Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.     

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.     

Characterization and function of catalytic subunit alpha of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. A study with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Subunit alpha (Mr 89,000) from vacuolar membrane H+-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido[alpha-32P]adenosine triphosphate. Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10(-6) to 10(-5) M and decreased in the presence of ATP and ADP. The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 microM causing half-maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP). The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with [14C]NBD-Cl, subunit alpha was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. From these results, it was concluded that subunit alpha of yeast vacuolar H+-ATPase has a catalytic site that contains a single, essential tyrosine residue. The kinetics of single site hydrolysis of [gamma-32P]ATP (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100) indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site. NBD-Cl inactivated the single site hydrolysis and inhibited the formation of an enzyme-ATP complex. Dicyclohexylcarbodiimide did not affect the single site hydrolysis, but inhibited the enzyme activity under steady-state conditions.     

Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.     

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.     

Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37 degrees C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N-ethylmaleimide and p-chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or beta-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37 degrees C, pH 8.3 in sucrose-citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

Effect of NBD chloride (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) on the pyridine nucleotide transhydrogenase of Escherichia coli.

Pyridine nucleotide transhydrogenases of bacterial cytosolic membranes and mitochondrial inner membranes are proton pumps in which hydride transfer between NADP(+) and NAD(+) is coupled to proton translocation across cytosolic or mitochondrial membranes. The pyridine nucleotide transhydrogenase of Escherichia coli is composed of two subunits (alpha and beta). Three domains are recognized. The extrinsic cytosolic domain 1 of the amino-terminal region of the alpha subunit bears the NAD(H)-binding site. The NADP(H)-binding site is present in domain 3, the extrinsic cytosolic carboxyl-terminal region of the beta subunit. Domain 2 is composed of the membrane-intrinsic carboxyl-terminal region of the alpha subunit and the membrane-intrinsic amino-terminal region of the beta subunit. Treatment of the transhydrogenase of E. coli with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) inhibited enzyme activity. Analysis of inhibition revealed that several sites on the enzyme were involved. NBD chloride modified two (betaCys-147 and betaCys-260) of the seven cysteine residues present in the transhydrogenase. Modification of betaCys-260 in domain 2 resulted in inhibition of enzyme activity. Modification of residues other than cysteine residues also resulted in inhibition of transhydrogenation as shown by use of a cysteine-free mutant enzyme. The beta subunit was modified by NBD chloride to a greater extent than the alpha subunit. Reaction of domain 2 and domain 3 was prevented by NADPH. Modification of domain 3 is probably not associated with inhibition of enzyme activity. Modification of domain 2 of the beta subunit resulted in a decreased binding affinity for NADPH at its binding site in domain 3. The product resulting from the reaction of NBD chloride with NADPH was a very effective inhibitor of transhydrogenation. In experiments with NBD chloride in the presence of NADPH it is likely that all of the sites of reaction described above will contribute to the inhibition observed. The NBD-NADPH adduct will likely be more useful than NBD chloride in investigations of the pyridine nucleotide transhydrogenase.     

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K(m) values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.     

Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochemistry, as a derivatization agent in analytical chemistry, and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines containing two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compound formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated aromatic systems for amine derivatization in analytical and biochemistry applications. The reaction with polyamines opens the way to libraries of NBD-tagged compounds.     

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.     

Site-directed fluorogenic modification of bacteriorhodopsin by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole

Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone-sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration-dependent being nearly maximum at 20 micrograms/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation. Hormone-modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin-treated cells was elevated 26-fold and 20-fold respectively, while the rate in homogenates from these cells was elevated only 2.8-fold. Insulin suppressed catecholamine-dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin-treated cells. Insulin suppression was only 56% when measured in homogenates. The hormone-sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin-permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration-dependent manner with 1 microM, 30 microM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 microM, 30 microM and 1.0 mM cyclic AMP. Also in digitonin-treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone. These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone-sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.     

Mitochondrial F1-ATPase will bind and cleave ATP but only slowly release ADP after N,N'-dicyclohexylcarbodiimide or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatization

The ATPase from the inner mitochondrial membrane is known to be inhibited by modification of one of the three catalytic subunits with N,N'-dicyclohexylcarbodiimide (DCCD) or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. An experimental approach described in this paper shows that most of the residual ATPase activity observed after the usual DCCD or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole modification is due to the presence of unmodified enzyme, although the large fraction of modified enzyme retains a weak catalytic activity. This weak catalytic activity can be stimulated by methanol or dimethyl sulfoxide. When the modified enzymes are exposed to Mg2+ and [3H]ATP, about equal amounts of [3H]ATP and [3H]ADP appear at catalytic sites. The turnover rate for these enzymes is less than 1/1000 that of the native enzyme when it is calculated from the rate at which the enzyme becomes labeled at the catalytic sites with [3H]ATP and [3H]ADP during steady state hydrolysis. In addition, a higher ATP concentration is required for steady state turnover and, after ATP binding, the principal rate-limiting step is the capacity of the derivatized enzyme to undergo the binding changes necessary for the release of ADP and Pi. When the modified enzymes are not hydrolyzing ATP, they convert to form(s) that show a distinct lag in the replacement of bound nucleotides at catalytic sites. The replacement of bound nucleotides is still promoted by MgATP, even though the enzymes have been converted to sluggish forms. Contrary to a recent suggestion based on the study of the DCCD-modified enzyme (Soong, K.S., and Wang, J.H. (1984) Biochemistry 23, 136-141), our data provide evidence for the existence of catalytic cooperatively between at least two alternating sites in the modified enzyme and are consistent with continued sequential participation of all three sites.     

Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Different fluorescent labels were tested in order to monitor conformational transitions of the four-hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four-hexamer was labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, the maximum wavelength lambda max of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for lambda max of the fully oxygenated haemocyanin were 531.5 nm (pH less than 7.8) and 530.0 nm (pH greater than 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH less than 7.2) and 535.2 nm (pH greater than 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891-1895]. Only four amino acids of the four-hexamer were labelled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. They were identified as lysine 484 on the purified peptide Leu-Arg-Lys-Phe-His-Arg. This amino acid is located on the surface of the four copies of subunit d. The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.     

Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site.

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.