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Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.

Equilibrium binding studies on the interaction between the anthracycline daunomycin and plasma membrane fractions from daunomycin-sensitive and -resistant murine leukemia P-388 cells are presented. Drug binding constants (KS) are 15,000 and 9800 M-1 for plasma membranes from drug-sensitive and drug-resistant cells, respectively. Drug binding to the membranes is not affected by either (i) thermal denaturation of membrane proteins or (ii) proteolytic treatment with trypsin, thus suggesting that the protein components of the membranes do not have a major role in determining the observed drug binding. Also, fluorescence resonance energy transfer between tryptophan and daunomycin in the membranes indicates that interaction of protein components with the drug should not be responsible for the observed differences in drug binding exhibited by plasma membranes from drug-sensitive and -resistant cells. Plasma membranes from drug-sensitive cells contain more phosphatidylserine and slightly less cholesterol than membranes from drug-resistant cells. Differences in the content of the acidic phospholipid between the two plasma membranes seem to produce a different ionic environment at membrane surface domains, as indicated by titration of a membrane-incorporated, pH-sensitive fluorescence probe. The possible role of membrane lipids in modulating drug binding to the membranes was tested in equilibrium binding studies using model lipid vesicles made from phosphatidylcholine, phosphatidylserine, and cholesterol in different proportions. The presence of phosphatidylserine greatly increases both the affinity and the stoichiometry of daunomycin binding to model lipid vesicles. The similarity between the effects of phosphatidylserine and other negatively charged compounds such as dicetyl phosphate, cardiolipin, or phosphatidic acid suggests that electrostatic interactions are important in the observed binding of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

changes cannot follow from changes in fluidity. More recently, we have shown that nonylphenol, which strongly inhibits ATPase activity, reduces the equilibrium level of phosphorylation of the ATPase by phosphate, again a result which cannot be explained in terms of effects of fluidity (F. Michelangeli, S. Orlowski, P. Champed, J. M. East & A. G. Lee, unpublished work). The idea that the activity of membrane-bound enzymes should be sensitive to membrane fluidity is intrinsically attractive for, after all, we know that enzymes undergo conformational changes and it seems reasonable that the more fluid the environment the easier will be these'changes. Nevertheless, for the (Ca"-Mg'+)-ATPase there is no evidence that the fluidity of the surrounding lipid has any significant effect on activity, while there is evidence that other effects must be important. There seems to be no reason to suppose that the ATPase is in any way unusual in this respect.