Site Of ATP Hydrolysis Research Articles

Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli.

A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five.

Mycochromone decreases ATP‐driven proton translocation in plant plasma membrane‐ and tonoplast‐enriched vesicles

Abstract Mycochromone, a metabolite produced by Mycosphaerella rosigena, inhibits the ATP‐dependent proton translocation and the ATP‐generated electrical potential in pea stem tonoplast‐enriched vesicles, without affecting the H+/K+ exchange induced by nigericin or an artificially imposed proton gradient. The inhibition is dependent on the time of pre‐incubation and mycochromone concentration. In addition, mycochromone inhibits the ATP‐dependent proton translocation in radish plasma membrane‐enriched vesicles, though it does not alter ATPase activity (evaluated by hydrolysis of ATP) in either type of plant vesicle. Mycochromone seems to act on the H+ channels for proton translocation of the H+‐pumping ATPase localized on plasmalemma and tonoplast, without affecting the catalytic site of ATP hydrolysis.

The family of human Na +K +-ATPase genes : A partial nucleotide sequence related to the α-subunit

The Na+,K+-activated adenosine triphosphatase (EC 3.6.1.3) localized in animal cell membranes represents a universal system for the active transport of Na+ and K+. The enzyme is an oligomer composed of two polypeptide chains. Its a-subunit (110 kDa) contains a site for ATP hydrolysis and a site for binding of cardiac glycosides which are specific inhibitors of the enzyme. There is still no evidence of the functional role of the glycosylated P-subunit (44 kDa). fn order to elucidate the molecular mechanism of the active cation transport we have analysed the structural organization of the enzyme from several species. The complete nucleotide sequences of cDNA clones corresponding to the coding regions of appropriate mRNAs were determined, and the primary structure of the (Yand ~-subunits of the pig kidney Na+,K+-ATPase could then be deduced [l-3].

The active site structure of Na+/K+-transporting ATPase: location of the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin.

When the dog kidney Na+/K+-transporting ATPase (EC 3.6.1.37, formerly EC 3.6.1.3) was labeled with an ATP analogue, 5'-(p-fluorosulfonyl)benzoyladenosine (FSBA), there was a concomitant loss of ATPase activity. The presence of ATP protected the enzyme from both labeling and inactivation. The ATP-sensitive incorporation of FSBA is associated only with modification of the alpha subunit from which two labeled tryptic peptides were purified and sequenced. To establish any regions of the enzyme protruding from the membrane, the native Na+/K+-transporting ATPase from the electric ray, Torpedo californica, was treated with trypsin; and four peptides, which were released into the water phase, were purified and sequenced. A comparison of the peptide sequences with the deduced amino acid sequences of the DNA coding for the alpha subunit of T. californica and sheep kidney reveal the following. (i) FSBA-labeled peptides from the dog kidney enzyme are located in the central hydrophilic domain and show almost complete sequence homology with the same region in the alpha subunit from the electric ray and sheep kidney. Furthermore, the sequence homology of one of the two labeled peptides can be extended to the sarcoplasmic Ca2+-transporting ATPase and B subunit of Escherichia coli K+-transporting ATPase. (ii) Three trypsin-exposed peptides are found in the central hydrophilic domain, and one peptide is in the hydrophilic segment near the C terminus of the alpha subunit. (iii) The active center of Na+/K+-transporting ATPase is likely to be constructed from at least four different stretches in the primary sequence and, irrespective of the different specificity of cations, the various cation transport ATPases that form phosphorylated enzyme appear to have a common structure at the catalytic site for ATP hydrolysis.

Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP.

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, label is incorporated primarily into the alpha, beta, and Mr = 78,000 chains; a much smaller amount is incorporated into the Mr = 69,000 chain. The light chains are not labeled. The incorporation is UV-dependent, ATP-sensitive, and blocked by preincubation of the enzyme with vanadate plus low concentrations of ATP or ADP. These results suggest that the alpha heavy chain contains the site of ATP binding and hydrolysis in the alpha subunit. In the beta subunit, the beta heavy chain and one or both intermediate chains may contain ATP-binding sites.

Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5'-triphosphate.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

The ATPase complex of Escherichia coli.

The ATPase (ATP synthase) complex of Escherichia coli is composed of an extrinsic membrane protein (ECF1), which contains the active site for ATP formation and hydrolysis, and is attached to ECF0, a transmembrane protein through which protons move to or from the active site on ECF1. ECF1 is composed of five subunits (alpha-epsilon) with a stoichiometry of alpha 3 beta 3 gamma delta epsilon. The stoichiometry of the three subunits (a-c) of ECF0 is probably a1b2c10-15. In addition to 3 mol tightly bound adenine nucleotide/mol ECF1, three other "exchangeable" nucleotide binding sites can be detected. These sites are still present in the alpha and beta subunit defective ECF1 of uncA401 and uncD412 mutants, although some changes in the tightness of binding are evident. The active sites of ECF1 require normal alpha and beta subunits and may be present at alpha beta subunit interfaces. Hydrolysis of ATP requires cooperative interactions between alpha and beta subunits. At low concentrations of ATP, in the absence of added divalent cations, hydrolysis of this substrate can occur at a single site without release of the product. This is consistent with alternating or sequential site mechanisms for ATP hydrolysis or synthesis. Predictions of secondary and tertiary structures from the known primary amino acid sequences of polypeptides a, b, and c have led to the following conclusions. Polypeptide a forms six or seven transmembrane alpha helices. The amino-terminal sequence of polypeptide b spans the membrane, but most of the protein is exposed on the cytoplasmic surface of the membrane where it can be cleaved by proteases in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity.

The 18 S dynein of the outer arm of Chlamydomonas flagella contains two different heavy polypeptide chains (Mr approximately equal to 340,000), two intermediate chains (Mr = 69,000 and 78,000), and eight light chains (Mr = 8,000-20,000). We report here that when purified 18 S dynein is dialyzed against a low ionic strength solution, it is dissociated into two smaller subunits which can then be separated and purified by sucrose density gradient centrifugation. One subunit contains one of the heavy chains and a Mr = 16,000 light chain; the other contains the other heavy chain and the remaining intermediate and light chains. Both subunits have ATPase activity. When recombined in the presence of 5-25 mM KCl, the subunits reassemble to form a particle similar to native 18 S dynein; neither subunit by itself can reform such a particle. 18 S dynein is therefore a heteropolymer containing two compositionally distinct subunits. Because the complete outer arm contains both 12 S and 18 S dyneins, the arm must have a total of three sites of ATP binding and hydrolysis: one associated with 12 S dynein and two with 18 S dynein.

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein.

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase.

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.

Spontaneous reactivation of covalently labeled proton adenosinetriphosphatase.

Bovine heart mitochondrial adenosinetriphosphatase selectively labeled by [14C]-N,N'-dicyclohexylcarbodiimide or [14C]-7-chloro-4-nitro-2,1,3-benzoxadiazole was used together with other components to form reconstituted submitochondrial particles. When assayed for ATP hydrolysis under normal hydrolysis condition, these labeled submitochondrial particles were found to increase slowly in specific activity with preincubation time, without losing the covalent label. But when assayed for oxidative phosphorylation, the ratio of the specific activity of the same labeled particles to that of the control particles was higher and was unaffected by preincubation. If the labeled particles had been treated by a simulated procedure for oxidative phosphorylation measurement before the ATPase assay, their specific activities for ATP hydrolysis were also found to be higher and unaffected by preincubation. These observations are difficult to reconcile with the alternating three-site model for proton adenosinetriphosphatase or any model which requires the sequential action of three identical sites for ATP hydrolysis and synthesis. A new model with one active and two latent interacting sites is proposed for interpreting the present data.

Identification of the nucleotide-binding site for ATP synthesis and hydrolysis in mitochondrial soluble F1-ATPase.

The binding of ADP to mitochondrial soluble F1-ATPase was studied by a membrane filtration method. One mol of F1, which contained about 1 mol of tightly bound adenine nucleotide, bound 4 mol of [alpha-32P]ADP at a saturating concentration of [alpha-32P]ADP. Two mol of the bound [alpha-32P]ADP was readily exchanged with medium nonradioactive ADP while the remaining 2 mol of the bound [alpha-32P] ADP was hardly exchanged (tightly bound [alpha-32P]ADP). F1 catalyzed the synthesis of [alpha-32P]ATP from the medium [alpha-32P]ADP and Pi. However, when exchangeably bound [alpha-32P]ADP was replaced by nonradioactive ADP, no [alpha-32P] ATP formation was observed. Furthermore, tightly bound [alpha-32P]ADP was not released from F1 during ATP hydrolysis catalyzed by the enzyme. These results indicate that both ATP synthesis and hydrolysis catalyzed by F1 occur at the exchangeable binding site and not at the tight binding site on the enzyme.

27] Methylated lysines and 3-methylhistidine in myosin: Tissue and developmental differences

Publisher Summary This chapter deals with tissue and developmental differences in methylated lysines and 3-methylhistidine (3-MeHis) in myosin. Four different methylated amino acids have been identified in myosins—namely, 3-MeHis, ɛ - N -monomethyllysine (MeLys), ɛ - N -dimethyllysine (Me 2 Lys), and ɛ - N -trimethyllysine (Me 3 Lys). All these unusual residues are in the heavy chain of subfragment 1, the globular head of myosin that carries the sites for ATP hydrolysis and actin combination. Distribution of methylated amino acids is investigated by isolating and sequencing the peptides which contain the 3-MeHis and the two Me 3 Lys in rabbit fast skeletal muscle myosin. Both 3-MeHis and Me 3 Lys are present as fully methylated and trimethylated amino acids in the myosin heavy chain, and there are no partially methylated Lys and His residues. The 3-MeHis containing tryptic peptide contains 13 amino acid residues, including the methylated histidine. The methylated amino acids, like other amino acids, are analyzed in the HCl hydrolyzates of proteins. Similar to other posttranslational amino acid side-chain modifications, lysines and histidines are enzymatically methylated after they are incorporated into the myosin peptide chain. The methyl donor is S -adenosyl-L-methionine (SAM). Methylation of myosin with SAM donors has been carried out in various experimental designs such as in muscle homogenates and cultures, in perfused hearts, and by in vitro methylation of nascent myosin chains still attached to polyribosomes.

Localization of endogenous ATPases at the nerve terminal.

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg2+-ATPase; (b) an ATPase active at high concentrations of Ca2+ in the absence of Mg2+ (CaH-ATPase); (c) a Ca2+ requiring Mg2+-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca2+-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and CaH-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.

Disruptiin of energy transductiin in sarcoplasmic reticulum by trypsin cleavage of (Ca2+ + Mg2+)-ATPase.

Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca2++Mg2+)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A1 and A2 fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca2+-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of “tightly bound” nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.

Cell and Membrane Physiology

Three of the four articles in this section deal with contractility and its relation to structure in a variety of cells. They are complemented by one reviewing a novel way of studying cell metabolism-a prerequisite of mo­ tion-by a nondestructive process. Goldman et al deal with the wide variety of filaments found in mam­ malian cells. In addition to filaments made up of proteins analogous to those found in the muscle cell, other filaments or fibers exist that differ in mor­ phology from actinand myosin-containing filaments. A number of proteins have been recognized as their constituents. The functional role of these filaments in maintaining cell structure and in cellular contractility-a fast growing field-is the main theme of the review. Tregear & Marston cover the more conventional contractile apparatus, i.e. that found in skeletal muscle. They review current views on the mecha­ nism by which the portion of the myosin molecule that carries the active site for the ATP hydrolysis interacts with actin. In the view of the large majority of workers in this field, myosin molecules interacting with actin form the so-called crossbridges, whose cyclic formation and breaking, cou­ pled to ATP hydrolysis, is the basis of force generation. Tregear & Marston present an up-to-date review on the topic, together with some suggestions of their own. While knowledge of the striated muscle system has reached a high level of development, information concerning smooth muscle has been lagging in many areas. Recent work has cleared up several uncertainties (particularly in regard to structure), has led to progress in energetics, and has brought to light some novel aspects of control. The chapter by Murphy is, therefore, a timely review of a rapidly developing field. The relation between cell metabolism and cell function, whether that function be muscle contraction, secretion, or the generation of an electrical signal, has been recognized as a fundamental problem of biology. Classical

The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site.

Fluorescein 5'-isothiocyanate (FITC) has been shown to specifically inactivate the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) at low concentrations (Karlish, S. J. D. (1979) Na+,K+ATPase Structure and Kinetics 115-128). The site of modification of purified dog kidney (Na,K)-ATPase by FITC has been investigated by enzymatic cleavage and fluorescence resonance energy transfer. The binding of FITC, which occurs at a stoichiometry of approximately one site per ATP binding site, causes an ATP-protectable inactivation of ATPase activity suggesting that it is reacting at the ATP hydrolysis site. The FITC reaction site apparently is located near the center of the COOH-terminal 77,000-dalton peptide fragment obtained by chymotryptic cleavage of the alpha subunit. Addition of ouabain to the native enzyme in the presence of chymotrypsin enhances cleavage at this site and releases the fluorescein moiety from the membrane. It is further shown that the distance from the FITC reaction site to the ouabain binding site, as judged by fluorescence resonance energy transfer from anthroyl ouabain to FITC, is approximately 74 A. These results demonstrate that ouabain inhibits the (Na,K)-ATPase by causing a protein conformational change which extends an unusually large distance across the membrane.

Preferential interaction parameters for F 1-ATPase from a thermophilic bacterium (PS3)

The proton translocating ATPase of energy transducing membranes couples the vectorial transport of protons through the membrane to synthesize ATP. The enzyme can be divided into an integral membrane portion, F,, and a peripheral portion, called the coupling factor or Fr-ATPase, which contains the catalytic site of ATP synthesis and hydrolysis [l-3]. The Fr portion of the H’-ATPase consists of 5 different subunit species, OL,/~,T,~ and E. The subunits have been isolated of Fr from Escherichia coli, PS3 and CFr [4-61, and their amino acid composition and Mr-values determined [7]. The subunit stoichiometry is still controversial because of incomplete reassembly to a functional Fr of the isolated subunits [7]. Also, the reported J&-values of Fr vary from 350 OOO380 000, e.g., for ECFr [9,10], due to differences in analytical methods and preparations. However, analytical ultracentrifugation data were not treated as a multicomponent system with c2 the mass of the protein (Fr), c3 the added solvent component, e.g., methanol or glycerol, and cr the principal solvent (buffer) with cr in g/ml solution. This paper reports on studies of TFr in 0.01 M Tris-HCI buffer at pH 7.0-8-O in the presence and absence of methanol and glycerol by means of light scattering and differential refractometry experiments, in order to detect any interactions of component three with the protein. Since these organic solvents

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity.

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.

Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores

The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phospho-rylation and the light-dependent ATP-P i exchange reactions of R. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3-butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.