An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis

Cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide‐binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di‐GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo‐complex, demonstrated the site of cyclic GMP binding that modulates cyclic di‐GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di‐GMP signalling.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

Analysis of warfarin enantiomers: Chromatographic separation of six stereoisomeric esters prepared from (−)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid

Reaction of rac-warfarin, (?)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid [(?)-HCA] and carbodiimide reagents gave two noncyclic ketonic diastereoisomeric derivatives whereas rac-warfarin and (?)-HCA acid chloride with 4-(dimethylamino)pyridine gave four cyclic hemiketal diastereoisomeric ester derivatives. The structure and stereochemistry of diastereoisomeric esters prepared from warfarin and p-chlorowarfarin were determined from ¹H- and ¹³C-NMR spectra, mass spectra, and hydrolysis to warfarin and p-chlorowarfarin enantiomers. The structure and stereochemistry of one of the cyclic hemiketal diastereoisomeric derivatives of warfarin are supported by an X-ray crystallographic determination. Mechanisms for the formation of all products are proposed. © 1994 Wiley-Liss, Inc.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10⁻² M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain]

A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.     

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin

Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.     

2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

Nucleosides and Nucleotides. 192. Toward the Total Synthesis of Cyclic ADP-Carbocyclic-Ribose. Formation of the Intramolecular Pyrophosphate Linkage by a Conformation-Restriction Strategy in a Syn-form Using a Halogen Substitution at the 8-Position of the Adenine Ring

Abstract

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N ⁶-trichloroacetyl-2′,3′-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5′,5′-diol derivatives 18. When 18 was treated with POCl₃ in PO(OEt)₃, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5′,5′-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.     

Ribonucleic Acid Synthesis in Plant Mitochondria: Effects of Some Inhibitors and Cyclic Nucleotides

Mitochondria isolated from 48-h germinating Vigna sinensis (L.) Savi can incorporate [³H]uridine into acid-insoluble material. The incorporation is highly sensitive to rifampicin and partially so to ethidium bromide, two specific inhibitors of template function. The inhibitory effect of rifampicin can be partly counteracted by cyclic 3′:5′-AMP but not by cyclic 3′:5′-GMP, if they are allowed to interact with the synthetic system before the treatment with rifampicin. This indicates that cyclic AMP and rifampicin compete for a common site on the RNA polymerase responsible for DNA-dependent RNA synthesis. Inhibition by ethidium bromide is unaffected by prior nucleotide interaction with the system.     

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.     

Platinum complexes with imino ethers or cyclic ligands mimicking imino ethers: synthesis,in vitro antitumour activity,and DNA interaction properties

Both trans- and cis-[PtCl₂(NH₃)(L)] compounds have been synthesized, L representing either the imino ether HN=C(OMe)Me having a Z or E configuration at the C=N double bond, or the cyclic ligands and (compounds 1–4 for trans geometry and 5–8 for cis geometry, respectively). The cyclic ligands mimic the imino ether ligands but, differently from imino ethers, cannot undergo change of configuration. In a panel of human tumor cells, trans compounds inhibit growth much more than transplatin. Moreover, compound 1 in most cases is less active than 2, and 1 and 2 are less active than 3 and 4, respectively. For cis compounds with imino ethers, the activity is reduced (5) or unaffected (6) with respect to cisplatin. Moreover, unlike trans compounds, substitution of cyclic ligands (7, 8) for imino ethers (5, 6) generally decreases the activity. This determines, for compounds with cyclic ligands, an unusual inversion of the cis geometry requirement for activity of platinum(II) species. Importantly, 1–4 and 5–8 partially circumvent the multifocal cisplatin resistance of A2780cisR cells, and 1–4 also overcome resistance from reduced uptake of 41McisR cells. DNA interaction regioselectivity of 1–4 and 5–8 is not substantially modified with respect to transplatin and cisplatin. However, both imino ethers and cyclic ligands slow down the DNA interstrand cross-link reaction, (E)-HN=C(OMe)Me and decreasing also its extent. Therefore, DNA interaction of 1–4 and 5–8 appears to be characterized by persistent monoadducts (1–4), and by monoadducts and/or intrastrand cross-links structurally different from those of cisplatin (5–8). This study demonstrates that ligand configuration modulates the activity of both trans and cis compounds, and supports the development of platinum drugs based on their coordination chemistry to combat cisplatin resistance.F.P. Intini and A. Boccarelli contributed equally to this work     

ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: EFFECTS OF α- AND β-ADRENERGIC AGENTS, HISTAMINE, SEROTONIN AND ADENOSINE

—Norepinephrine and epinephrine, in combination with either adenosine or histamine, enhanced the accumulation of cyclic AMP in guinea pig cerebral cortical slices. Isoproterenol had only marginal effects under the same conditions. Studies with d- and l-norepinephrine and with the α- and β-adrenergic blocking agents, phenoxybenzamine, phentolamine, dihydroergokryptamine, propranolol and sotalol, indicated that the effect of catecholamines on cyclic AMP levels in this tissue was stereo-specific and was mediated primarily via interaction with a classical α-adrenergic receptor. Studies with the antihistaminics, diphenhydramine and pheniramine, and the antiserotonin agent, methysergide, indicated that guinea pig cerebral cortical slices contain receptors for histamine and serotonin, whose activation also stimulates an enhanced accumulation of cyclic AMP in the presence of adenosine.     

Structural basis for the bifunctionality of the U5 snRNP 52K protein (CD2BP2)

The bifunctional protein U5-52K is associated with the spliceosomal 20 S U5 snRNP, and it also plays a role in immune response as CD2 receptor binding protein 2 (CD2BP2). U5-52K binds to the CD2 receptor via its GYF-domain specifically recognizing a proline-rich motif on the cytoplasmic surface of the receptor. The GYF-domain is also mediating the interaction of the proteins U5-52K and U5-15K within the spliceosomal U5 snRNP. Here we report the crystal structure of the complex of GYF-domain and U5-15K protein revealing the structural basis for the bifunctionality of the U5-52K protein. The complex structure unveils novel interaction sites on both proteins, as neither the polyproline-binding site of the GYF-domain nor the common ligand-binding cleft of thioredoxin-like proteins, to which U5-15K belongs, are involved in the interaction of U5-15K and U5-52K.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.     

The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in Stentor coeruleus.

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N⁶, 2′-0-dibutyryl adenosine cyclic 3′:5′-monophosphate (DBC) or theophylline begun at the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions. To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.     

The effects of anions on the solution structure of Na,K-ATPase

Anions interact with protein to induce structural changes at ligand binding sites. The effects of anion complexation include structural stabilization and promote cation-protein interaction. This study was designed to examine the interaction of aspirin and ascorbate anions with the Na+, K+-dependent adenosine triphosphatase (Na,K-ATPase) in H2O and D2O solutions at physiological pH, using anion concentrations of 0.1 microM to 1 mM with final protein concentration of 0.5 to 1 mg/ml. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the anion binding mode, binding constant, and the protein secondary structure in the anion-ATPase complexes. Spectroscopic evidence showed that the anion interaction is mainly through the polypeptide C=O and C-N groups with minor perturbation of the lipid moiety. Evidence for this came from major spectral changes (intensity variations) of the protein amide I and amide II vibrations at 1651 and 1550 cm(-1). respectively. The anion-ATPase binding constants were K=6.45 x 10(3) M(-1) for aspirin and K=1.04 x 10(4) M(-1) for ascorbate complexes. The anion interaction resulted in major protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated sheet 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38% in the free Na,K-ATPase to that of the alpha-helix 24-26%; beta-pleated 17-18%; turn 8%; beta-antiparallel 5-3% and random 45.0% in the anion-ATPase complexes.