Efflux pump (e.g., P-gp, MRP1, and BCRP) inhibition has been recognized as a strategy to overcome multi-drug resistance and improve drug bioavailability. Besides small-molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and Vitamin E TPGS (TPGS 1000) are known to modulate efflux pump activity. Competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase have been proposed as possible mechanisms. Focusing on TPGS 1000, the aim of our study was to unravel the inhibitory mechanism by comparing the results of inhibition experiments in a Caco-2 transport assay with data from electron spin resonance (ESR) and from ATPase activity studies. ESR results, on Caco-2 cells using 5-doxyl stearic acid (5-SA) as a spin probe, ruled out cell membrane fluidization as a major contributor; change of membrane fluidity was only observed at surfactant concentrations 100 times higher than those needed to achieve full efflux inhibition. Concurrently, TPGS 1000 inhibited substrate induced ATPase activity without inducing significant ATPase activity on its own. By investigating TPGS analogues that varied by their PEG chain length, and/or possessed a modified hydrophobic core, transport studies revealed that modulation of ATPase activity correlated with inhibitory potential for P-gp mediated efflux. Hence, these results indicate that ATPase inhibition is an essential factor in the inhibitory mechanism of TPGS 1000 on cellular efflux pumps.

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The glycyl radical (Gly-734) contained in the active form of pyruvate formate-lyase (PFL) of Escherichia coli is generated by the S-adenosylmethionine-dependent pyruvate formate-lyase-activating enzyme (PFL activase). A 5-deoxyadenosyl radical intermediate produced by the activase has been suggested as the species that abstracts the pro-S hydrogen of the glycine 734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V., and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we have worked out a convenient large scale preparation of functionally competent PFL activase from its apoform. The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. Cys 3Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. Gel filtration chromatography showed a monomeric structure (28 kDa) for both the apoenzyme and holoenzyme form. The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S] ؉ ) from axial to rhombic symmetry.Pyruvate formate-lyase (PFL) 1 is a key enzyme of the anaerobic glucose fermentation in Escherichia coli and other microorganisms, catalyzing the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate (1). In its active form, this enzyme contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center (Cys-418/Cys-419), where a thiyl radical initiates the homolytic cleavage of the pyruvate C-C bond (3, 4).The PFL radical is produced post-translationally by abstraction of the H si atom from the Gly-734 methylene group (5). This occurs by the action of pyruvate formate-lyase-activating enzyme (PFL activase), which employs adenosylmethionine (AdoMet) and dihydroflavodoxin (or artificial 1eϪ donors) as co-substrates, yielding 5Ј-deoxyadenosine and methionine as stoichiometric co-products (Equation 1).Because the abstracted H atom is recovered in the 5Ј-CH 3 of deoxyadenosine, a 5Ј-deoxyadenosyl radical intermediate has been proposed as the actual H abstracting species in this system. How this nucleoside radical is generated from AdoMet is an intriguing problem, requiring in the first place PFL activase to be fully characterized at the molecular level. Previous work has identified PFL activase as a monomer of 28 kDa, and its primary structure, as deduced from the DNAnucleotide sequence (246 amino acids), has been established by N-and C-terminal amino acid sequencing (6, 7). The enzyme has long since been recognized to require Fe 2ϩ for catalytic activity (6), and iron contents in the order of one iron/polypeptide chain were determined in purified prepara...

Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix-assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of < 112 kDa. The metal content was determined by X-ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)±Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal-binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high-spin Fe(III) in a rhombic environment. The Tyr±Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pK a of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.Keywords: EPR; Ipomoea batatas; metalloenzyme; purple acid phosphatase; X-ray fluorescence.Purple acid phosphatases (PAPs) with a Fe(III)±Me(II) center in their active site catalyze the hydrolysis of activated phosphoric acid esters and phosphoric acid anhydrides in the pH range 4±7. These metalloenzymes are resistant to inhibition by tartrate and show a characteristic Tyr±Fe(III) charge transfer (CT) band at <560 nm responsible for the characteristic purple color of the enzyme (for review see Refs 1±4).Mammalian PAPs or tartrate-resistant acid phosphatases (TRAPs) are monomeric glycoproteins with a molecular mass of <35 kDa containing a Fe(III)±Fe(II) unit in the active site. Recently it has been shown that the residual activity of oxidized bovine spleen purple acid phosphatase (bsPAP) originates from an`impurity' of bsPAP with iron at the ferric site and zinc at the ferrous site (FeZn-bsPAP) in the native form [5].For mammalian acid phosphatases multiple physiological functions, i.e. the degradation of aged erythrocytes [6,7] and active bone resorption [8,9] have been discussed. Although no crystal structure is available for any of these enzymes at present, the dimetal system has been characterized by several spectroscopic methods including Mo Èûbauer spectroscopy [5,10±13] A breakthrough was achieved with the determination of the three-dimensional structure of kbPAP [40,41]. Based on this crystal structure and the amino acid sequence alignment, it has been shown that the predicted secondary structure of the mammalian PAP uteroferrin (Uf) is similar in foldin...