Cell-based multiwell assays for the detection of substrate accumulation and oxidation.

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping (14)CO(2) produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling.

Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor.

Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and 0.068 microM for the homologous human and mouse apical transporters, respectively. Hybrid human-mouse and mouse-human transporters were engineered to identify regions involved in this 150-fold difference in the inhibition constant for 2164U90. A mouse-human chimera with only the most C-terminal hydrophobic domain and the C-terminus of the transporter originating from the human variant was found to have a sensitivity to 2164U90 inhibition similar to that of the human transporter. Conversely, a human-mouse hybrid transporter encompassing the same C-terminal region from the mouse sequence but now inserted into the human sequence demonstrated the greater inhibition seen with the mouse wild type ASBT. Amino acid substitutions, individually or in combinations, of six candidate nonconserved residues between mouse and human transporters in this C-terminal domain showed replacements of Thr294 by Ser and Val295 by Ile to be responsible for the difference in the sensitivity toward 2164U90 seen between the species. The hamster apical SBAT encompassing Ser/Ile in these positions shared the lower sensitivity to 2164U90, as seen with the human ASBT, even though it is identical to the mouse SBAT in the remaining four positions of this region. In addition, the rat ASBT which is identical to the mouse ASBT in this domain also had the high sensitivity to 2164U90 inhibition found for the mouse ASBT. Methanethiosulfonates (MTS) are known to inactivate the sodium/bile acid transporters through alkylation of a cysteine in the most C-terminal hydrophobic domain (1). Inactivation of the human ASBT due to MTS modification of cysteine 270 was shown to be largely abolished when the transporter was preincubated with 2164U90, suggesting that the binding of this benzothiazepine is in the vicinity of position 270. Thus, the domain containing the two most C-terminal putative transmembrane regions of the SBATs, H8-H9, previously shown to constitute part of the binding pocket for bile acids, interacts also with the bile acid transport competitive inhibitor, 2164U90.

Organization of the membrane domain of the human liver sodium/bile acid cotransporter.

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.

Na(+)/H(+) exchanger NHE3 has 11 membrane spanning domains and a cleaved signal peptide: topology analysis using in vitro transcription/translation.

The transmembrane topology of Na(+)/H(+) exchanger NHE3 has been studied using in vitro transcription/translation of two types of fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NHE3 membrane spanning domains (msds). These vectors encode N-terminal 101 (HKM0) or 139 (HKM1) amino acids of the H,K-ATPase alpha-subunit, a linker region and a reporter sequence containing five N-linked glycosylation consensus sites in the C-terminal 177 amino acids of the H,K-ATPase beta-subunit. The glycosylation status of the reporter sequence was used as a marker for the analysis of signal anchor and stop transfer properties of each putative msd in both the HKM0 and the HKM1 vectors. The linker region of the vectors was replaced by sequences that contain putative msds of NHE3 individually or in pairs. In vitro transcription/translation was performed using [(35)S]methionine in a reticulocyte lysate system +/- microsomes, and the translation products were identified by autoradiography following separation using SDS-PAGE. We propose a revised NHE3 topology model, which contains a cleaved signal peptide followed by 11 msds, including extracellular orientation of the N-terminus and intracellular orientation of the C-terminus. The presence of a cleavable signal peptide in NHE3 was demonstrated by its cleavage from NHE3 during translational processing of full-length and truncated NHE3 in the presence of microsomes. Of 11 putative msds, six (msds 1, 2, 4, 7, 10, and 11) acted as both signal anchor and stop transfer sequences, while five (msds 3, 5, 6, 8, and 9) had signal anchor activities when tested alone. Of the latter, 3, 5, 6, and 9 were shown to act as stop transfer sequences after C-terminal extension. The actual membrane orientation of each sequential transmembrane segment of NHE3 was deduced from the membrane location of the N- and C-termini of NHE3. The regions between putative msds 8 and 9 and between msds 10 and 11, which correspond to the fourth and fifth extracellular loops, did not act as msds when tested alone. However, the extension of the fifth extracellular loop with adjacent putative msds showed some membrane-associated properties suggesting that the fifth extracellular loop might be acting as a "P-loop"-like structure.

Inhibition of the human sodium/bile acid cotransporters by side-specific methanethiosulfonate sulfhydryl reagents: substrate-controlled accessibility of site of inactivation.

Mammalian sodium/bile acid cotransporters (SBATs) constitute a subgroup of the sodium cotransporter superfamily and function in the enterohepatic circulation of bile acids. They are glycoproteins with an exoplasmic N-terminus, seven or nine transmembrane segments, and a cytoplasmic C-terminus. They exhibit no significant homology with other members of the sodium cotransporter family and there is limited structure/function information available for the SBATs. Membrane-impermeant methanethiosulfonates (MTS) inhibited bile acid transport by alkylation of cysteine 270 (apical SBAT)/266 (basolateral SBAT) that is fully conserved among the sodium/bile acid cotransporters. The accessibility of this residue to MTS reagent is regulated by the natural substrates, sodium and bile acid. In experiments with the apical SBAT, sodium alone increases the reactivity with the thiol reagents as compared to sodium-free medium. In contrast, bile acids protect the SBATs from inactivation, although only in the presence of sodium. The inhibition and protection data suggest that cysteine 270/266 lies in a sodium-sensitive region of the SBATs that is implicated in bile acid transport.

Cytostar-T scintillating microplate assay for measurement of sodium-dependent bile acid uptake in transfected HEK-293 cells.

Real-time measurements of bile acid uptake into HEK-293 cell monolayers expressing the human sodium/bile acid cotransporters have been demonstrated using Cytostar-T microplates with an integral scintillating base. In these 96-well microplates, which permits culturing and observation of adherent cell monolayers, uptake of (14)C-labeled glycocholate and taurocholate into transfected HEK-293 cells was time-dependent, sodium-stimulated, and saturable. The sodium-activated uptake of 30 microM [(14)C]glycocholate (GC) via the ileal (IBAT) and liver (LBAT) transporters was 30-40 times higher than GC uptake in a sodium-free background. In addition, ouabain inhibition of the plasma membrane Na(+), K(+)-ATPase, causing the sodium gradient to collapse, resulted in total loss of glycocholate transport. Induction of gene expression by sodium butyrate showed that the amount of labeled bile acid accumulated in the cell monolayers at steady state was a function of the total amount of transporter expressed. Uptake of labeled bile acids was inhibited both by the specific IBAT inhibitor, 2164U90, and by various bile acids. No major difference was observed between IBAT and LBAT in their specificity for the bile acids tested while the dihydroxy bile acids had the highest affinity for both the transporters studied. The Cytostar-T proximity assay has been demonstrated to be an accurate and reproducible method for monitoring specific bile acid transport in transfected mammalian cells and the results are similar to those obtained by traditional methods. We conclude that the technique is an attractive approach to the cellular study of membrane transport of radiolabeled solutes in general and suggest a role in screening and characterization of novel transport inhibitors.

Membrane insertion scanning of the human ileal sodium/bile acid co-transporter.

Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.

In vitro translation analysis of integral membrane proteins.

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H+,K+ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method used vectors containing the N terminal region of the gastric H+,K+ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the beta-subunit of the gastric H+,K+ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 2 Ca2+ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the gastric H+,K+ ATPase provided evidence for only 7 transmembrane segments but coupled with other data established a 10 membrane segment model. The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane alpha-helices.

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.

Oxygen reaction and proton uptake in helix VIII mutants of cytochrome bo3.

The oxygen reaction of wild-type and helix VIII mutants of cytochrome bo3 from Escherichia coli, and the associated proton uptake during this reaction, has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. We have focused on mutations in the transmembrane helix VIII where protonatable residues have been exchanged, and mainly on the inactive mutants (i.e., T352A, T359A, and K362L, -M, and -Q). The kinetics for electron transfer during oxidation for the mutants are similar to the wild-type; two rate constants of 3.2 x 10(4) and 3.4 x 10(3) s-1 (at 1 mM oxygen) are detected. Proton uptake is observed for wild-type as well as for the mutant enzymes, but the mutations within helix VIII have affected the rate of proton uptake; it is significantly accelerated in the mutants. These results show that none of the protonatable residues in helix VIII are required in the reaction between the fully reduced cytochrome bo3 and oxygen. We have also studied electron redistribution after photolysis of CO from the mixed-valence compound; we found three kinetic components for wild-type and the mutants T352A and T359A, but for K362M only the first and third components are observed, with amplitudes that are lower than those for the corresponding components in the wild-type enzyme, suggesting that the characteristics of internal electron transfer in the K362M mutant are different from those of the wild-type enzyme.

Light-induced structural changes in cytochrome c oxidase: implication for the mechanism of electron and proton gating.

We have investigated electrogenic events and absorbance changes following pulsed illumination of partly reduced cytochrome c oxidase in the absence of dioxygen and carbon monoxide (Hallén et al. (1993) FEBS Lett. 318, 134-138). In both types of experiment similar kinetics were observed; a rapid (tau < 0.5 micros) change was followed by relaxations with time constants of approx. 7 micros and 80 micros. Both the time constant and the activation energy of the 80 micros component were, within the experimental error, the same as those of one of the steps in the reduction of dioxygen by reduced cytochrome c oxidase. The absorbance changes showed a rapid haem reduction, followed by reoxidation. They were affected by CN(-) and N(-)3, ligands which bind in the binuclear centre of cytochrome c oxidase; the absorbance changes were quenched by CN(-) and in the presence of N(-)3, the amplitude of the 7 micros component increased whereas that of the 80 micros decreased. Based on these findings, a model is proposed which involves electron transfer from Cu(+)B to Fe(3+)A3, as a response to structural changes upon pulsed illumination. The same structural changes are also suggested to take place in the oxygen reduction. These changes may play an important role in the gating of electrons as well as protons, an obligatory feature of a redox-linked proton pump.

Internal electron transfer in cytochrome c oxidase is coupled to the protonation of a group close to the bimetallic site.

Absorbance changes following CO dissociation by flash photolysis from mixed-valence cytochrome oxidase have been followed in the Soret and alpha regions. Apart from CO dissociation and recombination, three kinetic phases with rate constants in the range 10(5)-10(3) s-1 at pH 7.5 can be resolved in both spectral regions. The slowest one of these phases, which had earlier only been observed in the alpha region, has now been detected in the Soret region by the use of a low CO concentration to slow down the recombination reaction. This phase had been assigned to a structural change, but a kinetic difference spectrum demonstrates that it represents electron transfer from cytochrome a3 to cytochrome a. A kinetic deuterium isotope effect of 2-3 at pH 7.5 suggests that it involves proton transfer as well. The temperature dependence of the reaction gives an Arrhenius activation energy of 42 kJ.mol-1. The reaction is faster at low pH, and the equilibrium is shifted toward cytochrome a as the pH is raised. The rate and equilibrium changes can be described as involving acid-base groups with pKa values of approximately 7.7 and 8.7, respectively. The kinetic results can be simulated on the basis of a model in which one acid-base group interacts with cytochrome a3, so that its pKa drops on oxidation of this center. The group is in proton equilibrium with the solvent via a proton pathway, suggested to be a proton channel. The rate of a shift in the redox equilibrium between the two cytochromes reaches a high limit at low pH, where the channel is saturated with protons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome bo from E. coli does not exhibit the same proton transfer characteristics as the bovine cytochrome c oxidase during oxygen reduction.

The reaction where fully reduced cytochrome bo from E. coli partially reduces dioxygen has been characterized with respect to the kinetics of the associated proton uptake, and with respect to the pH- and D2O-sensitivity of the electron transfer reactions. A monophasic proton uptake with a rate constant of about 8 x 10(3) s-1 and a stoichiometry of 0.8 H+/bo were recorded, using the indicator dye, Cresol red, at pH 8.2. The electron transfer reactions were independent of pH in the range 6.0-9.5 and were not affected by exchanging H2O to D2O as solvent. Comparison of these results with those obtained in an earlier investigation of the bovine cytochrome c oxidase [(1992) Biochemistry 31, 11853-11859], indicates differences between the two oxidases with respect to the role of protons in oxygen reduction and/or the mechanism of proton uptake from the medium.

Light-induced structural changes in cytochrome c oxidase. Measurements of electrogenic events and absorbance changes.

We have investigated flash-induced electrogenic events and absorbance changes in cytochrome c oxidase in the absence of dioxygen and carbon monoxide. Electrogenic events were studied using a Teflon-bound layer of cytochrome c oxidase oriented in a phospholipid monolayer. Absorbance changes were observed exclusively in partly reduced cytochrome c oxidase; the largest changes were found in the one-electron-reduced species. Electrogenic events were detected in all reduction states of the enzyme. Both types of experiments displayed a rapid (< 0.5 microseconds) event followed by a biphasic relaxation. The time constants of the relaxation were 6 +/- 2 microseconds and 70 +/- 10 microseconds in the electrogenicity, and 9 +/- 3 microseconds in the absorbance changes (at approximately 22 degrees C). The kinetic absorbance difference spectrum was consistent with that of reduced minus oxidized haem. The experimental results are discussed in terms of structural changes in the vicinity of cytochrome a3. These changes may play an important role in all studies that involve flash photolysis of cytochrome c oxidase-ligand complexes.

Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.

The narrow proton beam therapy unit at the the Svedberg Laboratory in Uppsala.

The synchrocyclotron at the The Svedberg Laboratory (TSL) in Uppsala is now reconstructed and can presently operate with fixed frequency and proton energies up to 100 MeV. A first treatment room with a narrow proton beam unit for therapy of eye tumours is now in operation. Therapy of eye melanomas started in April, 1989 and during 1989 and 1990, 19 patients were treated with 72 MeV protons. The narrow beam unit provides a fixed horizontal beam and the patient is treated in a seated position. The present paper describes mainly the technical aspects of the unit which so far has been used only for eye melanomas. In the future, modifications of the unit will allow therapy of intracranial targets when higher proton energies are available. In its final form, the proton therapy facility at TSL will harbour a second treatment unit. Here a rotating gantry for 200 MeV protons will provide a broad beam, which will enable treatment of tumours located anywhere in the body.

Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation.

Changes in pH during the reactions of the fully reduced and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, using the pH indicator phenol red. Solubilized enzyme as well as enzyme reconstituted into phospholipid vesicles has been studied. With the solubilized enzyme, a biphasic uptake of one proton from the medium was observed, whereas the reconstituted enzyme gave release of 1.3 protons to the extravesicular medium. It is concluded from these results that a total of two to three protons are taken up during oxidation of the fully reduced enzyme. Kinetic analysis suggests that the proton uptake is initiated by the transfer of the third electron to the oxygen binding site. A reaction scheme that integrates proton transfers and oxygen chemistry is presented.