The pH dependences of reactions catalyzed by the complete proton-translocating transhydrogenase from Rhodospirillum rubrum, and by the complex formed from its recombinant nucleotide-binding domains.

Transhydrogenase couples the translocation of protons across a membrane to the transfer of reducing equivalents between NAD(H) and NADP(H). Using transhydrogenase from Rhodospirillum rubrum we have examined the pH dependences of the 'forward' and 'reverse' reactions, and of the 'cyclic' reaction (NADP(H)-dependent reduction of the analogue, acetyl pyridine adenine dinucleotide, by NADH). In the case of the membrane-bound protein in chromatophores, the imposition of a protonmotive force through the action of the light-driven electron-transport system, stimulated forward transhydrogenation, inhibited reverse transhydrogenation, but had no effect on the cyclic reaction. The differential response at a range of pH values provides evidence that hydride transfer per se is not coupled to proton translocation and supports the view that energy transduction occurs at the level of NADP(H) binding. Chromatophore transhydrogenase and the detergent-dispersed enzyme both have bell-shaped pH dependences for forward and reverse transhydrogenation. The cyclic reaction, however, is rapid at low and neutral pH, and is attenuated only at high pH. A mixture of recombinant purified NAD(H)-binding domain I, and NADP(H)-binding domain III, of R. rubrum transhydrogenase carry out the cyclic reaction with a similar pH profile to that of the complete enzyme, but the forward and reverse reactions were much less pH dependent. The rates of release of NADP+ and of NADPH from isolated domain III were pH independent. The results are consistent with a model for transhydrogenation, in which proton binding from one side of the membrane is consequent upon the binding of NADP+ to the enzyme, and then proton release on the other side of the membrane precedes NADPH release.

The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.

The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli.

Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD+ analogue AcPdAD+ by a combination of NADH and NADPH. At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD+ indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism. High concentrations of either NADH or AcPdAD+ led to substrate inhibition. These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD+ is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP+, in a cyclic process. When this reaction was carried out with [4A-2H]NADH replacing [4A-1H]NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting. In simple 'reverse' transhydrogenation, the reduction of AcPdAD+ was slower with [4B-2H]NADPH than with [4B-1H]NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0. This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP+ release from the enzyme is rate-limiting at low pH. The lack of a deuterium isotope effect in the reduction of thio-NADP+ by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions. A steady-state rate equation is derived for the reduction of AcPdAD+ by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E. coli transhydrogenase described in the literature.

Norfloxacin versus parenteral therapy in the treatment of complicated urinary tract infections and resistant organisms.

As part of a multicenter randomized trial of the treatment of complicated urinary infections in hospitalized patients, we treated 35 patients of whom 28 were evaluable (16 patients given parenteral therapy and 12 norfloxacin). The distribution of pathogens was similar in both groups as was the elimination of the organisms (12/12 norfloxacin and 14/16 parenteral therapy). The only complication of norfloxacin therapy was an episode of anxiety in a patient with obstructive lung disease who had experienced anxiety and difficulty with a variety of other medications. The largest difference between the parenteral and norfloxacin treated groups was in the ease of therapy and in cost, which for the former was a total of $5 091, i.e. $318 per patient and $30.1/day, exclusive of administration costs. The parenteral administration costs averaged $45/day. Analogous costs for norfloxacin were estimated at $3.00/day per patient and administration costs for 2 tablets norfloxacin/day were calculated at $7.5. The variety of drugs used in the parenteral arm included a nephrotoxic aminoglycosides in half the cases and a wide variety of beta-lactams as well as vancomycin. Thus, for simplicity of therapy as well as cost norfloxacin was judged superior to parenteral therapy of hospitalized patients with mild or moderately severe urinary tract infections including patients with underlying urinary tract abnormalities and prior to long term foley catheter use. An additional 15 cases were treated in an open trial or on a compassionate use basis. All but one patient, who was noncompliant, responded, including, several with chronic relapsing infections with antibody coated bacteria in the urine. Three such patients who were followed for 3-6 months maintained urine free of bacteria.

Empirically derived personality types among male and female college students.

Data on the 16 PF obtained from 130 male and female college students were cluster analyzed to produce an empirical personality typology. Two different clustering algorithms were compared. Seven personality types emerged: 1) Well-Adjusted Conservative, 2) Ego-Involved Neurotic, 3) Norm Independent, 4) Socially-Detached Neurotic, 5) Superego Controlled, 6) Self-Assured Experimenter, and 7) Tough-Minded Controlled. Not only did the types differ significantly in personality, but they also were found to be significantly different on nine different measures of interpersonal orientation. Since the types did give intuitive insight into the nature of personality as particular combinations of personality traits and also were different on variables other than those used for the classification, the scientific utility of the typological approach received support.