An Adenosinetriphosphate-Activated Hemolytic System : III. Effect of rabbit hemolytic factor on the membrane permeability of human red cells

Lysis of human red cells in vitro by an enzyme obtained from rabbit red cell hemolysates and the inhibition of this lytic activity by human stroma have been shown to require Mg⁺⁺ and ATP, and ATP utilization has been demonstrated in both reactions. We find that sodium or potassium ions are also required for the lytic phenomenon and that they enhance the inhibition. The rate of hemolysis is not affected by the internal concentrations of these ions but depends only on the external concentration. The rate of influx and efflux of Na²²Cl and K⁴²Cl in surviving red cells is greatly enhanced both during and after treatment with rabbit hemolytic factor whereas the entry of C²⁴-sucrose, a small foreign molecule, is mediated only in the presence of hemolytic factor. Glycolysis neither protects against lysis nor enhances the activity of this system, and cardiac glycosides which are known inhibitors of active transport of ions also have no effect. It appears that lysis in this system is not brought about by increased active transport of ions into the cell but that the rabbit factor degrades or combines with some membrane component, altering permeability and resulting in increased diffusion, first of sodium and potassium ions and other small molecules, and finally of large molecules (hemoglobin) out of the cell.     

An adenosinetriphosphate-activated hemolytic system. II. Utilization of adenosinetriphosphate in the reaction

Failure to demonstrate ATP¹ utilization in an ATP-activated hemolytic system had been previously reported. In the present study ATP utilization is shown to be associated with the hemolytic reaction and also with the ATP-dependent inactivation of the hemolytic factor of the system by crude, washed, human red cell stroma. Using crude stroma, relatively large ATP utilization occurs and continues, but at a decreasing rate, after inactivation of the hemolytic factor is complete. With purified stroma there is very slight uptake of ATP by the stroma in the presence of hemolytic factor and Mg⁺⁺. This uptake can only be demonstrated by radioactive ATP. Both C¹⁴- and P³²—labelled nucleotide were used for this purpose. In the presence of an excess of stroma the uptake seems to be dependent on the amount of hemolytic factor used. Evidence is given contraindicating the possibility that this uptake is non-specific.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

A microsomal ATP-activated pyridine nucleotide transhydrogenase

An ATP-activated transhydrogenase which catalyzes the reduction of TPN⁺ by DPNH has been demonstrated in the microsomal fraction from the endosperm of immature Echinocystis macrocarpa seeds. The activity is specifically dependent on the presence of ATP (K_m of approximately 0.1 mm) of several nucleotides tested. The reaction is stimulated by MgCl₂ addition up to concentrations of 6 mm. When 10^?2m EDTA is added to the assay mixture in the absence of added MgCl₂, a transhydrogenation reaction is observed which no longer shows any dependence on added ATP. A TPN⁺-dependent ATPase activity can be demonstrated in these preparations, but no fixed stoichiometry between ATP cleavage and TPNH formation could be established. A lag in attaining the maximal rate of transhydrogenation is seen unless the enzyme is preincubated for 10 min with ATP before initiating the reaction. It can further be shown that preincubation of the enzyme with ATP followed by removal of the ATP on a Dowex 1 column produces an enzyme capable of catalyzing the transhydrogenation without the further addition of ATP. 2,4-Dinitrophenol and thyroxin are effective inhibitors of the transhydrogenase and 2,4-dinitrophenol was shown to inhibit the activating effect of ATP during the preincubation period. It is concluded that the role of ATP is in the modification of the enzyme rather than direct participation in the transhydrogenation. The transhydrogenase is inhibited by ADP and AMP. This results in a response of the enzyme to adenylate energy charge in a manner characteristic for regulatory enzymes which participate in ATP-utilizing metabolic sequences.     

Author index

The effect of the accumulation of β-galactosides on the uptake of P_i into cells and cell nucleotides was examined in ML strains of Escherichia coli. Nonmetabolizable sulfur analogs of lactose, which are accumulated only in the presence of the product of y gene of the Lac operon, inhibited the uptake of P₁ into whole cells and into cell nucleotides. This inhibition was most pronounced in starved cells, those with a low rate of ATP production. When the cell membrane was disrupted by sonication or detergents, the inhibition was lost. No significant inhibition was seen in y^? strains or in inducible y⁺ strains which were not induced. Hence. inhibition of the uptake of phosphate into nucleotides is dependent on the presence of the product of the y gene and a β-galactoside.A technique using ³²P_i and ³³P₁ was developed for simultaneously measuring the turnover and level of nucleotides. β-Galactosides inhibited ATP synthesis in aerobic cells, but stimulated ATP synthesis in anaerobic cells, indicating that an intermediate of oxidative phosphorylation was the source of energy for β-galactoside accumulation.     

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Köhler P. B.,Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of ¹⁴CO₂ from 1,4-¹⁴C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg²⁺ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap₅A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO₂ and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO₂ or propionate. This system is inhibited in flukes incubated in vitro with mebendazole.It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Mitochondrial Swelling Induced by Glutathione

Reduced glutathione, in concentrations approximating those occurring in intact rat liver, causes swelling of rat liver mitochondria in vitro which is different in kinetics and extent from that yielded by L-thyroxine. The effect is also given by cysteine, which is more active, and reduced coenzyme A, but not by L-ascorbate, cystine, or oxidized glutathione. The optimum pH is 6.5, whereas thyroxine-induced swelling is optimal at pH 7.5. The GSH-induced swelling is not inhibited by DNP or dicumarol, nor by high concentrations of sucrose, serum albumin, or polyvinylpyrrolidone, in contrast to thyroxine-induced swelling. ATP inhibits the GSH swelling, but ADP and AMP are ineffective. Mn^-+ is a very potent inhibitor, but Mg⁺⁺ is ineffective. Ethylenediaminetetraacetate is also an effective inhibitor of GSH-induced swelling. The respiratory inhibitors amytal and antimycin A do not inhibit the swelling action of GSH, but cyanide does; these findings are consistent with the view that the oxidation-reduction state of the respiratory chain between cytochrome c and oxygen is a determinant of GSH-induced swelling. Reversal of GSH-induced swelling by osmotic means or by ATP in KCl media could not be observed. Large losses of nucleotides and protein occur during the swelling by GSH, suggesting that the action is irreversible. The characteristically drastic swelling action of GSH could be prevented if L-thyroxine was also present in the medium.     

Photoreduction of Pyridine Nucleotide by Subcellular Preparations from Rhodopseudomonas spheroides

We have investigated the photoreduction of pyridine nucleotides by crude extracts and chromatophores of Rhodopseudomonas spheroides.

Our findings are as follows:

NADP is preferentially photoreduced by crude extracts (37,000 × g supernatant fraction) and there is no requirement for the addition of exogenous substrates. Crude extracts also catalyze a nonphotosensitive reduction of NAD.

NADP photoreduction is completely inhibited if an NADH trapping system is present and indicates that NADH is required for NADP photoreduction.

Washed chromatophores (150,000 × g pellet) do not catalyze NADP photoreduction unless the supernatant fraction is added. The restoring effect of supernatant fraction is lost upon boiling and dialysis. However, supernatant materials can be replaced by an NADH generating system. There is no requirement for anaerobic conditions.

Evidence has been presented which suggests that Rhodopseudomonas spheroides contains an energy-linked transhydrogenase that can be driven by a high energy intermediate generated by light or ATP. This intermediate may also be functional in ATP synthesis. The synthesis of ATP and the ATP-supported transhydrogenase is inhibited by oligomycin. This inhibitor does not affect the light-mediated reaction.

     

Transport and metabolism of adenosine in human blood platelets

The uptake and metabolism of [¹⁴C]- or [³H]adenosine have been studied in suspensions of washed platelets and in platelet rich plasma. The appearance of radio-activity in the platelets and the formation of radioactive adenosine metabolites have been used to determine the uptake. Adenosine is transported into human blood platelets by two different systems: a low K_m system (9.8 μM) which is competitively inhibited by papaverine, and a high K_m system (9.4 mM) which is competitively inhibited by adenine. Adenosine transported via the low K_m system is probably directly incorporated into adenine nucleotides, while adenosine transported through the high K_m system arrives unchanged inside the platelet and is then converted into inosine and hypoxanthine or incorporated into adenine nucleotides.     

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1⁺Mac-1⁺ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca²⁺ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.     

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5′-triphosphate with azoferredoxin

The interaction of ATP with both iron-sulfur proteins of nitrogenase from Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, has been studied by low-temperature EPR spectroscopy. ATP in the presence of Mg²⁺ changes the rhombic EPR signal of azoferredoxin with g-values of 2.06, 1.94 and 1.87 to an axial signal, with g values of 2.04 and 1.93. The binding of two molecules of ATP per azoferredoxin dimer (mol. wt 55 000) is suggested. Comparative data with other purine and pyrimidine nucleotides and ATP analogues demonstrate the involvement of structural elements of the substrate in the conversion of the EPR signal of azoferredoxin. A similar effect is induced by 5 M urea, which suggests that ATP causes a conformation change of the protein. In contrast, no effect of ATP was observed on the EPR signal of molybdoferredoxin.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Membrane Biochemistry : by E. Sim Chapman and Hall; London, 1982

Adenine, cytidine and guanosine nucleotides were supplied to cultures of Rhodopseudomonas capsulata under aerobic heterotrophic and phototrophic growth conditions. Aerobic growth is not affected by exogenous nucleotides (up to 10 mM) whereas phototrophic growth is strongly inhibited by adenine but not by guanosine or cytidine nucleotides. During phototrophic growth there is an inverse relationship between the concentration of exogenous adenine nucleotides and photopigment synthesis. There are no statistically significant differences between the inhibitory effect of AMP, ADP and ATP on the growth rate and bacteriochlorophyll synthesis since adenine nucleotides are incorporated into the cell as AMP by means of the phosphoribosyl transferase system.     

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells

Summary Bovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na⁺/H⁺ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P_2Y and P_2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na⁺/H⁺ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue, ATPγS, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [³²P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

Glycine uptake into barley mesophyll vacuoles is regulated but not energized by ATP

[U-¹⁴C]glycine uptake into barley (Hordeum vulgare cv Hasso) vacuoles was investigated. Glycine (2 millimolar) transport was stimulated two- to fourfold by NaATP. Stimulation was saturable with respect to ATP (1 millimolar) and linear up to 20 millimolar glycine. Stimulation by NaATP was suppressed by Mg²⁺ in equimolar amounts. Neither MgATP nor Mg-inorganic pyrophosphate had any effect on basal transport rate. Thus, the proton motive force can be excluded as the driving force. Uncouplers (valinomycine/carbonylcyanide-m-chlorophenylhydrazone) inhibited the basal rate up to 30% but had no influence on NaATP-stimulated uptake. Vanadate had no effect on either basal or NaATP-stimulated uptake. Nonhydrolyzable ATP analogs (adenylyl(β, γ-methylen)-diphosphate or adenylyl-imidodiphosphate) stimulated comparable to NaATP. Other nucleotides (UTP, ADP) had no effect. Some evidence exists that other amino acids (arginine, alanine, isoleucine, phenylalanine) are transported to a certain extent by a similar mechanism. The results indicate a high capacity channel-like translocator that is regulated but not energized by ATP.     

Inhibition of chloroplast coupling factor by naphthylglyoxal

The trypsin-activated Ca²⁺ -ATPase of spinach chloroplast membranes was completely inhibited by treatment with naphthylglyoxal, a fluorescent compound that should bind covalently to arginine residues. The inhibition followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentration gave values near unity, suggesting that inactivation is a consequence of modifying one arginine residue per active site. Partial protection against naphthylglyoxal was afforded by ADP and ATP, with either less or no protection by other nucleotide bases. At inhibition levels less than complete, the K_m for ATP was not affected but the V_max of the enzyme was diminished. The light-dependent exchange of tightly bound nucleotides on the membrane-bound enzyme was not inhibited by naphthylglyoxal treatment, indicating significant retention of the conformational response of the enzyme to the membrane high-energy state. Using [³H]naphthylglyoxal, the extent of inhibition was a linear function of the amount of naphthylglyoxal bound up to 60% inhibition. The curves extrapolated to 2 mol naphthylglyoxal bound, associated with complete inhibition of ATPase. The radioactive naphthylglyoxal was distributed equally between α- and β-subunits.