Induced mono-(ADP)-ribosylation of rat liver cytosolic proteins by lipid peroxidant agents.

We have studied the effect of free radical generating agents on the mono-(ADP)-ribosylation of rat liver cytosolic proteins. Our results show that this post-translational modification, whose physiological significance is still unclear, is activated by lipid peroxidant agents via activation of cytoplasmatic mono-(ADP)-ribosyltransferases. The implication of free radicals in this process is demonstrated by the fact that mono-(ADP)-ribosylation can be prevented by melatonin, N-tert-butyl-alpha-phenylnitrone and dithiothreitol. On the basis of our results, we discuss the modification of proteins caused by free radicals as a possible mechanism by which they damage the cell.     

The effect of in vivo treatment with triiodothyronine on the in vitro synthesis of protein-poly(ADP)-ribose adducts by isolated cardiocyte nuclei and the separation of poly(ADP)-ribosylated proteins by phenol extraction and electrophoresis

In vivo treatment of rats with triiodothyronine (30 micrograms/100 g of body weight for 4 consecutive days) inhibited poly(ADP)-ribose polymerase activity of cardiocyte nuclei, but low enzymatic activity of nuclei of noncardiocyte origin remained unaffected. RNA synthesis in cardiocyte nuclei isolated from triiodothyronine-treated rats was augmented. A positive correlation was observed between the degree of inhibition of poly(ADP)-ribose polymerase and cardiac ventricular enlargement in triiodothyronine-treated animals. RNA synthesis in isolated cardiocyte nuclei was inhibited by in vitro poly(ADP)-ribosylation only when cardiocyte nuclei were obtained from triiodothyronine-treated animals. In vitro poly(ADP)-ribosylated proteins were isolated from cardiocyte nuclei by solvent partitioning between phenol and aqueous phases. About 90% of the protein-poly(ADP)-ribose adducts partitioned into the aqueous fraction, and the chain length of polymers in this phase was between medium (n = 4-9) and long (n greater than 32), whereas the phenol phase contained protein-oligomer and monomer adducts. Not only the chain length of oligomers but the nature of modified proteins appeared to participate in determining the partitioning of polymer-protein adducts, and different proteins were separated from the two phases by gel electrophoresis. More than 90% of protein-polymer adducts formed by cardiocyte nuclei were not extracted by 0.25 N HCl, indicating prevalence of nonhistone proteins as polymer acceptors. Gel electrophoresis and near quantitative recovery of adducts in a gel system that protected from degradation of adducts to free polymers confirmed the predominance of nonhistone proteins as main acceptors and demonstrated an artifact of autoradiography that seemed to indicate histone H1 as a significant acceptor. Treatment with triiodothyronine diminished poly(ADP)-ribosylation of certain groups of proteins more than others, implying some degree of selectivity of action of the hormone. Catabolism of the polymer in vitro was not affected by triiodothyronine treatment.     

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.     

Reaction of poly(ADP)-ribosylation of histone H1 in the presence of P1,P4-bis(5'-adenosyl)tetraphosphate and its phosphonate analogs

Effects of P1,P4-bis(5'-adenosyl)tetraphosphate and its phosphonate analogs on the ADP-ribosylation of H1 catalyzed by bovine testis ADP-ribose polymerase was investigated. Analogs App[CH(COCH3)]ppA and Ap[CH2]pppA as well as Ap4A inhibited poly(ADP)-ribosylation of histone H1 and at the same time accepted the ADP-ribosyl moiety of NAD. It was shown that inhibition of ADP-ribosylation of histone H1 is due to the competition of nucleotides with histone H1 for accepting ADP-ribosyl moiety of NAD on the one hand, and alteration of acceptor properties of the histone H1 on the other.     

Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa)

In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity.Key words: NADPH-diaphorase activity, protozoa, nitric oxide synthase.     

PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage. In contrast, although Adprt1b was dispensable for PARylation at DSBs, Adprt1a and, to a lesser extent, Adprt2 were required for this event. Disruption of adprt2 had a subtle impact on the ability of cells to perform NHEJ. However, disruption of adprt1a decreased the ability of cells to perform end joining with a concomitant increase in homologous recombination. PAR-dependent regulation of NHEJ was achieved through promoting recruitment and/or retention of Ku at DSBs. Furthermore, a PAR interaction motif in Ku70 was required for this regulation and efficient NHEJ. These data illustrate that PARylation at DSBs promotes NHEJ through recruitment or retention of repair factors at sites of DNA damage.     

Glutamate Regulates the Activity of Topoisomerase I in Mouse Cerebellum

Topoisomerase I (topo I) is a nuclear enzyme which participates in most DNA transactions. It was shown to be inhibited in depolarized neurons by poly adenosine diphosphate (ADP)-ribosylation of the enzyme protein. We demonstrated previously an age and sex dependent topo I activity and enzyme protein level in the various regions of mouse brain. A specific distribution pattern of topo I was observed and the inhibitory neurons exhibited the highest enzyme activity and protein level in both the nucleus and the cytoplasm. Here, we show that neurotransmitters (glutamate and gamma-aminobutyric acid (GABA)) regulate the activity of topo I in mouse cerebellum sections. Glutamate exhibited a significant time-dependent inhibition of topo I activity but no effect of the enzyme protein level. GABA in contrary only slightly and transiently inhibited topo I activity. The inhibitory effect of glutamate was mediated by Ca⁺² and by ADP-ribosylation of topo I protein and the glutamate ionotropic receptors were involved. Glutamate also diminished the inhibitory effect of topotecan on topo I. These results point to distinct and highly specific effects of the major neurotransmitters on topo I activity in the cerebellum suggesting that topo I possesses a specific role in the brain which differs from its known biological functions.     

Oxidative stress inhibits apoptosis in human lymphoma cells.

Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75-100 microM), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed.     

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.     

VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation

Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.     

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.     

Temporally different poly(adenosine diphosphate-ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of ³H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with ³H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased ³H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation. These results also indicate that a poly(ADP-ribosylation) signal is required for cell division; such signal is temporally different from that related to S phase initiation and occurs at the exit of S phase. © 1993 Wiley-Liss, Inc.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.     

Adiponectin acts in the brain to decrease body weight

Adiponectin (ADP) is an adipocyte hormone involved in glucose and lipid metabolism. We detected a rise in ADP in cerebrospinal fluid after intravenous (i.v.) injection, consistent with brain transport. In contrast to leptin, intracerebroventricular (i.c.v.) administration of ADP decreased body weight mainly by stimulating energy expenditure. Full-length ADP, mutant ADP with Cys39 replaced with serine, and globular ADP were effective, whereas the collagenous tail fragment was not. Lep(ob/ob) mice were especially sensitive to i.c.v. and systemic ADP, which resulted in increased thermogenesis, weight loss and reduction in serum glucose and lipid levels. ADP also potentiated the effect of leptin on thermogenesis and lipid levels. While both hormones increased expression of hypothalamic corticotropin-releasing hormone (CRH), ADP had no substantial effect on other neuropeptide targets of leptin. In addition, ADP induced distinct Fos immunoreactivity. Agouti (A(y)/a) mice did not respond to ADP or leptin, indicating the melanocortin pathway may be a common target. These results show that ADP has unique central effects on energy homeostasis.     

Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase

The adenine binding domain of the ADP site within human glutamate dehydrogenase (GDH) was identified by cassette mutagenesis at the Tyr187 position. The wild type GDH was activated 3-fold by ADP at a concentration of 1 mM at pH 8.0, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of the size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of the mutant enzymes revealed essentially unchanged apparent K(m) values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V(max) values. The binding of ADP to the wild type or mutant GDH was further examined by photoaffinity labeling with [alpha-(32)P]8-azidoadenosine 5'-diphosphate (8N(3)ADP). 8N(3)ADP, without photolysis, mimicked the stimulatory properties of ADP on GDH activity. Saturation of photoinsertion with 8N(3)ADP occurred with apparent K(d) values near 25 microM for the wild type GDH, and the photoinsertion of [alpha-(32)P]8N(3)ADP was decreased best by ADP in comparison to other nucleotides. Unlike the wild type GDH, essentially no photoinsertion was detected for the Tyr187 mutant GDH in the presence or absence of 1 mM ADP. For the wild type GDH, photolabel-containing peptide generated by tryptic digestion was identified in the region containing the sequence EMSWIADTYASTIG, and the photolabeling of this peptide was prevented >95% by the presence of 1 mM ADP during photolysis, whereas no such a peptide was detected for the Tyr187 mutant GDH in the presence or absence of ADP. These results with cassette mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide is within the ADP binding domain of the human GDH and that Tyr187 is responsible for the efficient base binding of ADP to human GDH.     

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

脂联素后处理对大鼠心肌缺血/再灌注损伤的影响及ADP/PI3K/Akt信号通路的作用

目的：探讨脂联素（ADP）后处理对大鼠心肌缺血/再灌注损伤（MIRI）的影响以及脂联素/磷脂酰肌醇-3激酶/蛋白激酶B （ADP/PI3K/Akt）通路的作用。方法：SD大鼠麻醉后气管插管连接呼吸机,开胸暴露心肌,在左心耳和肺动脉圆锥之间用带线圆针对冠脉左前降支（LAD）穿线,LAD结扎断流30 min后松线再灌注120 min,建立大鼠MIRI模型。大鼠随机分为以下5组（n=12）：①假手术组（Sham组）：LAD仅穿线不结扎;② MIRI组;③ADP后处理组（ADP组）：LAD断流10 min时静注ADP继续断流20 min,然后再灌注120 min;④ADP+LY294002组：LAD断流10 min时静注ADP和LY294002,其余同ADP组;⑤LY294002组：LAD断流10 min时静注LY294002,其余同ADP组。各组取血检测LDH和cTnI含量,取左心室心肌测定PI3k、Akt、p-Akt、ADPmRNA、ADPR1mRNA和PI3KmRNA表达。结果：与Sham组比较,MIRI组血浆LDH和cTnI均明显升高（P<0.05）;和MIRI组相比,ADP组心肌损伤指标明显下降（P<0.05）,而应用LY294002的两组心肌损伤比ADP组加重（P<0.05）。ADP组心肌PI3K、p-Akt、ADPmRNA、ADPR1mRNA和PI3kmRNA表达比MIRI组升高（P<0.05）,应用LY294002两组上述5个指标比MIRI组降低（P<0.05）。结论：ADP后处理对大鼠MIRI有保护作用,ADP/PI3K/Akt通路参与了以上作用。     

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.