Classical molecular dynamics computer simulations have been used to investigate the thermodynamics and kinetics of sodium chloride association in polarizable water. The simulations make use of the three-site polarizable water model of Dang [J. Chem. Phys. 97, 2659 (1992)], which accurately reproduces many bulk water properties. The model’s static dielectric constant and relaxation behavior have been calculated and found to be in reasonable agreement with experimental results. The ion–water interaction potentials have been constructed through fitting to both experimental gas-phase binding enthalpies for small ion–water clusters and to the measured structures and solvation enthalpies of ionic solutions. Structural properties and the potential of mean force for sodium chloride in water have been calculated. In addition, Grote–Hynes theory has been used to predict dynamical features of contact ion-pair dissociation. All of the calculated ionic solution properties have been compared with results from simulations using the extended simple point charge (SPC/E), nonpolarizable water model [J. Phys. Chem. 91, 6296 (1987)]. The dependence on polarizability is found to be small, yet measurable, with the largest effects seen in the solvation structure around the highly polarizable chlorine anion. This work validates the use of some nonpolarizable water models in simulations of many condensed-phase systems of chemical and biochemical interest.

An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos . In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrixpore complex-lamina fraction . These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections . The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock . The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development . In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide . These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.A method for the affinity purification of antibodies from diazotized paper blots ofSDS polyacrylamide gels has recently been published by Olmsted (1) . Although a detailed quantitative assessment of the efficiency of this technique was not presented in her report, Olmsted did note that in order to achieve signals with the affinity-purified IgG that were of comparable intensity to those seen with the unfractionated serum, she eluted antibodies from approximately 10 times the amount ofpaper used to characterize those eluates in the reprobe . This, in conjunction with data showing significant residual signal remaining on the original blot after elution, suggested a relatively poor recovery of affinity-purified IgG. Further limitations of the procedure reported by Olmsted include the selection ofdiazotized paper for the original blotnitrocellulose is easier to use, gives higher resolution, and is hence preferred for routine blotting applications-as well as the use of a radiolabeled protein A probe to detect the first antibody bound to the blot. This detection method necessi-

The crystalline swelling properties and interlayer structure of a cesium montmorillonite clay were investigated using molecular computer simulations. Two classes of dry-clay structures, proposed previously to explain X-ray diffraction and NMR experiments, were identified using Monte Carlo annealing calculations. Hydrated clays with water contents ranging from 0.044 to 0.440 gH 2 O/gclay were investigated using molecular dynamics simulations. Layer spacings calculated as a function of water content were found to be similar to experimental swelling curves, showing a distinct plateau at the monolayer-hydrate spacing. Hydration energies were calculated as a function of water content and expressed in three complementary forms. The immersion energy form was found to be most useful, revealing an apparently global minimum in the swelling-coordinate energy that corresponds to the monolayer hydrate. This is in agreement with experimental measurements and may help clarify the energetic origins of discrete, crystalline swelling processes in clay minerals. For two- and four-layer hydrates, cesium ions readily formed two different types of inner-sphere complexes with the clay surface. Ions associated with negatively charged tetrahedral substitution sites formed exclusively inner-sphere complexes and occupied hexagonal cavities adjacent to the substitutions. Other cesium ions occupied both inner-sphere and outer-sphere configurations with roughly equal probability. The ease with which cesium associates with the clay surface may be responsible for the formation of monolayer hydrates in cesium-substituted clays and for selective binding of cesium to many clay minerals.

Hydrophobic interactions are investigated by molecular dynamics computer simulations of the free energy, entropy, and internal energy of association of two methane molecules in water. Recently we reported a computer simulation calculation of the entropy of association of methane in water [J. Am. Chem. Soc. 114, 5875 (1992)], where entropy drives nonpolar solutes together at short distances. Here this method is compared with two other general methods for the calculation of the entropic contribution to the free energy. The calculated thermodynamic quantities for methane association are in good agreement with available experimental measurements. Solute contact configurations are found to be of greater importance than solvent-separated configurations, in conflict with earlier theoretical and simulation studies of similar systems. In some cases, this conflict may be understood in terms of differences in the assumed, model intermolecular potential energies.

Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.

A Monte Carlo method for grand canonical and grand isoshear ensemble simulations has been used to characterize the free energy, energy, and entropy of clay mineral swelling. The Monte Carlo approach was found to be more efficient at simulating water content fluctuations in the highly constrained clay environment than a previously developed molecular dynamics method. Swelling thermodynamics calculated for Cs-, Na-, and Sr-montmorillonite clays indicate a strong dependence of swelling on the interlayer ion identity, in agreement with various experimental measurements. The Sr clay swells most readily, and both the Na and Sr clays prefer expanded states (two-layer hydrate or greater) when in contact with bulk water. In contrast, swelling is inhibited in the Cs clay. Differences in swelling behavior are traced directly to the tendency of the different ions to hydrate. The swelling free energies are decomposed into their energetic and entropic components, revealing an overall energetic driving force for the swelling phenomena. Entropic effects provide a smaller, mediating role in the swelling processes. The results provide a unique molecular perspective on experimentally well-characterized swelling phenomena.

The dependence of the crystalline swelling and hydration properties of clay minerals on interlayer ion size and charge was investigated using molecular dynamics computer simulations of Na-, Cs-, and Sr-substituted montmorillonites. For all clays studied, layer spacings measured as a function of water content exhibited plateaus at the one-layer hydrate spacing. Calculated immersion energies exhibited minima for integer-layer hydrates up through the three-layer hydrate, with apparent global minima identified with the one-layer hydrate for Cs-montmorillonite and with the two-layer hydrates for the Na-and Sr-montmorillonites. In addition, for Sr-montmorillonite, layer spacings jumped discontinuously between one-layer and two-layer separations and showed a second plateau at the two-layer hydrate spacing. The immersion energy curve for Sr-montmorillonite showed similar discontinuities. These results provide evidence for a constant water content swelling transition between one-layer and two-layer spacings. This transition was further characterized using pressure versus layer-spacing isotherms. The isotherms showed a loop structure with peaks that correlated with the coordination number of the interlayer ion. Integration of the Sr-clay isotherms yielded Gibbs energy curves with minima at both one-layer and two-layer spacings. The results indicate that the mechanism of swelling and hydration depends upon the interlayer ion charge. For monovalent ions studied, swelling and hydration were coincidental, with water occupying all available interlayer volume. In contrast, expansion of Sr-montmorillonite, driven by formation of a first and partial second hydration shell for Sr 2+ , occurred initially without the filling of the interlayer volume. Trapping in metastable expanded states was also observed for Sr-montmorillonite and identified as a possible mechanism for layer-spacing hysteresis.

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of ∼90 kDa. PEPT2 antisera detected a specific broad band of ∼85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).