Inhibition by pertussis toxin of guanyl nucleotides exchange on transducin in bovine rod cell membranes

The effect of pertussis toxin on GTP-binding protein of bovine rod cell outer segments (transducin) was studied. Pertussis toxin was shown to ADP ribosylate either alpha subunit of free transducin or transducin-GDP complex, whereas GTP and its analogue Gpp(NH)p strongly inhibit ADP ribosylation of transducin. Pertussis toxin inhibits rod outer segment membrane GTPase and GTPase of homogeneous transducin by 40% and 70-80%, respectively. Activation of rod cell cyclic nucleotide phosphodiesterase by transducin is reduced after its preincubation with pertussis toxin. In transducin modified by pertussis toxin, 83% of GDP becomes tightly bound and cannot be exchanged with Gpp(NH)p. The stabilization of complex transducin-GDP after ADP ribosylation can explain the inhibitory effect of pertussis toxin on GTP hydrolysis by transducin, and on phosphodiesterase activation by guanyl nucleotides.     

Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate, Gn

The subcellular distribution of G protein subunits in the neutrophil was examined. Cells were nitrogen cavitated and subcellular organelles fractionated on discontinuous sucrose gradients. The presence of GTP-binding regulatory protein (G protein) alpha and beta/gamma subunits in each organelle was determined using three methods of analysis: specific binding of guanine nucleotide, ADP ribosylation by pertussis toxin, and immunoblot analysis with subunit-specific G protein antibodies. Both plasma membrane and cytosolic G protein components were detected. In contrast, neither the specific nor the azurophilic granules contained detectable G protein. Based on the ability of exogenous G protein beta/gamma subunits to increase the ADP ribosylation of the cytosolic form of G protein and upon the hydrodynamic behavior of the cytosolic protein, it is likely that this represents an uncomplexed G protein alpha subunit. Proteolytic mapping with Staphylococcus aureus V8 protease suggests the soluble alpha subunit is from Gn, the major pertussis toxin substrate of human neutrophils. Using quantitative analysis, the levels of the 40-kD G protein alpha subunit and of the 35/36-kD beta subunit in the neutrophil membrane were determined.     

Crystal structure of the C3bot-RalA complex reveals a novel type of action of a bacterial exoenzyme

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.     

The carboxyl terminus of the S1 subunit of pertussis toxin confers high affinity binding to transducin.

The kinetic constants for the ADP-ribosylation of transducin were determined for the recombinant S1 subunit of pertussis toxin (rS1, composed of 235 amino acids) and two genetically derived deletion peptides, C180 and C195, which are composed of the 180 and 195 amino-terminal residues of the S1 subunit, respectively. Titration of NAD in the presence of a constant concentration of transducin (0.5 microM) showed that the KmappNAD in the ADP-ribosylation of transducin were similar, approximately 20 microM, for rS1, C195, and C180. In contrast, titration of transducin in the presence of a constant concentration of NAD (25 nM) showed that rS1 possessed a lower Kmapp(transducin) and greater kcat than either C195 or C180. Previous studies (Cortina, G., and Barbieri, J.T. (1991) J. Biol. Chem. 266, 3022-3030) showed that the 16 carboxyl terminal residues of the S1 subunit did not function in the ADP-ribosylation of transducin. It thus appears that residues between 195 and 219 of the S1 subunit are required for high affinity transducin binding and may be involved in the transfer of ADP-ribose to transducin. To localize the defect in the recognition of transducin by C180, rS1 and C180 were assayed for the ability to ADP-ribosylate either transducin or the purified alpha subunit of transducin (T alpha). Upon saturation of the target protein, rS1 ADP-ribosylated equivalent moles of transducin or T alpha, with the linear velocity of rS1-mediated ADP-ribosylation of transducin approximately 16-fold more rapid than the rate of ADP-ribosylation of T alpha. In contrast, the initial linear velocity of C180-mediated ADP-ribosylation of transducin was only 1.7-fold more rapid than the rate of ADP-ribosylation of T alpha. These data indicate that the amino-terminal 180 amino acids of S1 confer the specificity for ADP-ribosylation primarily through the interaction with T alpha, while residues between 195 and 219 of S1 confer high affinity binding to transducin primarily through the interaction, either directly or indirectly, with T beta gamma.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

Localization of a region of the S1 subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity

Purified recombinant S1 subunit of pertussis toxin (rS1) possessed similar NAD glycohydrolase and ADP-ribosyltransferase activities as S1 subunit purified from pertussis toxin. Purified rS1 and C180 peptide, a deletion peptide which contains amino acids 1-180 of rS1, had Km values for NAD of 24 and 13 microM and kcat values of 22 and 24 h-1, respectively, in the NAD glycohydrolase reaction. In contrast, under linear velocity conditions, the C180 peptide possessed less than 1% of the ADP-ribosyltransferase activity of rS1 using transducin as target. Radiolabeled tryptic peptides of transducin that had been ADP-ribosylated by either rS1 or C180 peptide were identical which suggested that both rS1 and C180 peptide ADP-ribosylated the same amino acid within transducin. To extend the functional primary amino acid map of the S1 subunit, two carboxyl-terminal deletions were constructed. One deletion, C195, removed the 40 carboxyl-terminal amino acids and the other, C219, removed the 16 carboxyl-terminal amino acids of the S1 subunit. Both C195 and C219 migrated in reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular masses of 22,000 and 27,500 Da, respectively. Relative to the C180 peptide C195 possessed 10-20-fold increase and C219 possessed 100-150-fold increase in ADP-ribosyltransferase activities. In addition, C219 appeared to have the same ADP-ribosyltransferase activity as rS1. These studies indicate that (i) rS1, purified from Escherichia coli, possesses biochemical properties similar to S1 subunit purified from pertussis toxin, (ii) amino acids 1-180 of the S1 subunit contain residues required for NAD binding, N-glycosidic cleavage, and transfer of ADP-ribose to transducin, and (iii) residues between 181 and 219 of the S1 subunit are required for efficient ADP-ribosyltransferase activity.     

Effects of GTP on choleragen-catalyzed ADP ribosylation of membrane and soluble proteins

Choleragen-dependent ADP ribosylation of soluble proteins from bovine thymus, using [32P]NAD as substrate, was increased 3- to 4-fold by GTP. The effect was specific for nucleoside triphosphate, with GTP approximately equal to ITP greater than CTP greater than ATP greater than UTP. Half-maximal enhancement was observed with 0.5 mM GTP. The magnitude of the GTP effect decreased with increasing NAD concentration; GTP had no effect on hydrolysis of NAD at low NAD concentrations. Digestion of ADP-ribosylated proteins with snake venom phosphodiesterase yielded primarily 5'-AMP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins from thymus after incubation with choleragen and [32P]NAD separated numerous ADP-ribosylated proteins; radioactivity in all bands was increased by nucleoside triphosphate. Choleragen catalyzed the ADP ribosylation of several purified proteins; depending on the protein, GTP either increased, decreased, or had no effect on the extent of ADP ribosylation. Choleragen-dependent ADP ribosylation of a wide variety of proteins is consistent with the possibility that intoxication results in covalent modification of more than one cellular protein and perhaps alters the activity of other enzymes in addition to adenylate cyclase.     

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions

In this study, we have examined the interactions of the beta gamma subunit complex of the retinal GTP-binding protein transducin (beta gamma T) with its alpha subunit (alpha T) using fluorescence spectroscopic approaches. The beta gamma T subunit complex was covalently labeled with 2-(4'-maleimidylanilino)napthalene-6-sulfonic acid (MIANS), an environmentally sensitive fluorescent cysteine reagent. The formation of the MIANS beta gamma T complexes (two to five MIANS adducts per beta gamma T) resulted in 2-3-fold enhancements in the MIANS fluorescence, and 20-25-nm blue shifts in the fluorescence emission maxima, relative to the emission for identical concentrations of MIANS-labeled MIANS complexes. The addition of alpha T.GDP to these MIANS beta gamma T complexes resulted in an additional enhancement in the MIANS fluorescence (typically ranging from 20 to 40%) and a 5-10-nm blue shift in the wavelength for maximum emission. These fluorescence changes were specifically elicited by the GDP-bound form of alpha T and were not observed upon the addition of purified alpha T.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) complexes to the MIANS beta gamma T species. Conditions which resulted in the activation of the alpha T.GDP subunit (i.e. the addition of AlF4- or the addition of rhodopsin-containing vesicles and GTP gamma S) resulted in a reversal of the alpha T.GDP-induced enhancement of the MIANS beta gamma T fluorescence. Thus the MIANS beta gamma T fluorescence provided a spectroscopic monitor for transducin-subunit association and transducin-activation. Based on the results from studies using this spectroscopic read-out, it appears that the association of the alpha T.GDP species with the beta gamma T subunit complex to form the holotransducin molecule is rapid and does not limit the rate of the rhodopsin-stimulated activation of holotransducin. However, either the dissociation of the activated alpha T subunit from the beta gamma T complex, or a conformational change in beta gamma T which occurs as a result of the subunit dissociation event, appears to be slow relative to the G protein-subunit association event.     

Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy.

In this work we have used fluorescence spectroscopic approaches to examine the binding of the beta gamma T subunit complex of transducin to the photoreceptor, rhodopsin. To do this, we have covalently labeled the beta gamma T subunit complex with the environmentally sensitive fluorescent cysteine reagent 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). By using the MIANS moiety as a fluorescent reporter group, we were able to monitor directly the binding of the MIANS-beta gamma T complex to light-activated rhodopsin, which was reconstituted into phosphatidylcholine vesicles, through an enhancement (30-50%) in the MIANS fluorescence. Phosphatidylcholine vesicles, alone, elicited only minor changes in the MIANS-beta gamma T fluorescence (i.e. less than 10% enhancement), whereas the addition of rhodopsin in the absence of lipid vesicles and in minimal detergent fully mimicked the effects of reconstituted rhodopsin and caused a significant enhancement of the MIANS fluorescence. The interactions between the MIANS-beta gamma T complex and rhodopsin also resulted in a quenching of the rhodopsin tryptophan fluorescence (approximately 30%), which most likely reflected resonance energy transfer between the tryptophan residues and the MIANS moieties. The binding of the MIANS-beta gamma T species to the alpha T subunit was accompanied by an enhancement of the MIANS fluorescence (30-50%) and a slight blue shift of the emission maximum, as described previously (Phillips, W. J., and Cerione, R. A. (1991) J. Biol. Chem. 266, 11017-11024). However, the alpha T-induced enhancement of the MIANS-beta gamma T fluorescence was not additive with the enhancement elicited by rhodopsin. Conditions which resulted in the activation of the alpha T subunit reversed the alpha T-induced enhancement of the MIANS emission, whereas the rhodopsin-induced enhancement persisted, thereby suggesting that the rhodopsin-beta gamma T complex can remain intact throughout the G protein activation event. Studies with synthetic peptides representing different regions of the cytoplasmic domain of rhodopsin demonstrated that a portion of the putative carboxyl-terminal tail (amino acid residues 310-324) was capable of eliciting changes in the MIANS-beta gamma T fluorescence as well as inhibiting the MIANS-beta gamma T-induced quenching of the rhodopsin tryptophan fluorescence. These results suggest that this region of the rhodopsin molecule may constitute a portion of the binding domain for the beta gamma T complex.     

Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS complexes, relative to their rates of dissociation from alpha(T)-GTPgammaS. The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha(T), indicating that only a subtle movement of the Switch 2 domain on alpha(T) accompanies the binding of this GTPgammaS analogue. Nevertheless, the BOD-GTPgammaS-bound alpha(T) subunit is able to bind with high affinity to the recombinant, purified gamma subunit of PDE (gamma(PDE)) labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS (K(d) approximately 13 nM)), as well as bind to and stimulate the activity of PDE, albeit less efficiently compared to alpha(T)-GTPgammaS. Taken together, these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha(T), and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha(T) in this conformational state with its biological target/effector.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Extracellular nicotinamide adenine dinucleotide induces t cell apoptosis in vivo and in vitro.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3(+), CD4(+), and CD8(+) cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69(+) T cells.     

Characterization of oxidized nicotinamide adenine dinucleotide (NAD(+)) analogues using a high-pressure-liquid-chromatography-based NAD(+)-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD(+))-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD(+)) and products (adenosine 5'-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD(+) analogue, 2'-F-ribo-NAD(+), was a competing substrate or a competitive inhibitor for this toxin. This NAD(+) analogue was hydrolyzed at a rate of 0.2% that of NAD(+) yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD(+) analogue, epsilon-NAD(+), is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD(+) as determined using HPLC. The rate of hydrolysis of epsilon-NAD(+) calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD(+)-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD(+) and its analogues.     

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.     

An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP-binding protein, transducin. Effects on transducin function

An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the alpha subunit of transducin (alpha T) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified alpha T and beta/gamma (beta gamma T) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or alpha T) to AS/7 approximately equal to 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the alpha T subunit and the beta gamma T complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free alpha T, holo-transducin, or alpha T in the presence of excess beta gamma T, prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of alpha T to bind or hydrolyze GTP and, in fact, the incubation of alpha T with AS/7 results in a stimulation of the intrinsic GTPase activity for alpha T alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-alpha T interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated alpha T subunit. Specifically, when the pure alpha T-guanosine 5-O-(3-thiotriphosphate) (alpha TGTP gamma S) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with alpha TGTP gamma S, alone, or with alpha TGTP gamma S preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of alpha T; the inactive alpha TGDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the alpha T subunit highlight the existence of distinct functional domains on alpha T.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups

The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.     

Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.     

Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin.

Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)