Guanosine 5'-O-(3-thiotriphosphate) as an analog of GTP in protein biosynthesis. The effects of temperature and polycations on the accuracy of initial recognition of aminoacyl-tRNA ternary complexes by ribosomes

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) is a good analog of GTP in the reactions leading to the formation of a peptide bond in protein biosynthesis. It forms binary and ternary complexes with elongation factor Tu (EF-Tu), and with EF-Tu and aminoacyl-tRNA (aa-tRNA). In addition, it stimulates aa-tRNA binding to ribosomes. Although GTP gamma S hydrolysis is more than three orders of magnitude slower than GTP hydrolysis, both reactions are dependent on the formation of a noncovalent complex (RS X TC) between mRNA-programmed ribosomes and ternary complex, and the complexes resulting from that hydrolysis are intermediates in peptide formation. The rate of dissociation of the ribosome X EF-Tu X GTP gamma S X aa-tRNA complex was determined from the rate of labeled peptide formation in the presence of an unlabeled ternary complex chase. This rate (2.2 X 10(-3) s-1) is similar to that determined previously (Thompson, R.C., and Karim, A.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4922-4926) from the progress of GTP gamma S hydrolysis. The effects of temperature and polycation concentration on this rate constant and that for GTP gamma S hydrolysis are reported. The rate constants measured are consistent with a kinetic rather than thermodynamic limit on the accuracy of the aa-tRNA selection in vivo.     

Solid-phase acyl donor as a substrate pool in kinetically controlled protease-catalysed peptide synthesis

Recently we have demonstrated the advantage of solid- phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Reactivity of the P-site-bound donor in ribosomal peptide-bond formation

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)-site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat). If we assume that the puromycin-reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P-site binding of the donor substrates AcPhe-tRNA or fMet-tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3 = 0.8 min-1 versus 8.3 min-1). On the other hand there are conditions, under which the proportion of P-site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min-1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell-free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.     

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.     

Kinetics of Ampicillin Synthesis Catalyzed by Penicillin Acylase from E. coli in Homogeneous and Heterogeneous Systems. Quantitative Characterization of Nucleophile Reactivity and Mathematical Modeling of the Process

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme–nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous (aqueous solution–precipitate) systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous aqueous solution–precipitate systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.     

Quantitative and qualitative differences in guanine nucleotide binding protein activation by formyl peptide and leukotriene B4 receptors.

Formyl peptides and leukotriene B4 (LTB4) stimulate disparate neutrophil functional responses and second messenger generation. The hypothesis that differences in receptor-guanine nucleotide-binding proteins (G protein) interaction account for the disparate responses was examined using HL-60 granulocyte plasma membranes. The quantity of receptor-coupled G proteins was determined by guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) equilibrium binding in the presence or absence of f-Met-Leu-Phe and/or LTB4. About one-third of the total GTP gamma S binding sites were coupled to f-Met-Leu-Phe receptors, to LTB4 receptors, and to receptors when both ligands were added simultaneously. The dissociation constant of GTP gamma S-binding sites in the presence of LTB4 was significantly greater than that in the presence of f-Met-Leu-Phe. f-Met-Leu-Phe shifted the GDP dose-inhibition curve for GTP gamma S binding further to the right than did LTB4. The apparent initial rate of GTP hydrolysis and GTP gamma S binding stimulated by f-Met-Leu-Phe was significantly greater than that stimulated by LTB4. There were significantly more formyl peptide receptors than LTB4 receptors, however, formyl peptide and LTB4 receptor density did not differ under GTP gamma S binding assay conditions. The rate of GTP hydrolysis stimulated by LTB4 was not increased in membranes containing twice the LTB4 receptor density. We conclude that formyl peptide receptors stimulate more rapid activation of a common pool of G proteins than LTB4 receptors because of a significantly reduced affinity of formyl peptide receptor-activated G proteins for GDP.     

Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine

An important nutritional dipeptide precursor, benzoyloxycarbonyl protected L-alanyl-L-glutamine (Z-Ala-Gln), was successfully prepared through a kinetically controlled enzymatic peptide synthesis method. A commercially available and low-cost protease (papain) was used as biocatalyst with Z-Ala-OMe and Gln as acyl donor and nucleophile, respectively. The dipeptide yield was 35.5% under the optimized reaction conditions: 35°C, pH 9.5, and the ratio of acyl donor/nucleophile is 1:10. Based on the reaction mechanism and experimental data, the kinetic model was established, which was in accordance with the Michaelis-Menten equation, and the apparent Michaelis constant K(m)(app) and the apparent maximum reaction rate r(max)(app) were calculated as 1.71 mol/L and 6.09 mmol/(L Min), respectively.     

Increased nucleophile reactivity of amino acid beta-naphthylamides in alpha-chymotrypsin-catalyzed peptide synthesis

Alpha-chymotrypsin-catalyzed acyl transfer from Boc-L-MetONp, Ac-L-TyrOEt, Bz-L-TyrOMe, Mal-L-PheOMe to the C-protected amino acids (L-AlaNH2, L-LeuNH2, L-ArgOMe and beta-naphthylamides of L-Arg, L-Leu, L-Ala and L-Glu) has been studied. Modification of the carboxylic groups with beta-naphthylamide was shown to increase the reactivity of nucleophiles in these reactions by a factor of more than 100 in comparison with amides and esters of the same amino acids. This effect can be accounted for by the effective formation of the nucleophile-acylenzyme complex due to hydrophobic interactions of the beta-naphthylamide moiety with the corresponding subsite of alpha-chymotrypsin. The reaction kinetics follows the scheme involving hydrolysis of the nucleophile-acylenzyme intermediate. The contribution of this pathway depends on the structures of both the acyl-group donor and the added nucleophile. The competitive inhibition by amino acid beta-naphthylamides is also observed. The results obtained show that modification of the COOH-group of added nucleophiles by beta-naphthylamide strongly affects the reactivity of these compounds in the alpha-chymotrypsin-catalyzed peptide synthesis.     

Mechanism of Carboxypeptidase Y Catalyzed Hydrolysis and Aminolysis Reactions

The reaction mechanism of carboxypeptidase Y catalyzed reactions is investigated. Presteady state and steady state kinetic measurements are performed on the hydrolysis and aminolysis of an ester and an amide substrate. It is found that deacylation is the rate determining step in hydrolysis of the ester, pivalic acid 4-nitrophenol and acylation in that of the amide, succinyl-L-alanyl-L-alalyl-L-propyl-L-phenylalanine 4-nitroanilide.

The kinetic effects observed in the presence of a nucleophile, L-valine amide, where aminolysis occurs in parallel to the hydrolysis reaction are analysed in details. The results are described satisfactorily by a reaction scheme which involves the binding of the added nucleophile, (i) to the free enzyme, resulting in a simple competitive effect, and (ii) to the acyl-enzyme with the formation of a complex between the enzyme and the aminolysis product, the dissociation of which is rate determining. That scheme can account for both increases and decreases of kinetic parameter values as a function of the nucleophile concentration. There is no indication of binding of the nucleophile to the enzyme-substrate complex before acylation takes place.     

Effect of mass-transfer limitations on the selectivity of immobilized alpha-chymotrypsin biocatalysts prepared for use in organic medium

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.     

Inhibition of Cyclooxygenase Activity of Prostaglandin-H-Synthase by Excess Substrate (Molecular Oxygen)

For the cyclooxygenase reaction of prostaglandin-H-synthase isolated from ram vesicular glands, dependences of the initial reaction rate, the maximal yield of the product, and the rate constant of enzyme inactivation in the course of reac- tion on oxygen concentration were studied in the absence and in the presence of electron donor in the reaction medium. It is shown that in the absence of electron donor the cyclooxygenase reaction is strictly governed by Michaelis-Menten kinet- ics over a wide range of oxygen concentrations (5–800 μM). In the presence of electron donor in the reaction medium it was found that cyclooxygenase reaction is inhibited by an excess of dissolved oxygen: the maximal values of the initial reaction rate and yield of the product are attained at oxygen concentration 50 μM, and its increase to 500 μM causes twofold decrease in the initial rate and maximal yield. The rate constant of enzyme inactivation in the course of reaction increases on increase in oxygen concentration both in the presence and in the absence of electron donor.     

Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.     

Catecholamine-stimulated guanosine 5'-O-(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase: kinetic analysis in reconstituted phospholipid vesicles

The stimulatory GTP-binding protein of adenylate cyclase, Gs, and beta-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasiirreversible binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gs, equivalent to Gs activation by nucleotide, was studied with respect to the stimulation of this process by beta-adrenergic agonists and Mg2+. The rate of GTP gamma S binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+ concentrations. In the absence of agonist, the apparent first-order rate constant, kapp, was 0.17-0.34 min-1 and did not vary significantly with the concentration of nucleotide. At 50 mM MgCl2, kapp increased somewhat, to 0.26-0.41 min-1, and remained invariant with the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At 10(-9) M GTP gamma S, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kapp. However, kapp measured in the presence of isoproterenol increased as an apparently saturable function of the GTP gamma S concentration, such that isoproterenol caused a 17-fold increase in kapp at 1 microM GTP gamma S. The effect of isoproterenol on kapp also appeared to saturate at high isoproterenol concentration, yielding a kapp approximately 6 min-1 at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity Gs-GTP gamma S complex to the stable activated state.     

Acyl group transfer by proteases forming acyl-enzyme intermediate: kinetic model analysis

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as p(max)). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of p(max).The analysis has demonstrated that at a given ratio of initial S and N concentrations, p(max) is affected only by (i) the ratio of the second-order rate constants for enzymatic hydrolysis of S and P(alpha) and (ii) the ratio of rate constants for an attack of the acyl-enzyme intermediate by the nucleophile and water (beta). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (p(max)) can be calculated a priori from values of alpha and beta that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of p(max) for a broad spectrum of S and N initial concentrations.     

Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cytochrome c,d1)

The dissimilatory nitrite reductase (cytochrome c,d1) from Pseudomonas aeruginosa was observed at pH 7.5 to catalyze nitrosyl transfer (nitrosation) between [15N]nitrite and several N-nucleophiles or H2 18O, with rate enhancement of the order of 10(8) relative to analogous chemical reactions. The reducing system (ascorbate, N,N,N',N'-tetramethylphenylenediamine) could reduce nitrite (but not NO) enzymatically and had essentially no direct chemical reactivity toward nitrite or NO. The N-nitrosations showed saturation kinetics with respect to the nucleophile and, while exhibiting Vmax values which varied by about 40-fold, nevertheless showed little or no dependence of Vmax on nucleophile pKa. The N-nitrosations and NO-2/H2O-18O exchange required the reducing system, whereas NO/H2O-18O exchange was inhibited by the reducing system. NO was not detected to serve as a nitrosyl donor to N-nucleophiles. These and other kinetic observations suggest that the enzymatic nitrosyl donor is an enzyme-bound species derived from reduced enzyme and one molecule of nitrite, possibly a heme-nitrosyl compound (E-FeII X NO+) for which there is precedence. Nitrosyl transfer to N-nucleophiles may occur within a ternary complex of enzyme, nitrite, and nucleophile. Catalysis of nitrosyl transfer by nitrite reductase represents a new class of enzymatic reactions and may present another example of electrophilic catalysis by a metal center. The nitrosyl donor trapped by these reactions is believed to represent an intermediate in the reduction of nitrite by cytochrome c,d1.     

The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)