The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta-glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.

We have investigated the phase behavior of dipalmitoylphosphatidylcholine-cholesterol bilayers using both the fluorescence of bilayer-associated 1,6-diphenyl-1,3,5-hexatriene (DPH) and freeze-fracture electron microscopy to elucidate specimen structure. Arrhenius analysis of the fluorescence-derived "microviscosity" parameter reveals temperature-induced structural changes in these membranes. In addition, isotherms of DPH fluorescence anisotropy and total intensity are used to detect alterations in membrane structure with varying cholesterol content. Freeze-fracture electron microscopic studies, utilizing rapid "jet-freezing" techniques, show strikingly different fracture-face morphologies for different combinations of sample cholesterol content and temperature. A phase diagram is proposed that offers a unifying interpretation of the fluorescence and freeze-fracture results. In this interpretation, inflections in temperature-scanning and isothermal fluorescence measurements reveal phase lines in the dipalmitoylphosphatidylcholine-cholesterol membranes Two-phase regions of the proposed phase diagram correspond to samples showing two coexisting fracture-face morphologies, while single-phase regions produce membranes having only one clearly identifiable structure. The proposed phase diagram provides an explanation for several conflicting literature proposals of stoichiometries for phosphatidylcholine-cholesterol complexes in membranes. These stoichiometric complexes correspond to the boundaries of two-phase areas in the gel region of the phase diagram. To better approximate the effect of cholesterol on natural membranes, the structure of egg phosphatidylcholine-cholesterol multilamellar vesicles was also investigated by using DPH fluorescence. The results for this complex natural phospholipid system are interpreted by comparison with the synthetic phospholipid results.

Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-cl), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and three dimensions concomitantly, and its diffusion rate, unlike that of all other redox components, is modulated along with its activity by ionic strength. The diffusion coefficients established in this study reveal that the theoretical diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum (uncoupled) turnover numbers. Since electron transport is slower than the theoretical limit set by the lateral diffusion of the redox components, ordered chains, assemblies, or aggregates of redox components are not necessary to account for electron transport. Rather, mitochondrial electron transport is diffusion coupled, consistent with a "random-collision model" for electron transport.A number of recent studies suggest that the oxidation-reduction (redox) components of the mitochondrial inner membrane are not arranged as an ordered macromolecular assembly or chain but are free to diffuse laterally and independently of one another in the membrane plane (1-5). These components include the dehydrogenase-containing complexes I and II, cytochrome b-cl complex III, cytochrome oxidase complex IV, cytochrome c, and ubiquinone. Implicit in these studies, and consistent with a "random-collision model," the rate of electron transfer between the membrane redox components may be diffusion coupled and, indeed, diffusion controlled (2).It is the purpose of this paper to report the rates of lateral diffusion for the inner membrane redox components as de-termined by fluorescence recovery after photobleaching and to evaluate such diffusion with respect to electron transport.Based on the diffusion coefficients established experimentally by our study, the calculated diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum turnover numbers. Thus electron transfer between all redox components is diffusion coupled in the mitochondrial inner membrane, consistent with a random-collision model (2) (7), and purified mitoplasts (7) were washed in isolation medium diluted 1:7.5 (40 mOsm) to give spherical inner membranes (8), then washed twice with 0.15 M KCI/1 mM Hepes, pH 7.4, to remove native cytochrome c. Calcium-induced fusion of these inner membranes was carried out in a chamber x100 1Lm high formed between a glass coverslip and slide with Scotch double-stick tape shims. Twenty microliters of the spherical membranes (5 mg/ml) was added to the chamber from one side, and 20 ,ul of 10 mM CaC12/5 mM Hepes, pH 6.4, was added from the other side and then incubated at 35C for 7 min. Fusion was stopped by washing the chamber with 0.05 ml of 10 mM potassium phospha...

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A complex forms when bovine P2 protein is added to single-bilayer vesicles created by sonicating myelin lipids. The complex was studied by biochemical analysis, freeze-fracture (FF) and thin-section electron microscopy (EM), and by X-ray diffraction. Smaller amounts of P2 cause the vesicles to aggregate and fuse whereas larger amounts (greater than or equal to 4 wt%) cause multilayers to form. Binding saturates at 15 wt% P2. FF EM shows that large, flat multilayers form within 15 min of addition of P2. Only smooth fracture faces are seen, as expected for a peripheral membrane protein. X-ray diffraction shows a constant repeating distance in the multilayers: 86.0 +/- 0.7 A between the centers of bilayers in the range 4 wt% less than or equal to P2/(P2 + lipid) less than or equal to 15 wt%. Assuming a 53 A-thick bilayer, the space between bilayers is 33 A wide. This is a wider space than for myelin basic protein (MBP) (20-25 A wide). The respective widths are consistent with a compact, globular structure for P2 and a flattened shape for MBP. Calculated electron-density profiles of the lipids with and without P2 reveal the protein largely in the interbilayer spaces, with a small part possibly inserted into the lipid headgroup layers. The different proportions of P2 in the sciatic nerve of various species are tentatively correlated with the different average widths observed by X-ray diffraction for the cytoplasmic space (major period line) between bilayers in the respective sciatic myelins.