Biomedical subjects

A E Senior

Publications and source records attributed to A E Senior.

At least 37 records · Page 2Linked to original sources

PubMed1998

Residues in P-glycoprotein catalytic sites that react with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a specific covalent inhibitor of P-glycoprotein ATPase activity (M. K. Al-Shawi, and A. E. Senior, 1993, J. Biol. Chem. 268, 4197-4206). Complete inhibition occurs at a reaction stoichiometry of 1 mol NBD/mol P-glycoprotein, and the reagent has proved valuable in understanding catalytic mechanisms, particularly in relation to catalytic site cooperativity (A. E. Senior, and S. Bhagat, 1998, Biochemistry 37, 831-836). The actual location of reaction in the amino acid sequence has not yet been determined. Using a combined mutagenesis and biochemical approach we establish here that the initial reaction of NBD-Cl is with Cys within the Walker A consensus sequence of the N- or C-terminal nucleotide site (Cys-431 or Cys-1074 of human P-glycoprotein). Reaction with either Cys yields full inhibition. It was further found that inhibition consists of dithiothreitol (DTT)-reversible and DTT-irreversible components. The former predominates at low pH and the latter at higher pH. This demonstrates that, at higher pH, intramolecular transfer of NBD from Cys to Lys occurs, probably to the proximate Walker A Lys (Lys-433 or Lys-1076 of human P-glycoprotein). After transfer of NBD to Lys, P-glycoprotein ATPase remains fully inhibited.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase.

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.

Adenine Nucleotides↗

PubMed1998

Catalytic site nucleotide binding and hydrolysis in F1F0-ATP synthase.

F1F0-ATP synthase was purified from Escherichia coli beta Y331W mutant. The beta-Trp-331 provided a specific fluorescent probe of catalytic site nucleotide binding. Physiological (mM) concentration of substrate MgATP filled all three catalytic sites. With MgATP or MgADP the catalytic sites showed marked binding cooperativity and asymmetry, which was dependent on Mg2+. Nucleotide binding was fast, with kon = approximately 6 x 10(5) M-1 s-1. Pi at physiological concentration (5 mM) did not bind to catalytic sites. Measurement of MgATP hydrolysis and binding under identical conditions as a function of MgATP concentration revealed that Vmax was achieved only when all three catalytic sites were filled in every enzyme molecule. The enzyme species with two catalytic sites occupied and one site empty displayed low, nonphysiological catalytic rate. This is the first characterization of nucleotide binding parameters in F1F0. The fact that the behavior of purified F1F0 was similar in most respects to that of isolated F1 demonstrated that the presence of the additional F0 subunits a, b, and c, and also fixed stoichiometric amounts of epsilon and delta, does not affect catalytic site properties. The results impact on possible catalytic mechanisms, namely, they emphasize that Pi cannot simply bind spontaneously, that an enzyme species with all three sites occupied is the only catalytically competent species, and that release of product and binding of substrate cannot be simultaneous, rather the former must precede the latter.

Adenosine Diphosphate↗

PubMed1998

Effects of cardiovascular drugs on ATPase activity of P-glycoprotein in plasma membranes and in purified reconstituted form.

Drug interactions with P-glycoprotein (Pgp) were quantitatively assessed using ATPase assay. Two experimental systems were used, (i) plasma membranes isolated from a multidrug-resistant cell line, which contained 30% Pgp as fraction of total membrane protein, and (ii) purified reconstituted Pgp. The cardioactive drugs verapamil, quinidine, diltiazem, nifedipine, and a series of digitalis analogs, interacted directly with Pgp as shown on ATPase in both systems. Apparent affinities of drug binding were calculated. Direct competition was shown between digitoxin and verapamil. Drug-drug interaction in vivo at the level of Pgp is expected from the results. This approach seems well-suited for empirical determination of drug interactions with Pgp, and prediction of drug-drug interactions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites.

P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites. The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp. We show that, at reaction stoichiometry of < or = 1 mol/mol, both nucleotide sites become labeled in relatively nonselective fashion. There is therefore strong interaction between the two nucleotide sites because (a) reaction of one site with NBD-Cl severely impedes reaction of reagent with the other site, and (b) reaction of one site inhibits steady-state ATPase, i.e. both sites are inhibited. Vanadate-trapping experiments revealed that when one nucleotide site was reacted with NBD-Cl, not even a single ATPase turnover event could occur in the other, intact, nucleotide site. The data demonstrate therefore that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

Mg2+ coordination in catalytic sites of F1-ATPase.

Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of betaThr-156 is a direct ligand of Mg2+, whereas the carboxyls of betaGlu-181, betaGlu-185, and betaAsp-242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that betaThr-156, betaGlu-185, and betaAsp-242 are all involved in Mg2+ coordination, whereas betaGlu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.

Adenine Nucleotides↗

PubMed1998

ATPase activity of Chinese hamster P-glycoprotein.

We have developed two defined experimental systems for biochemical investigation of P-glycoprotein, namely, plasma membranes highly enriched in Pgp, obtained from the CR1R12 Chinese hamster ovary cell line, and pure, reconstituted Pgp, obtained by solubilization of Pgp from CR1R12 plasma membranes, Reactive Red 120 chromatography, and reconstitution in liposomes. Studies of the ATPase catalytic mechanism by kinetic methods and covalent inactivation have been greatly facilitated by the availability of these experimental systems. The technique of vanadate trapping of nucleotide has been particularly useful. As a result of these studies, we now have explicit, testable, proposals for (1) the normal catalytic pathway of ATP hydrolysis, (2) a postulated alternating catalytic site cycle, and (3) coupling of ATP hydrolysis to drug transport. The experimental methods described here should prove valuable for future studies of Pgp and of ABC transporters in general.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Catalytic mechanism of P-glycoprotein.

We generated Chinese hamster ovary cells which are highly multidrug-resistant by selection in colchicine. Purified plasma membranes from these cells are enriched in P-glycoprotein (Pgp), up to 32% w/w of membrane protein. From plasma membranes we purified Pgp to homogeneity and reconstituted it in proteoliposomes. Both plasma membranes and purified reconstituted Pgp show drug-stimulated ATPase activity (approximately 20 s-1), comparable to other transport ATPases. These materials enable investigation and characterization of the catalytic sites and mechanism. Various approaches have been used, notably enzyme kinetics, photoaffinity and other covalent labelling, use of vanadate as transition-state analog, and inhibition by beryllium and aluminum fluoride. Both Pgp nucleotide sites hydrolyse MgATP and are of relatively low specificity and affinity for nucleotides. Trapping of nucleotide by vanadate in either site blocks catalysis at both sites; covalent inactivation of either site completely blocks turnover. Therefore the catalytic sites interact strongly, and it appears that when one site enters the transition-state conformation the other site is prohibited from doing so. A minimal reaction scheme for ATP hydrolysis has been determined. We have proposed an alternating catalytic sites scheme, in which drug-transport is coupled to relaxation of a high chemical potential conformation of the catalytic site (Pgp.MgADP.Pi) which is generated by the hydrolysis step itself. Photoaffinity labelling of Pgp catalytic sites has revealed equivalent Tyr residues which lie close to the adenine ring of bound MgATP in both sites.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Photoaffinity labelling of P-glycoprotein catalytic sites.

Photoaffinity labelling of hamster P-glycoprotein was carried out after trapping of radioactive Mg-8-azido-ADP in the catalytic sites by vanadate or beryllium fluoride. With either trapping agent the same labelled peptide was obtained in homogeneous form, with the sequence -FNEVVFNxPTRPDI-, corresponding to residues 1034-1037 in the C-terminal nucleotide binding site. The missing residue 'x' corresponds to Tyr-1041, which is therefore a primary reaction target of 8-azido-ADP. This tyrosine is conserved in all hamster, mouse and human P-glycoproteins. A second major labelled peptide fraction was also identified. The major sequence in this fraction was -NIHFSxPSR-, corresponding to residues 393-401 of hamster P-glycoprotein, where 'x' corresponds to Tyr-398 in the N-terminal nucleotide binding site. Therefore Tyr-398, which is also conserved in other P-glycoproteins, is also a reaction target for 8-azido-ADP. In sequence alignment of the two nucleotide binding sites, Tyr-398 exactly corresponds to Tyr-1041. The data indicate that these two tyrosines lie close to the adenine ring of bound substrate MgATP in the respective catalytic sites of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Binding of TNP-ATP and TNP-ADP to the non-catalytic sites of Escherichia coli F1-ATPase.

Using site-directed-tryptophan fluorescence, parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to non-catalytic sites of Escherichia coli F1-ATPase were determined. All three non-catalytic sites showed the same affinity for MgTNP-ATP (Kd = 0.2 microM) or MgTNP-ADP (Kd = 6.5 microM) whereas even at concentrations of 100 microM no binding of uncomplexed TNP-ATP or TNP-ADP was observed. The results demonstrate that the three non-catalytic sites bind TNP-nucleotides non-cooperatively, and emphasize the importance of Mg2+ for non-catalytic-site nucleotide binding. Parameters for binding of (Mg)TNP-ADP to the three catalytic sites were also determined, and showed marked cooperativity. This work completes the set of thermodynamic parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to all six nucleotide sites of F1, providing essential information to fully exploit the potential of these nucleotide analogs in studies of F1-ATPase.

Adenosine Diphosphate↗

PubMed1997

Inhibition of P-glycoprotein ATPase activity by beryllium fluoride.

ATPase activity of P-glycoprotein (multidrug-resistance protein) was found to be potently inhibited by beryllium fluoride (BeFx) in combination with MgATP, MgADP, or corresponding Mg-8-azido-nucleotides. Inhibition was due to trapping of nucleoside diphosphate at catalytic sites. Full inhibition was achieved on trapping of 1 mol of nucleotide per mol of Pgp. Reactivation was slow (t(1/2) = 32 min at 37 degrees C), and release of trapped nucleotide correlated with recovery of ATPase. Trapping of 8-azido-ADP followed by UV irradiation yielded permanent inactivation and specific labeling of Pgp in plasma membranes. Both N- and C-terminal nucleotide binding sites were labeled. These findings give strong confirmation of the concepts that in intact Pgp both nucleotide sites are active in MgATP hydrolysis, and that they interact strongly. The characteristics of inhibition by BeFx were similar in general to those seen with vanadate. However, PPi gave strong protection against BeFx inhibition, and in this respect, inhibition by BeFx was clearly different from vanadate inhibition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Inhibition of P-glycoprotein ATPase activity by procedures involving trapping of nucleotide in catalytic sites.

Fluoroaluminate in combination with nucleotide inhibited ATPase activity of P-glycoprotein (Pgp) in plasma membranes and in pure reconstituted form. Low nucleotide concentrations were effective, e.g., half-maximal inhibition was obtained with 10 microM MgATP. With MgATP or MgADP, reactivation occurred with t1/2 = 7 min at 37 degrees C. With 8-azido-ATP, UV irradiation of inhibited Pgp gave specific photolabeling of both nucleotide sites. Fluoroaluminate therefore provides a valuable tool for functional and structural characterization of P-glycoprotein and probably of other ABC transporters. 2-Azido-ATP, in combination with vanadate, fluoroaluminate, or beryllium fluoride, inhibited Pgp ATPase activity. Low concentrations of 2-azido-ATP were effective. However, after UV irradiation of the inhibited Pgp species, in no case was there evidence of covalent labeling of nucleotide sites. Therefore in the Pgp catalytic sites, under conditions of nucleotide trapping, there is no suitable amino acid side chain adjacent to the photoactivated 2-position of bound 2-azido-nucleotide, and 8-azido-ATP is the preferred photolabeling analog.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Catalytic mechanism of F1-ATPase.

The structure of the core catalytic unit of ATP synthase, alpha 3 beta 3 gamma, has been determined by X-ray crystallography, revealing a roughly symmetrical arrangement of alternating alpha and beta subunits around a central cavity in which helical portions of gamma are found. A low-resolution structural model of F0, based on electron spectroscopic imaging, locates subunit a and the two copies of subunit b outside of a subunit c oligomer. The structures of individual subunits epsilon and c (largely) have been solved by NMR spectroscopy, but the oligomeric structure of c is still unknown. The structures of subunits a and delta remain undefined, that of b has not yet been defined but biochemical evidence indicates a credible model. Subunits gamma, epsilon, b, and delta are at the interface between F1 and F0; gamma epsilon complex forms one element of the stalk, interacting with c at the base and alpha and beta at the top. The locations of b and delta are less clear. Elucidation of the structure F0, of the stalk, and of the entire F1F0 remains a challenging goal.

Adenosine Diphosphate↗

PubMed1997

Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions.

Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites were filled in the E. coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure. However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e.g. Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.

Adenosine Triphosphate↗

PubMed1997

F1-ATPase, roles of three catalytic site residues.

Three critical residues, beta-Lys-155, beta-Asp-242, and beta-Glu-181, situated close to the gamma-phosphate of MgATP in F1-ATPase catalytic sites, were investigated. The mutations betaK155Q, betaD242N, and betaE181Q were each combined with the betaY331W mutation; the fluorescence signal of beta-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated. The following conclusions were made. The major functional interaction of beta-Lys-155 is with the gamma-phosphate of MgATP and is of primary importance at site 1 (the site of highest affinity) and site 2. Release of MgATP during oxidative phosphorylation requires conformational re-positioning of this residue. The major functional interaction of beta-Asp-242 is with the magnesium of the magnesium nucleotide at site 1; it has little or no influence at site 2 or 3. In steady-state turnover, the MgATP hydrolysis reaction occurs at site 1. beta-Glu-181 contributes little to nucleotide binding; its major catalytic effect derives apparently from a role in reaction chemistry per se. This work also emphasizes that nucleotide binding cooperativity shown by the three catalytic sites toward MgATP and MgADP is absolutely dependent on the presence of magnesium.

Adenosine Diphosphate↗

PubMed1997

ATP hydrolysis cycles and mechanism in P-glycoprotein and CFTR.

P-glycoprotein and CFTR are prominent members of the ABC Transporter family. Both use ATP, the former to drive extrusion of drugs from cells and confer multidrug-resistance, the latter to drive opening and closing of anion channels. We compare current working models of catalytic cycle and mechanism of the two proteins. In Pgp the NBDs appear functionally equivalent, in CFTR they appear functionally distinct. In both proteins, ATP hydrolysis occurs in both NBDs, and it is proposed that the two NBDs alternate in catalysis. Other differences and similarities are noted, fostering ideas for future developments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis.

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (betaF148W) showed normal enzymatic characteristics. The fluorescence responses of beta-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP.BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (Vmax conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful.

Adenosine Diphosphate↗

PubMed1996

F1F0-ATP synthase: development of direct optical probes of the catalytic mechanism.

Using strategically-placed tryptophan (Trp) residues as optical probes to monitor nucleotide binding and hydrolysis, we demonstrate that all three catalytic nucleotide binding sites in F1-ATPase must be filled to obtain physiological (Vmax) MgATP hydrolysis rates. At Vmax hydrolysis rates, the predominant enzyme species has one of the three catalytic sites filled with unhydrolyzed substrate MgATP, the other two sites are filled with product MgADP. A specifically-inserted Trp probe was also developed to characterize nucleotide binding to the noncatalytic sites, and a model to explain the specificity of these sites is shown. These sites appear to play no role in ATP hydrolysis.

Adenosine Triphosphate↗