Cardiolipin is essential for higher proton translocation activity of reconstituted Fo

The Fo membrane domain of FoF1-ATPase complex had been purifiedfrom porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of Fo was about 85% and the sample contained no subunits of F1-ATPase. The purified Fo was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H+ translocation activity of Fo was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of Fo with the order of CL＞PA＞＞PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of Fo vesicles containing CL, which suggested that CL's stimulation of the activity of reconstituted Fo might correlate with its non-bilayer propensity. After Fo was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of Fo vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling Fo at cysteine indicated that CL could induce Fo to a suitable conformation resulting in higher proton translocation activity.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F0

The F_o membrane domain of F_oF₁-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F_o was about 85% and the sample contained no subunits of F₁-ATPase. The purified F_o was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H⁺ translocation activity of F_o was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F_o with the order of CL＞PA＞＞PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F_o vesicles containing CL, which suggested that CL’s stimulation of the activity of reconstituted F_o might correlate with its non-bilayer propensity. After F_o was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F_o vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling F_o at cysteine indicated that CL could induce F_o to a suitable conformation resulting in higher proton translocation activity.     

Cardiolipin is essential for higher proton translocation activity of reconstituted F0

The F₀ membrane domain of F₀F₁-ATPase complex had been purified from porcine heart mitochondria. SDS-PAGE with silver staining indicated that the purity of F₀ was about 85% and the sample contained no subunits of F₁-ATPase. The purified F₀ was reconstituted into liposomes with different phospholipid composition, and the effect of CL (cardiolipin), PA (phosphatidic acid), PI (phosphatidylinositol) and PS (phosphatidylserine) on the H⁺ translocation activity of F₀ was investigated. The results demonstrated that CL, PA and PI could promote the proton translocation of F₀ with the order of CLPA>PI, while PS inhibited it. Meanwhile ADM (adriamycin) severely impaired the proton translocation activity of F₀ vesicles containing CL, which suggested that CL’s stimulation of the activity of reconstituted F₀ might correlate with its non-bilayer propensity. After F₀ was incorporated into the liposomes containing PE (phosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) as well as DEPE (dielaidoylphosphatidylethanolamine), it was found that the proton translocation activity of F₀ vesicles increased with the increasing content of PE or DOPE, which has high propensity of forming non-bilayer structure, but was independent of DEPE. The dynamic quenching of the intrinsic fluorescence of tryptophan by HB (hypocrellin B) as well as fluorescent spectrum of acrylodan labeling F₀ at cysteine indicated that CL could induce F₀ to a suitable conformation resulting in higher proton translocation activity.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Modulation of vitronectin receptor binding by membrane lipid composition.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.     

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.     

Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Characterization of reconstituted Fo from wild-type Escherichia coli and identification of two other fluxes co-purifying with Fo

We purified the ATPase Fo sector from a nonoverexpressing strain of Escherichia coli, reconstituted it into lipid vesicles made of either asolectin or two different mixtures of purified lipids, and measured proton flux through the reconstituted proton channel. We measured single-channel conductances and found that Fo activity depends on both lipids and reconstitution methods. In asolectin vesicles, Fo has a single-channel conductance of about 0.2 fS. Additionally, the relatively impure Fo prepared from cells carrying single-copy ATPase genes allowed us to observe two other fluxes, a nonselective cation leak (C(L)) and a slow H+ flux (Hs). Unlike the Fo flux, these fluxes could not be blocked by the Fo inhibitor DCCD. The C, reduces the total apparent trapped volume inside vesicles and therefore must equilibrate both H+ and K+ in the vesicles that contain it. When reconstituted into bilayers, these Fo preparations displayed a 120 pS cation channel with characteristics consistent with C(L) flux. The Hs conducts only H+ but at a slower rate than the Fo. We were therefore able to: 1) quantitate the single-channel conductance of the Fo, 2) demonstrate that our Fo purification method co-purified other membrane proteins that have ion-conduction properties, and 3) show that certain lipids are necessary for functional reconstitution of Fo.     

猪心线粒体F_o的纯化、重建及其质子转运功能

比较了猪心线粒体FoF1-ATPase膜部分Fo的四种纯化方法 .结果表明,用NaBr从亚线粒体除去FoF1-ATPase的水溶性部分F1-ATPase后,再以C HAPS增溶,并经蔗糖梯度离心,可获得高纯度的Fo.SDS-聚丙烯酰胺凝胶电泳鉴定表明, 纯化的Fo含有b、OSCP（寡霉素敏感授予蛋白）、d、a、e、F6、IF1、A6L和c等9种亚 基.用去污剂稀释法将纯化的Fo在脂质体上重建后,重建Fo表现较高的被动转运质子活 性. 这为在体外深入研究Fo的活性、构象与膜脂的关系,以及Fo与F1-ATPase的组装等提 供了很好的实验模型.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.     

Further study on the role of Mg2+ in lipid-protein interaction in reconstituted porcine heart mitochondrial H+-ATPase

Porcine heart mitochondrial H+-ATPase was reconstituted by cholate dialysis method in liposomes containing neutral (PC, PE), acidic (PG, PI, PA, PS, DPG) or neutral and acidic phospholipids. The Mg2+ effect on the ATPase activity and its sensitivity to oligomycin, ATP-induced delta psi and delta pH formation was observed for the proteoliposomes containing acidic but not neutral phospholipids. Maleimide spin labels with varying arm lengths or bromoacetamide spin probe were used to monitor the conformational difference of H+-ATPase in the Mg2+-containing and Mg2+-'free' samples. A difference in W/S ratio (weakly immobilized/strongly immobilized component in the ESR spectra) could be detected for the F0.F1-containing and F1-depleted, (F0)-containing proteoliposomes, suggesting conformational difference in the F0-F1 complex and F0 portion induced by the Mg2+ effect. A conformational change of the beta-subunits in the F1 portion was also deduced from the ATP-induced fluorescence quenching of aurovertin-complex for Mg2+-containing samples. The results obtained are in favor of our previous assumption that Mg2+ may play its role by altering the physical state of the lipid bilayer, which would induce a conformational change in F0 (buried in the lipid core), which in turn is transmitted to the catalytic F1, resulting in a higher enzyme activity.     

Cerebral Phospholipid Content and Na+, K+-ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+,K+-ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane-bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to less than 50% of control values but returned to near-normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15-45 min). Na+,K+-ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30-240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (-29%, p less than or equal to 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (-51% at 60 min, p less than or equal to 0.05) and remained below preischemic values even after 180 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton translocation by ATPase and bacteriorhodopsin.

Stable membrane proteins and lipids are convenient to study biomembranes. Two stable proton translocating proteins were purified and reconstituted into vesicles capable of proton translocation. One was a thermostable ATPase (TF0-F1) of thermophilic bacterium PS3 and the other was rhodopsin of Halobacterium halobium. TF0-F1 was composed of a proton pump moiety (TF1) and a proton channel moiety (TF0). TF1 was the first membrane ATPase which was crystallized and reconstituted from its five polypeptides. Like TF0 and TF1, the rhodopsin in purple membrane was highly stable against dissociating agents, acids and alkali. Phospholipids of these biomembranes were also stable and contained no unsaturated fatty acyl groups. The molecular species of the phospholipids of PS3 were determined by mass chromatography. Measurements were made of the difference in electrochemical potential of protons (deltamicronH+) across the membrane of the reconstituted vesicles. The deltamicronH+ attained was 312 mV in TF0-F1 vesciles and was 230 mV in the rhodopsin vesicles. To conclude that electron transport components are not necessary for ATP synthesis in energy yielding biomembranes, two experiments were performed: The ATP synthesis was observed i) on acid-base treatment of TF0-F1 vesicles, and ii) on illumination of the rhodopsin-TF0-F1 vesicles.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N'-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30-65% inactivation over a concentration range of 5-50 microM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5 X 10(5) M-1 X min(-1). The correlation between the initial binding of [14C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F0F1-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [14C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K+-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F0F1-ATPase complex.     

Proton translocating ATPase of a thermophilic bacterium. Morphology, subunits, and chemical composition.

1. A stable membrane-bound ATPase [EC 3.6.1.3] (TF0-F1) capable of proton translocation in reconstituted vesicles was purified from the thermophilic bacterium PS3 cultured in medium containing L-[U-14C]amino acids. 2. TF0-F1 was composed of a catalytic moiety (TF1) and a hydrophobic moiety (TF0). TF1 contained 3 polypeptide chains with molecular weights of 56,000, 3 of 53,000, 1 of 32,000, 1 of 15,500, and 1 of 11,000. TF0 contained 1 chain of 19,000, 2 of 13,500, and 5 of 5,400 daltons. TF1 was dissociated into subunits much less readily than F1. 3. TF1 consisted of 95A particles arrayed in hexagonal microcrystals. TF0-F1 consisted of a sphere (TF1) and a stalk plus base (TF0) which was buried in the membrane of the proton translocating vesicles. 4. Vesicles capable of energy transformation were formed when TF1 came in contact with the surface of liposomes containing TF0. On addition of phospholipids, the helix content of TF0 increased 3-fold. The role of F0 in forming channels for protons is discussed. 5. The amino acid compositions of TF0, TF1, and TF0-F1 were compared. TF0 was not hydrophobic, despite its interaction with phospholipids. The phospholipid composition and other properties of the proton translocating vesicles were examined. Vesicles reconstituted from a mixture of phosphatidylethanolamine, phosphatidylgly-cerol, and cardiolipin in the same ratio as in the membranes had the highest activity.     

DOPE、DOPC对重组去脂肌质网Ca~(2+)-ATPase活力和构象的影响

经磷脂酶Ａ２ 去脂的肌质网Ｃａ２ ＋ － ＡＴＰａｓｅ 重组于不同比例的二油酰磷脂酰胆碱（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ,ＤＯＰＣ） 和二油酰磷脂酰乙醇胺（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ,ＤＯＰＥ） 形成脂酶体,研究了不同磷脂环境中Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ 水解和Ｃａ２ ＋ 转运活力。结果表明,ＤＯＰＣ 和ＤＯＰＥ 分别有利于ＡＴＰ 水解和Ｃａ２ ＋ 的转运,ＤＯＰＥ 可以增强Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ水解和Ｃａ２ ＋ 转运之间的偶联效率。利用内源荧光、荧光淬灭及Ｆｏｒｓｔｅｒ 能量转移原理测定Ｃａ２ ＋ －ＡＴＰａｓｅ 相应的构象变化, 发现随着ＤＯＰＥ／ ＤＯＰＣ 比例的改变使Ｃａ２ ＋ － ＡＴＰａｓｅ 构象发生相应的变化。     

On the subunit composition of the Neurospora plasma membrane H+-ATPase

The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small.     

Effects of phospholipids on sphingomyelin hydrolysis induced by intestinal alkaline sphingomyelinase: an in vitro study

Digestion of dietary sphingomyelin (SM) is catalyzed by intestinal alkaline sphingomyelinase (SMase) and may have important implications in colonic tumorigenesis. Previous studies demonstrated that the digestion and absorption of dietary SM was slow and incomplete and that the colon was exposed to SM and its hydrolytic products including ceramide. In the present work, we studied the influences of glycerophospholipids and hydrolytic products of phosphatidylcholine (PC; i.e., lyso-PC, fatty acid, diacylglycerol, and phosphorylcholine) on SM hydrolysis induced by purified rat intestinal alkaline SMase in the presence of 10 mM taurocholate. It was found that various phospholipids including PC, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA) inhibit alkaline SMase activity in a dose-dependent manner, with the degree of inhibition being in the order PA > PS > PI > PC > PE. Similar inhibition was also seen in a buffer of pH 7.4, which is close to the physiologic pH in the middle of the small intestine. When the effects of hydrolytic products of PC were studied, lyso-PC, oleic acid, and 1,2-dioleoyl glycerol also inhibited alkaline SMase activity, whereas phosphorylcholine enhanced SMase activity. However, in the absence of bile salt, acid phospholipids including PA, PS, and PI mildly stimulated alkaline SMase activity whereas PC and PE had no effect. It is concluded that in the presence of bile salts, glycerophospholipids and their hydrolytic products inhibit intestinal alkaline SMase activity. This may contribute to the slow rate of SM digestion in the upper small intestine.