Muscular tissue oxygenation has a stronger association with postoperative complications and length of hospital stay than cerebral tissue oxygenation after major spine surgery.

Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical ␤␣␤␣␤ dinucleotide binding fold is responsible for binding NADP(H). A model defining ␤A, ␣B, ␤B, ␤D, and ␤E of this domain is presented. To test this model, four single cysteine mutants (cf␤A348C, cf␤A390C, cf␤K424C, and cf␤R425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined.The Membrane-bound transhydrogenase is composed of three domains. In the Escherichia coli enzyme, domain I (ecI, 1 ␣1 to ϳ␣404) and domain III (ecIII, ϳ␤260 to ␤462) are exposed to the cytosol and contain the binding sites for NAD(H) and NADP(H), respectively. Domain II (ϳ␣405 to ␣510 and ␤1 to ϳ␤260) spans the membrane. Domain I (dI) from E. coli (4, 5), Rhodospirillum rubrum (rrI) (6), and bovine (7), and domain III (dIII) from E. coli (5), R. rubrum (8, 9), and bovine (7, 9) have been overexpressed, purified, and partially characterized. So far, domain II has not been expressed as a separate entity. Interestingly, dII is not required for transhydrogenation to occur (decoupled from proton translocation), as initially shown by Yamaguchi and Hatefi (7). Mixtures of recombinant dI and dIII from the same species or from different species catalyze decoupled "forward" and "reverse" reactions (cf. Reaction 1) and the so-called "cyclic reaction" (which involves the reduction of bound NADP ϩ by NADH, followed by the oxidation of bound NADPH by AcPyAD ϩ ) (5,8,9). From a recent study, it was observed that mixtures of rrI plus rrIII and rrI plus ecIII behaved similarly (10). They catalyzed high cyclic reaction rates (about the same as those observed in the complete E. coli and R. rubrum enzymes) that were limited by the transfer of hydride equivalents (10) and slow reverse reaction rates that, with excess rrI under usual assay conditions, were limited by the release of NADP ϩ (5, 8). With this knowledge at hand, it is now possible to use the rrI plus ecIII system to complement mutagenesis experiments performed on the complete enzyme. In addition to substrate binding affinities and hydride equivalent transfer rates, release rates of NADP ϩ and relative affinities between domains are properties that can be studied in mixtures of rrI and ecIII.A three-dimensional model of the NAD(H)-binding site in E.

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.

Background Previous research has linked elevated low-density lipoprotein cholesterol (LDL-C) and remnant cholesterol (RC) with diabetes mellitus (DM). The present study aims to estimate the RC-related DM risk beyond LDL-C, and to investigate the extent to which the association of RC and DM is mediated via insulin resistance and inflammation. Methods We enrolled 7308 individuals without previous history of DM into the present study from the China Health and Nutrition Survey. Fasting RC was calculated as total cholesterol minus LDL-C and high-density lipoprotein cholesterol. Subjects were divided into four groups according to their LDL-C (100 mg/dL) and RC (24 mg/dL) levels to evaluate the role of LDL-C vs. RC on DM. A logistic regression analysis was then employed to evaluate the relationships between the discordant/concordant LDL-C and RC and DM. A mediation analysis was undertaken to identify potential mediators. Results Of all the participants, a total of 625 (8.55%) patients were newly diagnosed with DM. Compared to the high LDL-C/low RC group, the low LDL-C/high RC group was more common in DM patients. After a multivariate adjustment, elevated LDL-C and RC were associated with DM. Moreover, the low LDL-C/high RC group and the high LDL-C/low RC group manifested a 4.04-fold (95% CI 2.93–5.56) and 1.61-fold (95% CI 1.21–2.15) higher risk of DM, relative to those with low LDL-C/low RC. The subgroup analysis indicated that low LDL-C/high RC was more likely to be related to DM in females. Similar results were also shown when the sensitivity analyses were performed with different clinical cut-points of LDL-C. Insulin resistance and inflammation partially mediated the association between RC and DM. Conclusions Our findings provided evidence for RC beyond the LDL-C associations with DM that may be mediated via insulin resistance and the pro-inflammatory state. In addition, women are more susceptible to RC exposure-related DM.