A method which involves the measurement of bioelectrical resistive impedance (R) for the estimation of human body composition is described. This method is based upon the principle that the electrical conductivity of the fat-free tissue mass (FFM) is far greater than that of fat. Determinations of R were made in 37 healthy men aged 28.8 +/- 7.1 yr (mean +/- SD) using an electrical impedance plethysmograph with a four electrode arrangement that introduces a painless signal (800 microA at 50 kHz) into the body. FFM was assessed by hydrodensitometry and ranged from 44.6-98.1 kg. Total body water (TBW) determined by D2O dilution and total body potassium (TBK) from whole body counting were 50.6 +/- 10.3 L and 167.5 +/- 38.1 g, respectively. Test-retest correlation coefficient was 0.99 for a single R measurement and the reliability coefficient for a single R measurement over 5 days was 0.99. Linear relationships were found between R values and FFM (r = -0.86), TBW (r = -0.86), and TBK (r = -0.79). Significant (p less than 0.01) increases in the correlation coefficients were observed when the predictor Ht2/R was regressed against FFM (r = 0.98), TBW (r = 0.95), AND TBK (r = 0.96). These data indicate that the bioelectrical impedance technique is a reliable and valid approach for the estimation of human body composition. This method is safe, noninvasive, provides rapid measurements, requires little operator skill and subject cooperation, and is portable. Further validation of this method is recommended in subjects with abnormal body composition.

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.

We have used a combined approach of NMR spectroscopy and isothermal titration calorimetry (ITC) to determine the ligand-binding mechanism employed by a cocaine-binding aptamer. We found that the length of the stem containing the 3' and 5' termini determines the nature of the binding mechanism. When this stem is six base pairs long, the secondary structure of the aptamer is fully folded in the free form and only putative tertiary interactions form with ligand binding. If this stem is shortened by three base pairs, the free form of the aptamer contains little secondary structure, and ligand binding triggers secondary structure formation and folding. This binding mechanism is supported by both NMR spectral changes and the ITC measured heat capacity of binding (ΔC(p)°). For the aptamer with the long stem the ΔC(p)° value is -557 ± 29 cal mol(-1) K(-1) and for the aptamer with the short stem the ΔC(p)° value is -922 ± 51 cal mol(-1) K(-1). Chemical shift perturbation data and the observation of intermolecular NOEs indicate that the three-way junction is the site of ligand binding.

The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.