Molecular mechanism of the binding of neuropeptide achatin-I (Gly-D-Phe-Ala-Asp) to large unilamellar vesicles of zwitterionic egg-yolk phosphatidylcholine (EPC) was investigated by means of natural-abundance (13)C and high-resolution (of 0.01 Hz order) (1)H NMR spectroscopy. The binding equilibrium was found to be sensitive to the ionization state of the N-terminal NH(3)(+) group in achatin-I; the de-ionization of NH(3)(+) decreases the bound fraction of the peptide from approximately 15% to nearly none. The electrostatic attraction between the N-terminal positive NH(3)(+) group and the negative PO(4)(-) group in the EPC headgroup plays an important role in controlling the equilibrium. Analysis of the (13)C chemical shifts (delta) of EPC showed that the binding location of the peptide within the bilayer is the polar region between the glycerol and ester groups. The binding caused upfield changes Delta delta of the (13)C resonance for almost all the carbon sites in achatin-I. The changes Delta delta for the ionic Asp at the C-terminus are more than five times as large as those for the other residues. The drastic changes for Asp result from the dehydration of the ionic CO(2)(-) groups, which are strongly hydrated by electrostatic interactions in bulk water. The side-chain conformational equilibria of the aromatic d-Phe and ionic Asp residues were both affected by the binding, and the induced changes in the equilibria appear to reflect the peptide-lipid hydrophobic interactions.

The drug delivery (DD) process of benzene derivatives, n-propylbenzene (PrBe) and benzyl alcohol (BzOH), from water to phospholipid vesicles is first monitored by noninvasive NMR technique. The bilayer interface and interior as delivery sites are unambiguously specified by taking advantage of the site selectivity of NMR. Chemical shift differences of the ring proton signals provide direct evidence for the penetration of the "drugs" into the bilayer within a few minutes. PrBe is deeply penetrated into the hydrophobic chain region of the bilayer core. In contrast, BzOH is preferentially trapped in the interfacial region near the carbonyl group of the phospholipid, with the methylene group oriented toward the inside of the bilayer. The delivery site of BzOH is characterized by the doublet of the ring proton NMR signal, which is ascribed to BzOH delivered into the outer and inner layers of the vesicle. This is also confirmed by 13 C NMR, for the first time applied to specify the delivery site. UV absorption spectra of PrBe and BzOH in vesicles are consistent with the delivery sites determined by NMR. Application of the molecular level study of the DD processes to recent severe problems of endocrine disruptors (EDs) is finally proposed as a basis for the comprehensive understanding of the molecular mechanism of the membrane disrupting action and for the purpose of detoxication and the prevention of ED accumulation.

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The binding maximum of apoA-1 (N) in triolein (TO)-egg yolk phosphatidylcholine (PC) emulsions was 10-fold larger than that in PC large unilamellar vesicles (LUV) of similar size (100 nm) with no significant difference in the affinity. Replacement of the long-chain triglyceride, TO, by medium-chain triglycerides or cholesteryl oleate in emulsion cores significantly decreased the N value. The 13 C NMR chemical shifts of the PC carbonyl carbon at the surface layers indicated that PC polar headgroups are more separated and exposed to water molecules in emulsions than in vesicles. The N values were satisfactorily correlated with the chemical shift, that is, the degree of separation between the carbonyl groups at the surface. Although apoA-1 binding to the PC monolayers of emulsions brings about bending of the surface layers and creates local defects in the hydrocarbon regions in a similar manner as PC LUV, the surface-core interaction seems to fill the defects with the core neutral lipids, compensates for the bending stress, and eventually increases the N value. Dependence of the core effect upon the acyl chain length of triglycerides implied important roles of the acyl chains in the surface-core interaction between PC and triglycerides.

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Drug binding and mobility in fluid lipid bilayer membranes are quantified in situ by using the multinuclear solution NMR combined with the pulsed-field-gradient technique. One-dimensional and pulsed-field-gradient (19)F and (1)H NMR signals of an anticancer drug, 5-fluorouracil (5FU) are analyzed at 283-313 K in the presence of large unilamellar vesicles (LUVs) of egg phosphatidylcholine (EPC) as model cell membranes. The simultaneous observation of the membrane-bound and free 5FU signals enables to quantify in what amount of 5FU is bound to the membrane and how fast 5FU is moving within the membrane in relation to the thermal fluctuation of the soft, fluid environment. It is shown that the mobility of membrane-bound 5FU is slowed down by almost two orders of magnitude and similar to the lipid movement in the membrane, the movement closely related to the intramembrane fluidity. The mobility of 5FU and EPC is, however, not similar at 313 K; the 5FU movement is enhanced in the membrane as a result of the loose binding of 5FU in the lipid matrices. The membrane-bound fraction of 5FU is approximately 0.1 and almost unaltered over the temperature range examined. It is also independent of the 5FU concentration from 2 to 30 mM with respect to the 40-50 mM LUV. The free energy of the 5FU binding is estimated at -4 to -2 kJ/mol, the magnitude always close to the thermal fluctuation, 2.4-2.6 kJ/mol.

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