, respectively, GTP and cGMP was measured using time-resolved microcalorimetry. With only GTP present, the heat pulse comes from the activation of transducin and its subsequent deactivation by endogenous GTP hydrolysis. The nonhydrolyzable analog guanine 5'-[gamma-thio]triphosphate was used to distinguish between these two processes: about 40% of the total heat is due to activation. From the time course of the deactivation heat, the active lifetime of transducin is less than 1 s at 22 degrees C. With both GTP and cGMP present, the highly amplified hydrolytic activity of the PDE is responsible for most of the heat produced; its rate of release is directly proportional to the amount of activated PDE. Measurements of this rate at low photoexcitation levels (e.g., 30 molecules of photoexcited rhodopsin per rod) provide much kinetic information about the cascade. Notably, deactivation of the PDE takes 0.6 s (at 23 degrees C) and absolutely requires GTP hydrolysis. This concurs with the subsecond lifetime of active transducin and means that, once GTP hydrolysis has occurred, the hitherto active PDE is quickly inhibited.
3',5'-Cyclic-GMP Phosphodiesterases↗
Choleratoxin ADP-ribosylates transducin only when it is bound to photoexcited rhodopsin and depleted of its nucleotide.
The sensitivity of transducin (T) to choleratoxin (CT) in retinal cells depends on illumination and on the presence of GTP or analogs. Low concentrations of GPP-NH-P or GPP-CH2-P increase ADP-ribosylation while GTP gamma S inhibits it. We show that GTP analogs permanently activate an ADP-ribosylating factor (ARF) which mediates CT action on retinal cell membranes: when transducin-depleted membranes were pre-activated by GTP analogs, re-added transducin became sensitive to CT in the absence of nucleotide, and presence of photoexcited rhodopsin (R*). Any subsequent G-nucleotide addition (even GDP) decreased ADP-ribosylation. Thus nucleotide-free transducin molecule in R*-Tempty complex is the CT substrate.
Adenosine Diphosphate Ribose↗
GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants.
We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.
Amino Acid Sequence↗
A novel magnesium-dependent mechanism for the activation of transducin by fluoride.
Activation of transducin-GDP by NaF is mainly mediated by aluminofluorde or beryllofluoride complexes acting as GTP gamma-phosphate analogs. In millimolar magnesium, NaF at concentrations above 3 mM is active even in the absence of aluminium or beryllium. This activation has a Hill coefficient of 3 with respect to F-, and its rate is linear with respect to Mg2+ concentrations above 2 mM. Upon fluoride dilution, inactivation rate is hundreds of times faster than for aluminofluoride-activated T alpha GDP. We propose that at high NaF concentrations, 3 hydrogen-bonded fluorides in the gamma-phosphate site of T alpha GDP entrap a magnesium counterion and this induces the transconformation to the T alpha GTP form.
Aluminum↗
Subsecond deactivation of transducin by endogenous GTP hydrolysis.
The response of a retinal rod cell to a weak flash of light is mediated by a receptor/GTP-binding protein (rhodopsin/transducin) signal transduction system and terminates within a second. The T alpha subunit of transducin (composed of subunits T alpha, T beta and T gamma) is triggered by photoexcited rhodopsin (R*) to release GDP and bind GTP. The binding of GTP causes release of the T alpha unit from T beta gamma and allows it to modulate the activity of an enzyme that generates a second messenger. Termination of the response requires the hydrolysis of the GTP by intrinsic GTPase. As with other G proteins, the GTPase activity of transducin seems to be slow. Reported in vitro turnover rates of a few molecules of GTP hydrolysed per molecule of transducin per minute imply a T alpha-GTP deactivation time of many seconds. But this time might be only a small fraction of that of the GTPase cycle. We have now used time-resolved microcalorimetry in bovine rod outer segments (ROS) to monitor the heat release due to the hydrolysis of GTP by a transducin population that had been quickly activated by flash illumination of rhodopsin. The enthalpy of GTP hydrolysis is released within 1 s at 23 degrees C. This deactivation time seems to be independent of any diffusible factor in the preparation and concurs with the termination kinetics of the rod's response. Thereafter, transducin seems unable to reload GTP for many seconds. This refractory 'resetting' time may account for the low steady-state GTPase rates in vitro.
Animals↗