Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Inhibitory effect of membrane active compounds on the uptake of 14C-alpha-aminoisobutyric acid (AIB) in cultured chick embryo liver cells.

High concentrations of beta-adrenoceptor blocking drugs with membrane active properties and of the membrane active compounds quinidine and lidocaine inhibit the uptake of ∝-aminoisobutyric acid by chick embryo liver cells in culture. Beta-adrenoceptor blockers without membrane active properties were without effect. These results are in accordance with previous findings which showed partial inhibition of incorporation of amino acids into proteins caused by membrane active drugs in this system.     

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.