A rapid and high-throughput quantitation assay of the nuclear factor κB activity using fluorescence correlation spectroscopy in the setting of clinical laboratories.

BACKGROUND: Transcription factor nuclear factor-κB (NF-κB) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-κB activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-κB activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-κB to fluorescent-labeled DNA probes, which contain NF-κB binding sites. METHODS: Non-fluorescent competitive probes are employed to normalize the influence of the viscosity of the nuclear extracts between samples and to eliminate the influence of nonspecific binding of the fluorescent probes. To confirm accurate quantitation, human recombinant NF-κB p50 was mixed into U937 cell nuclear extracts, and the binding fraction of the fluorescent probes to NF-κB in the mixture was calculated for quantitation. To evaluate whether this method can be applied to measure the NF-κB activity in human lymphocytes, the NF-κB activity levels of systemic inflammatory response syndrome patients during perioperative periods were measured. RESULTS: The percentage recovery was 88.9%. The coefficients of variation of the intra-assay were approximately 10%. NF-κB activity levels during the perioperative period can were successfully measured. The assay time for the FCS measurement was within 20 minutes. CONCLUSIONS: This assay system can be used to quantitate NF-κB activity levels in a timely manner in the setting of hospital laboratories.

Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase.

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q2) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q2 (DQ) -Q2H2 (DQH2)-NAD+ in contrast to the order determined using Q1 (Q1-NADH-NAD(+)-Q1H2) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11-19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q2 and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q1 (one isoprenoid unit) is too short to induce the conformational change.

Effect of pH on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase.

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ-DQH2-NAD+. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH2 formation in the active site and releases of DQH2 and NAD+ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc1 complex. Physiological significances of these results are discussed.

The second coenzyme Q1 binding site of bovine heart NADH: coenzyme Q oxidoreductase.

The rotenone sensitivity of bovine heart NADH: coenzyme Q oxidoreductase (Complex I) depends significantly on coenzyme Q1 concentration. The rotenone-insensitive Complex I reaction in Q1 concentration range above 300 microM indicates an ordered sequential mechanism with Q1 and reduced Q1 (Q1H2) as the initial substrate to bind to the enzyme and the last product to be released from the enzyme product complex, respectively. This is the case in the rotenone-sensitive reaction although both Km and Vmax values of the rotenone-insensitive reaction for Q1 are significantly higher than those of the rotenone-sensitive reaction (Nakashima et al., 2002, J. Bioenerg. Biomemb. 34, 11-19). This rigorous control mechanism between the nucleotide and ubiquinone binding sites strongly suggests that the rotenone-insensitive reaction is also physiologically relevant.

Steady-state kinetics of NADH:coenzyme Q oxidoreductase isolated from bovine heart mitochondria.

Steady-state kinetics of the bovine heart NADH:coenzyme Q oxidoreductase reaction were analyzed in the presence of various concentrations of NADH and coenzyme Q with one isoprenoid unit (Q1). Product inhibitions by NAD+ and reduced coenzyme Q1 were also determined. These results show an ordered sequential mechanism in which the order of substrate binding and product release is Q1-NADH-NAD+-Q1H2. It has been widely accepted that the NADH binding site is likely to be on the top of a large extramembrane portion protruding to the matrix space while the Q1 binding site is near the transmembrane moiety. The rigorous controls for substrate binding and product release are indicative of a strong, long range interaction between NADH and Q1 binding sites.