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1.
The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.  相似文献   

2.
The electrophoretic mobility of liposomes containing a negatively charged derivative of phosphatidylethanolamine with a large headgroup composed of the hydrophilic polymer polyethylene glycol (PEG-PE) was determined by Doppler electrophoretic light scattering. The results show that this method is improved by the use of measurements at multiple angles to eliminate artifacts and that very small mobilities can be measured. The electrophoretic mobility of liposomes with 5 to 10 mol% PEG-PE is approximately -0.5 mu ms-1/Vcm-1 regardless of PEG-PE content compared with approximately -2 mu ms-1/Vcm-1 for similar liposomes but containing 7.5% phosphatidylglycerol (PG) instead of PEG-PE. Measurements of surface potential by distribution of an anionic fluorescent probe show that the PEG-PE imparts a negative charge identical to that by PG, consistent with the expectation of similar locations of the ionized phosphate responsible for the charge. The reduced mobility imparted by the surface bound PEG is attributed to a mechanism similar to that described for colloidal steric stabilization: hydrodynamic drag moves the hydrodynamic plane of shear, or the hydrodynamic radius, away from the charge-bearing plane, that of the phosphate moities. An extended length of approximately 50 A for the 2,000 molecular weight PEG is estimated from the reduction in electrophoretic mobility.  相似文献   

3.
A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.  相似文献   

4.
1. Cytochrome oxidase was incorporated into preformed liposomes containing phosphatidylserine. When confronted with a mixture of liposomes, some containing phosphatidylserine and some without it, the enzyme was incorporated only into the phosphatidylserine-containing liposomes. 2. The hydrophobic proteins of the oligomycin-sensitive ATPase incubated in the presence of a mixture of liposomes with and without cytochrome oxidase were preferentially incorporated into cytochrome oxidase-containing liposomes. This selectivity was abolished by either cytochrome c or ascorbate. 3. Cytochrome oxidase incubated in the presence of a mixture of liposomes with and without the hydrophobic proteins of the ATPase was preferentially incorporated into liposomes that did not contain the hydrophobic proteins. 4. Cytochrome oxidase and the oligomycin-sensitive ATPase were preferentially incorporated into pure liposomes over bacteriorhodopsin-containing vesicles. 5. Reduced coenzyme Q (QH2)-cytochrome c reductase was incorporated randomly when incubated in the presence of a mixture of pure liposomes and liposomes containing the hydrophobic proteins of the ATPase complex. 6. The significance of the incorporation procedure as a model for membrane biogenesis is discussed.  相似文献   

5.
Electrokinetic measurements are carried out in suspensions of liposomes made from mixtures of charged (cardiolipin, CL) and neutral (phosphatidylcholine, PC) lipids in the presence of lysine and lysine-based polypeptides. Neither monolysine nor polylysines adsorbed on neutral (PC) membranes. In the case of negatively charged membranes (CL/PC) all polypeptides showed a sharp dependence of liposome electrophoretic mobility on the amount of polymer added to the cell. In suspension of cardiolipin liposomes the position of zero charge point coincided for all high-molecular polylysines; thus, pentalysine neutralizes the membrane surface, whereas polycations with a higher polymerization degree change a sign of the surface charge. Electrophoretic mobility of liposomes in plateau range depended on the molecular weight of polylysines and composition of liposomes; for large macromolecules the absolute value came close to its value for the initial liposomes. Adsorption of polycations on planar bilayer lipid membranes (BLM) resulted in alteration of the boundary potential measured by the method of intramembranous field compensation (IFC). The electrokinetic measurements and IFC method gave close results in the case of lysine monomers; their surface concentration could be fitted by an isotherm of the molecule distribution between the membrane surface and solution. Considerable differences of the surface and boundary potentials found in the case of pentalysine, correspond to changes in the dipole component of boundary potential induced by the adsorbed molecules. Using the IFC method, the kinetics of the adsorption process before saturation was studied. The adsorption of polylysines was markedly slower (more than hour) than that of pentalysine (tens of min) or monolysine (minutes). Washout experiments showed that adsorption of penta-and monolysine on planar BLM was reversible, while that of high-molecular polylysines was practically irreversible.  相似文献   

6.
Effect of sterol incorporation on head group separation in liposomes   总被引:1,自引:0,他引:1  
Electrophoretic mobilities of multilamellar liposomes of varying composition have been measured to determine the effect of incorporated sterols on surface charge density. Liposomes made from mixtures of zwitterionic egg phosphatidylcholine (PC) and anionic egg phosphatidylglycerol (PG) in varying proportions were shown to have electrophoretic mobilities consistent with the anticipated surface charge density. Incorporation of cholesterol up to 50 mole per cent in the bilayer produced no detectable change in surface charge density. Similar results were obtained for lanosterol and epicoprostanol. These results are interpreted to mean that incorporation of the sterols into the bilayers produced no detectable change (less than 3%) in the spacing of charged phospholipids. It is inferred that sterols are incorporated among the fatty acyl chains of these phospholipid bilayers with little or no displacement of the head groups at the surface.  相似文献   

7.
《BBA》1972,275(3):485-490
Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB), a penetrating anion probe, was used.

The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB uptake by liposomes. The PCB response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.

Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F1and Fc, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.

The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).  相似文献   


8.
S J Comiskey  T D Heath 《Biochemistry》1990,29(15):3626-3631
An enzyme inhibition assay was developed to determine methotrexate-gamma-aspartate leakage from liposomes at lipid concentrations as low as 43 nM phospholipid. When negatively charged liposomes prepared with phosphatidylglycerol/cholesterol 67:33 or phosphatidylinositol/cholesterol 67:33 were incubated in 10% (v/v) newborn calf serum, they leaked over 90% of their contents in 2 min. In contrast, liposomes prepared from phosphatidylcholine/cholesterol 67:33 leaked 18% of their contents under the same conditions. The amount of negative charge required to induce liposome leakage was determined by preparing liposomes with varying amounts of phosphatidylglycerol and phosphatidylcholine. Extensive leakage was observed only from liposomes prepared with greater than 50 mol of phosphatidylglycerol per 100 mol of phospholipid. The effect of the phase transition temperature on leakage of negatively charged liposomes in 10% (v/v) serum was investigated by using a series of phosphatidylglycerols with varying acyl chain lengths. Liposomes prepared from distearoylphosphatidylglycerol or dipalmitoylphosphatidylglycerol leaked less than 18% of their contents in 10% serum, whereas liposomes prepared with dilauroylphosphatidylglycerol or unsaturated lipids leaked more than 70% of their contents. Lipoprotein removal from serum followed by treatment with lipid to remove residual apoproteins reduced the leakage from phosphatidylglycerol liposomes in 10% serum. Phosphatidylglycerol liposomes leaked 73% in the presence of human low-density lipoproteins, but only 29% in the presence of bovine apolipoprotein A-I, and 25% in the presence of human high-density lipoproteins. Phosphatidylglycerol/cholesterol and phosphatidylserine/cholesterol liposomes leaked 67% in 4 mg/mL bovine serum albumin purified by cold ethanol extraction. The leakage of liposomes in albumin solutions could be substantially reduced by treating the albumin with lipid.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.  相似文献   

10.
The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extemely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Cholesterol was shown to suppress the polymyxin-induced response in liposomes.  相似文献   

11.
Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.  相似文献   

12.
1. A new method for the isolation of the oliogomycin-sensitive ATPase from beef-heart mitochondria is described. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 mumol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 mumol for the oligomycin-insensitive complex. 6. The preparation of the oligomycin-sensitive complex contains at least 6--7 polypeptides in addition to those derived from F1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. Some of these polypeptides have been identified and isolated.  相似文献   

13.
It was found that mitochondrial oligomycin-sensitive ATPase (OS-ATPase) possesses the esterase activity with respect to some carboxylic acid esters with phenols and arylalcane alcohols. The substrate specificity of the esterase found was studied. The effects of some inhibitors and activators of ATPase on the enzyme activity were demonstrated. It was found that ADP inhibits the enzyme from submitochondrial particles containing factor F1 and does not inhibit the enzyme from the particles devoid of this factor. The data obtained suggest that esterase is localized in the hydrophobic part of the oligomycin-sensitive ATPase complex and are indicative of the functional interrelationship between the esterase and ATPase activities.  相似文献   

14.
In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.  相似文献   

15.
We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.  相似文献   

16.
The highly-purified, oligomycin-sensitive mitochondrial adenosine triphosphatase has been reconstituted with phosphatidylserine. Treatment of the phosphatidylserine-reconstituted ATPase with phosphatidylserine decarboxylase produced a 3-fold decrease in the specific activity of the resulting phosphatidylethanolamine-enriched ATPase complex. Subsequent control experiments indicated that the resulting phosphatidylethanolamine was responsible for the lowered ATPase specific activity. These observations indicate that acidic phospholips do more than facilitate an interaction between the highly-purified, lipid-depleted ATPase and phospholipid. The negatively charged phospholipid appears to be essential for maintaining high levels of oligomycin-sensitive activity even after the initial interaction between phospholipid and the ATPase complex has occurred.  相似文献   

17.
1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.  相似文献   

18.
A D Retzios  D R Thatcher 《Biochimie》1979,61(5-6):701-704
The amino acid substitution responsible for the different electrophoretic mobility of the ADHs alleloenzyme and the ADHf alleloenzyme of the alcohol dehydrogenase from a Nigerian population of Drosophila melanogaster has been established as lysine (ADHs) for threonine (ADHf). This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distinguishing many alleloenzymes which have identical isoelectric points without recourse to explanations involving conformational variability.  相似文献   

19.
The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography–mass spectrometry analysis of peptide–lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH 7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3 h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.  相似文献   

20.
The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extremely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Chlolesterol was shown to suppress the polymyxin-induced response in liposomes.  相似文献   

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