Hypothesis. Cytochrome c oxidase as a proton pump. A transition-state mechanism

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.     

Net sulfatide synthesis, galactosylceramide sulfotransferase and arylsulfatase A activity in the developing cerebrum and cerebellum of normal mice and myelin-deficient jimpy mice

Net sulfatide synthesis, galactosylceramide sulfotransferase (EC 2.8.2.11) and arylsulfatase A (EC 3.1.6.1) activities were measured in two brain regions, cerebrum and cerebellum, of normal and jimpy mice during postnatal development. In normally myelinating mice, two phases of increasing rates of net sulfatide synthesis were observed, the first coinciding with oligodendrocyte proliferation and the second with myelination. Net sulfatide synthesis was quantitatively higher in the cerebellum than in the cerebrum. In both brain regions, the developmental patterns of net sulfatide synthesis were related to the activity patterns of both galactosylceramide sulfotransferase and arylsulfatase A. In jimpy mice, a neurological mutant showing hypomyelination in brain, the first phase of net sulfatide synthesis was preserved in both brain regions and galactosylceramide sulfotransferase and arylsulfatase A activities were normal up to 12 days. However, during the phase in which myelination occurred in controls, the net sulfatide synthesis in both brain regions of jimpy mice was zero or even negative. The sulfatide deficit was larger in the cerebellum than in the cerebrum. In both mutant brain parts, galactosylceramide sulfotransferase activity increased up to 12 days showing about 50% of the maximal activities observed in normal brain regions. Thereafter up to 15 days, enzyme activity decreased to about 25% of that of controls and remained low in both brain regions. The developmental patterns and the activities of arylsulfatase A were, however, normal in the cerebrum and cerebellum of jimpy mice. These results suggest that the enzyme activities and the developmental patterns of galactosylceramide sulfotransferase and arylsulfatase A as measured in vitro reflect to a high degree their functional activity in vivo. Furthermore, sulfatide degradation by arylsulfatase A seems to be important in regulating net sulfatide synthesis during normal and impaired myelination.     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

Human annexin V binds to sulfatide: contribution to regulation of blood coagulation

Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and some properties of the glycolipid transfer protein from pig brain

A glycolipid-specific lipid transfer protein has been purified to apparent homogeneity from pig brain post-mitochondrial supernatant. The purified protein was obtained after about 6,000-fold purification at a yield of 19%. Evidence for the homogeneity of the purified protein includes the following: (i) a single band in acidic gel electrophoresis, in sodium dodecyl sulfate-gel electrophoresis, (ii) a single band in analytical gel isoelectric focusing, (iii) exact correspondence between the glycolipid transfer activity and stained protein absorbance in the acidic gel electrophoresis, and (iv) coincidence between the transfer activity and protein absorption at 280 nm in gel filtration through Ultrogel AcA 54. The protein has an isoelectric point of about 8.3 and a molecular weight of 22,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of 15,000 was calculated from AcA 54 gel filtration. The amino acid composition has been determined. The protein binds [3H]galactosylceramide but not [3H]phosphatidylcholine. Under the conditions used, 1 mol of the transfer protein bound about 0.13 mol of [3H]galactosylceramide. The glycolipid transfer protein-[3H]galactosylceramide complex was isolated by a Sephadex G-75 chromatography. An incubation of the complex with liposomes resulted in the transfer of [3H]galactosylceramide from the complex to the acceptor liposomes. The result indicates that the complex functions as an intermediate in the glycolipid transfer reaction. The protein facilitates the transfer of [3H]galactosylceramide from donor liposomes to acceptor liposomes lacking in glycolipid as well as to acceptor liposomes containing galactosylceramide.     

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.     

Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes

Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.     

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.     

Binding of pulmonary surfactant protein A to galactosylceramide and asialo-GM2.

The binding of pulmonary surfactant protein A (SP-A) to glycolipids was examined in the present study. The direct binding of SP-A on a thin-layer chromatogram was visualized using 125I-SP-A as a probe. 125I-SP-A bound to galactosylceramide and asialo-GM2, but failed to exhibit significant binding to GM1, GM2, asialo-GM1, sulfatide, and Forssman antigen. The study of 125I-SP-A binding to glycolipids coated onto microtiter wells also revealed that SP-A bound to galactosylceramide and asialo-GM2. SP-A bound to galactosylceramides with non-hydroxy or hydroxy fatty acids, but showed no binding to either glucosylceramide or galactosylsphingosine. Excess native SP-A competed with 125I-SP-A for the binding to asialo-GM2 and galactosylceramide. Specific antibody to rat SP-A inhibited 125I-SP-A binding to glycolipids. In spite of chelation of Ca2+ with EDTA or EGTA, SP-A retained a significant binding to glycolipids. Inclusion of excess monosaccharides in the binding buffer reduced the glycolipid binding of SP-A, but failed to achieve complete abolishment. The oligosaccharide isolated from asialo-GM2 is also effective at reducing 125I-SP-A binding to the solid-phase asialo-GM2. From these data, we conclude that SP-A binds to galactosylceramide and asialo-GM2, and that both saccharide and ceramide moieties in the glycolipid molecule are important for the binding of SP-A to glycolipids.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.     

Unusual partitioning behavior of CaATPase in dipalmitoylphosphatidylethanolamine/dielaidoylphosphatidylcholine++ + mixtures.

CaATPase from rabbit sarcoplasmic reticulum has been isolated, purified, stripped of its native lipids, and reconstituted into binary lipid mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoylphosphatidylethanolamine (DPPE) or acyl-chain perdeuterated DPPE (DPPE-d62). The partitioning properties of the protein were determined from differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. Acyl-chain perdeuteration allows the separate determination of the order and melting characteristics of each lipid species with FT-IR. The binary lipid mixture has been shown to be phase separated in the gel state (Brauner, J. W., and R. Mendelsohn, 1986, Biochim. Biophys. Acta, 861:16-24). The solid phases present at low temperatures correspond to a pure DEPC phase and a mixed phase of DEPC/DPPE-d62. Insertion of protein at 37 degrees C leads to a domain of relatively protein-free DPPE-d62 and a phase containing both lipids plus CaATPase. We suggest that CaATPase selects a fixed composition (60% DEPC, 40% DPPE-d62) for its immediate environment. The composition of the lipid in the immediate vicinity of protein is largely independent of the initial DEPC/DPPE-d62 ratios in the reconstitution protocol. The relevance of these results to observations of discrete domains in native membranes is discussed.     

Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP

(Na+ + K+)-ATPase from shark rectal glands reconstituted into lipid vesicles and oriented inside out catalyses an ouabain-sensitive Na+-Na+ exchange in the absence of intravesicular K+ when ATP is added extravesicularly. Intravesicular ouabain inhibited the exchange completely. This was also the case with digitoxigenin added to the vesicles. Intravesicular oligomycin inhibited the Na+-Na+ exchange partly in a fashion which was ATP dependent. The exchange is accompanied by a net hydrolysis of ATP with an apparent Km of 2.5 microM. ADP was found to give no stimulation of the Na+-Na+ exchange, contrarily, ADP inhibited the ATP-dependent exchange of Na+ both at optimal and supraoptimal ATP concentrations. When initial influx and efflux of 22Na was measured and the hydrolysis of ATP concomitantly determined a coupling ratio of 2.8:1.3:1 was found, i.e. 2.8 moles of Na+ were taken up (cellular efflux) and 1.3 moles of Na+ extruded (cellular influx) for each mole of ATP hydrolyzed. The electrogenic Na+-Na+ exchange generated a transmembrane potential which was measured with the fluorescent probe ANS (8-anilino-1-naphthalenesulfonic acid) to be 60 mV positive inside the liposomes (extracellular).     

Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.     

Interaction of benzene with bilayers. Thermal and structural studies

The thermal and structural properties of saturated phosphatidylcholine liposomes are significantly altered by benzene. Upon the addition of benzene, the liposomes first swell and then disperse into small multilamellar vesicles. At 20 degrees C these vesicles contain striations or ripples in the plane of the bilayer. Major changes in the thermal behavior of DSPC-benzene liposomes occur near mole ratios of 2:1 and 1:1. At a 2:1 mole ratio, the area under the main endothermic peak, delta Hm, essentially disappears; however, the total heat absorbed, delta Hf, remains approximately equal to that of the control. This occurs because for benzene mole fractions 0.12 less than x less than 0.50, benzene increases the apparent molar heat capacity, Cp, of the gel phase to about 1.2 kcal/(mol . deg). We interpret this increase in heat capacity to be due to an increase in the concentration of defects (or disorder) in the gel phase. At mole fractions of benzene between 0.5 and 0.9, the transition temperature decreases by 20-30 degrees C, and broad, multiple transitions are observed. From 0.5 less than or equal to x less than or equal to 0.9, the apparent molar heat capacity of the liquid-crystal phase increases to that of the defected rippled gel phase. The value of delta Hf approaches the heat of fusion for 2 mol of n-octadecane, suggesting that benzene uncouples the liquid-crystalline acyl chains. The lipids affected by benzene or "boundary lipids" have higher heat capacity than nonperturbed lipids. The apparent molar specific heat, Cp, of 1,2-distearoyl-sn-glycero-3-phosphorylcholine (and 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine) multilamellar vesicles is 0.20 +/- 0.05 kcal/(mol. deg) in the L beta', P beta, and L alpha phases. Cp fluctuates about this value in all three phases upon repeated phase transitions in the same sample. However, the value of Cp in the P beta (rippled) phase exhibits much greater fluctuations in Cp than that in the L alpha phase. We attribute these fluctuations to crystal packing defects.     

A transport model for the (Na+/K+)ATPase in liposomes including the (Na+/K+)-channel function

(Na+/K+)ATPase liposomes of various degrees of reconstitution are formed by varying the amount of phosphatidylcholine added to the soluble (Na+/K+)ATPase before vesicles are formed by cholate removal. In the presence of ATP, the reconstituted sodium pump effectuates (Na+/K+) antiport. In the absence of ATP, the reconstituted sodium pump forms a (Na+/K+) channel. The stable plateaus formed by (1) the active Na+ transport, (2) the active K+ transport, (3) the 'passive' Na+ flux, and (4) the 'passive' K+ flux are determined in the optimally and the partially reconstituted liposomes. The activities of all four vectorial functions vary in a tightly correlated fashion, suggesting that they are mediated by the same transport-active configuration of (Na+/K+)ATPase. A transport model which includes the active and the passive (Na+/K+) fluxes mediated by the sodium pump in liposomes is outlined.