Conformational analysis of CCK-B agonists using 1H-nmr and restrained molecular dynamics: Comparison of biologically active Boc-Trp-(N-Me)Nle-Asp-Phe-NH2 and inactive Boc-Trp-(N-Me)Phe-Asp-Phe-NH2

The tetrapeptide Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ is a potent CCK-B agonist. Replacement in this analogue of the norleucine residue by a phenylalanine, to yield Boc-Trp-(N-Me)Phe-Asp-Phe-NH₂, led to a 740-fold decrease in affinity whereas the same decrease in affinity was not observed in their nonmethylated counterparts. In order to ascertain the conformational preferences of these two N-methylated tetrapeptides, a study by two-dimensional (2D) nmr spectroscopy and molecular modeling was undertaken. The solution conformation of the two peptides was examined by ¹H-nmr in a d₆-DMSO/H₂O (80 : 20) mixture. A cis-trans equilibrium, induced by N-methylation, was observed for both analogues, and the proton spectra of the two retamers were fully characterized in each case. ¹H-¹H distance constraints, derived from 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy experiments, were used as inputs for subsequent restrained molecular dynamics simulations. Comparisons of the nmr and molecular modeling data point toward distinct conformational preferences for these two peptides with an opposite spatial orientation of the Trp residue, and could explain the large difference in their biological activities. Furthermore, the tridimensional structure of Boc-Trp-(N-Me)Nle-Asp-Phe-NH₂ could serve as a model for the design of nonpeptide CCK-B agonists. © 1994 John Wiley & Sons, Inc.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

Acetate transport in the S3 segment of the rabbit proximal tubule and its effect on intracellular pH

We monitored intracellular pH (pHi) in isolated perfused S3 segments of the rabbit proximal tubule, and studied the effect of acetate (Ac-) transport on pHi. pHi was calculated from the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein trapped intracellularly. All solutions were nominally HCO3(-)-free. Removal of 10 mM Ac- from bath and lumen caused pHi to rapidly rise by approximately 0.2, and then to decline more slowly to a value approximately 0.35 below the initial one. Removal of only luminal Ac- caused pHi changes very similar to those resulting from bilateral removal of Ac-. When Ac- was removed from bath only, pHi rose rapidly at first, and then continued to rise more slowly. Readdition of Ac- to bath caused pHi to rapidly fall to a value slightly higher than the one prevailing before the removal of Ac- from the bath. In experiments in which Ac- was first removed from both bath and lumen, readdition of 10 mM Ac- to only lumen caused a rapid but small acidification, followed by a slower alkalinization that brought the pHi near the value that prevailed before the bilateral removal of Ac-. The alkalinizing effects caused by the readdition of 10 or 0.5 mM Ac- were indistinguishable. When Ac- was returned to only lumen in the absence of luminal Na+, there was a small and rapid pHi decrease, but no pHi recovery. Removal of Na+ from bath did not affect the pHi transients caused by the addition of Ac- to lumen. In experiments in which Ac- was first removed bilaterally, readdition of Ac- to only bath caused a large and sustained drop in pHi, whereas the subsequent removal of Ac- from the bath caused a slight alkalinization. These pHi changes caused by readdition or removal of Ac- from baths were unaffected by the absence of external Na+. We conclude that there is a Na+/Ac- cotransporter at the luminal membrane, and pathways for acetic acid transport at both luminal and basolateral membranes. The net effect of Ac- transport on pHi is to alkalinize the cell as a result of the luminal entry of Na+/Ac-, which is followed by the luminal and basolateral exit of acetic acid.