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An in vitro study of alpha/ delta and alpha/b interaction is done by expressing an isolated alpha subunit in an ATP synthase alpha subunit deletion strain, purifying and using it together with pure delta subunit and pure cytoplasmic portion of b subunit. The achievement of the expression and purification of wild-type and mutant isolated alpha subunit in native functional state is described.

The study is conducted to determine nucleotide binding and structural transitions within the multidrug resistance P-glycoprotein, P-gp by use of electron spin resonance (ESR) spectroscopy using spin-labeled ATP. The result reveals that SL-ATP is an excellent substrate analogue that would allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.

The occluded nucleotide conformation in mouse MDR3 E552A/E1197A P-glycoprotein (Pgp) in respect to its involvement in the catalytic pathway is characterized. Results suggest that small molecules or peptides that interact at the nucleotide-binding domains (NBD) dimer interface might effectively disable Pgp catalysis.

The glutamine-471 and glutamine-1114 residues of P-glycoprotein apparently function as signaling molecules between the catalytic site and the drug-binding site. P-glycoprotein is a drug resistance protein that pumps drugs out of the cell.

Data are provided to show that F(sub 1)-ATPase has two catalytic sites that can form a transition state simultaneously. This was done by combining MgADP with fluoroscandium to form a noncovalent complex at the active site.

Research has been conducted on the ATP synthase F (sub)1 sector's catalytic and noncatalytic nucleotide sites. The characterization of the sites by tryptophan fluorescence technique is described.

Research was conducted on the role of alpha-Arg-376 in the binding properties of the F(sub)1-ATPase catalytic site employing enyme mutants derived from Escherichia coli. Results revealed impaired ATP synthesis and hydrolysis in all the mutants suggesting that alpha-Arg-376 is required for the stabilization of the enzyme's catalytic activity.

The role of Beta-Arg-182 in Escherichia coli F1-ATPase is investigated by characterizing two mutants, BetaR182Q and BetaR182K. Results reveal that Beta-Arg-182 interacts with the gamma-phosphate of MgATP, has no function interaction with the Beta-phosphate of MgADP and plays a direct role in the stabilization of the catalytic transition state. Results also suggest another role of Beta-Arg-182, which is in the conformational change at the catalytic site Beta-alpha subunit interface.

ATP synthesis in the final stage of oxidative phosphorylation in membranes of chloroplasts, mitochondria and bacteria is catalyzed by ATP synthase, which, in bacteria, can hydrolyze ATP to initiate a transmembrane proton gradient, under some circumstances. A new study investigates the effects of substitution of Trp residues in Eschericia coli F1 at positions surrounding nucleotides in catalytic sites of F1-ATPase and seeks new information about the catalytic site environment and enzyme mechanism.

A study was conducted to investigate catalytic site nucleotide binding and hydrolysis in F1F0-ATP synthase. The dependence of enzymatic activity on substrate concentrate was determined at 23 degrees C in a buffer solution. Enzymes were reconstituted into asolectin proteoliposimes to analyze wild type properties. Results indicated that the strong asymmetric and cooperative binding of catalytic sites depended on the presence of Mg2+.

A study was conducted on the interactions between P-Glycoprotein (Pgp) nucleotide sites using the covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results indicate strong interaction between the two nucleotide sites. The reaction of one site with NBD-Cl severely hinders reaction of reagent with the other site. Therefore, the catalytic cooperativity between the two Pgp nucleotide sites is very strong and mandatory for catalysis.

Research was conducted to examine the Mg2+ coordination in catalytic sites of F1-ATPase. A detailed analysis of the environment of the Mg2+ ion in the catalytic sites of Escherichia coli was performed by generating mutations that removed potential Mg2+-coordinating side chains and then combining them with the beta-Y331W mutation. The elimination of any of the Mg2+-coordinating residues resulted in a complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.

The effect beryllium fluoride on the P-glycoprotein ATPase activity was described through utilization of kinetic assay of Pgp-ATPase activity. Results have shown that P-glycoprotein (Pgp) in plasma membranes was strongly inhibited by beryllium fluoride (BeFx) in the presence of Mg-nucleotide. Furthermore, it was shown that PPi protected Pgp ATPase from inhibition of BeFx through competitive inhibition.

The reaction rate of residue beta-Cys-339 of E. coli F1-ATPase in beta-Ser339Cys mutant F1 with N-ethylmaleimide (NEM) is increased by the presence of MgATP and MgADP but not of MgAMPPNP. The inactivation of beta-S339C F1-ATPase is induced by NEM when MgATP or MgADP exists. An enzyme conformation is induced by MgATP or MgADP when residue beta-Ser-339 is exposed.

A simple high-speed method facilitates the purification of Chinese hamster P-glycoprotein (Pgp) which displays MgATPase activity that is activated by verapamil. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide terminate the activity of MgATPase. The high-speed technique involves chromatography on reactive Red 120 agarose and octylglucoside solubilization of Pgp from plasma membranes of Chinese hamster ovary cell lines.

Fluorescence properties of purified F1 from betaR398C and betaR398W mutant residues of Escherichia coli F1F0-ATP synthase were examined. The fluorescence spectrum of the F1 of betaR398W exhibited a major red-shift compared to those of the wild-type and the other mutant that suggested its position in a polar environment. A specific charged donor-acceptor H-bond between betaR398 and the 7-hydroxyl group of aurovertin was suggested to be involved in aurovertin binding to F1-ATPase. A 23 Angstrom distance separated the aurovertin binding site from the catalytic nucleotide binding site.

The mechanism of Escherichia coli F1-ATPase was studied. It was demonstrated that reversible ATP synthesis/hydrolysis, with high-affinity binding of ATP at a single site and slow release of hydrolysis products, occurs on soluble F1 during unisite catalysis. Rate constants for ATPbinding/release, ATP hydrolysis/resynthesis, Pi release and ADP binding/releasewere measured. The results showed that Pi is not required for unisite catalysisin E. coli F1 and that unisite ATP hydrolysis is pH-independent. In contrast, multisite ATP hydrolysis is pH-dependent in the range pH 5.0-9.5.

The effects of dimethyl sulfoxide (DMSO) on the Escherichia coli F1-ATPase were reported. It was shown that DMSO markedly inhibits multisite catalysis in E. coli F1-ATPase. The results suggest that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. E. coli F1 does not display significant Pi binding either in aqueous buffer or in 40 percent DMSO. Moreover, it was inferred that the deltaG for ATP hydrolysis in 40 percent DMSOis between -9.2 and -16.8 kJ/mol.