Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.     

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c reductases

The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.     

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial cytochrome c oxidase and adenosine triphosphate synthase complex.

The interaction between cytochrome c oxidase complex and adenosine triphosphate synthase (F1F0) complex in the purified, dispersed state and embedded in phospholipid vesicles was studied by differential scanning calorimetry and by spin-label electron paramagnetic resonance. The detergent-dispersed cytochrome oxidase and F1F0 complexes undergo endothermic thermodenaturation. However, when these complexes are embedded in phospholipid vesicles, they undergo exothermic thermodenaturation. The energy released is believed to result from the collapse of a strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by the removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both cytochrome oxidase complex and F1F0 was smaller than that of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex. This suggests specific interaction between cytochrome oxidase complex and F1F0 in the membrane. Further evidence for interaction between these two complexes is provided by saturation transfer EPR studies in which the rotational correlation time of spin-labeled cytochrome oxidase increases significantly when the complex is mixed with F1F0 prior to being embedded in phospholipid vesicles. From these results, it is concluded that at least a part of cytochrome oxidase and a part of F1F0 form a supermacromolecular complex in the inner mitochondrial membrane. No such supermacromolecular complex is detected between F1F0 and ubiquinol--cytochrome c reductase.     

Immunochemical study of subunit VI (Mr 13,400) of mitochondrial ubiquinol-cytochrome c reductase.

A preparation containing the Mr 13,400 protein (subunit VI), phospholipid, and ubiquinone was isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea solubilization, calcium phosphate-cellulose column chromatography at different pHs, acetone precipitation, and decanoyl-N-methylglucamide-sodium cholate extraction. The protein in this preparation corresponds to subunit VI of ubiquinol-cytochrome c reductase resolved in the sodium dodecyl sulfate-polyacrylamidce gel electrophoresis system of Sch?gger et al. (1987, FEBS Lett. 21, 161-168) and has the same amino acid sequence as that of the Mr 13,400 protein reported by Wakabayashi et al. (1985, J. Biol. Chem. 260, 337-343). The phospholipid and ubiquinone present in the preparation copurify with but are not intrinsic components of, the Mr 13,400 protein. This preparation has a potency and behavior identical to that of a free phospholipid preparation in restoring activity to delipidated ubiquinol-cytochrome c reductase. Antibodies against Mr 13,400 react only with Mr 13,400 protein and complexes which contain it. They do not inhibit intact, lipid-sufficient ubiquinol-cytochrome c reductase. However, when delipidated ubiquinol-cytochrome c reductase is incubated with antibodies prior to reconstitution with phospholipid, a 55% decrease in the restoration activity is observed, indicating that the catalytic site-related epitopes of the Mr 13,400 protein are buried in the phospholipid environment. Antibodies against Mr 13,400 cause an increase of apparent Km for ubiquinol-2 in ubiquinol-cytochrome c reductase. When mitoplasts or submitochondrial particles are exposed to a horseradish peroxidase conjugate of the Fab' fragment of anti-Mr 13,400 antibodies, peroxidase activity is found mainly in the submitochondrial particles preparation; little activity is detected in mitoplasts. This suggests that the Mr 13,400 protein is extruded toward the matrix side of the membrane.     

Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase

Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c1 in the reductase. DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.     

Proton currents and protein motion in membranes

A specific interaction between purified liver transglutaminase and small unilamellar phospholipid vesicles at the lipid phase transition have been revealed. The enzyme-induced perturbation of the bilayer is sufficient for phase transition release of encapsulated carboxyfluorescein from the vesicles. The size of the enzyme-phospholipid recombinants depends upon the protein-phospholipid ratio as shown on Sepharose 4B elution profile. The activity of transglutaminase inserted into the bilayer is greatly reduced. The interaction does not occur when the phospholipid vesicle are in the solid or liquid phase and it requires the structural integrity of the enzyme.     

Hematoporphyrin-promoted photoinactivation of mitochondrial ubiquinol-cytochrome c reductase: selective destruction of the histidine ligands of the iron-sulfur cluster and protective effect of ubiquinone.

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. this photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.     

The nature of the inhibition of 4,7-dioxobenzothiazole derivatives on mitochondrial ubiquinol-cytochrome c reductase

To investigate the inhibitory action and binding site of a quinone-like molecule, 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), a series of 4,7-dioxobenzothiazole derivatives were synthesized and their inhibitory efficiencies studied. Replacing the 6-hydroxyl or 2-hydrogen of UHDBT with a bromo or a methoxy group causes only a slight decrease in inhibitory efficiency, indicating that the 6-hydroxyl or the 2-hydrogen of UHDBT is not a structural requirement for inhibition. 5-Undecyl-6-bromo (or methoxy)-4,7-dioxobenzothiazole shows a pH-dependent inhibition similar to that observed with UHDBT, suggesting that the pH dependence is due to the presence of a dissociable group in the protein complex and not to the deprotonation of the hydroxyl group of the inhibitor. Replacing the 6-hydroxyl group with an azido group causes changes similar to those observed with UHDBT; the inhibition is accompanied by alteration of the epr characteristics of reduced iron-sulfur protein in ubiquinol-cytochrome c reductase. The extent of inhibition is not changed upon illumination of the treated reductase. When the photolyzed, 6-azido-5-(1',2'-[3H] undecyl)-4,7-dioxobenzothiazole [( 3H]6-azido-UDBT)-treated reductase is subjected to organic solvent extraction, no radioactivity is found in the reductase protein. Rather, the radioactivity is located in the phospholipid fraction. A [3H]azido-UDBT-cardiolipin adduct, identified after separation of the phospholipid fraction by high performance liquid chromatography, has 6-azido-UDBT linked to an acyl group, not to the head group of the cardiolipin molecule. These results suggest that inhibition by UHDBT is due to perturbation of specific cardiolipin molecules in ubiquinol-cytochrome c reductase. Since UHDBT and 6-azido-UDBT also inhibit the ubiquinol-cytochrome c reductase activity of delipidated reductase (10% of the original lipid remaining) assayed after reconstitution with ubiquinone and phospholipid, and the [3H]azido-UDBT-cardiolipin adduct is also found in the delipidated reductase, the UHDBT-perturbed cardiolipin molecule is structurally indispensable to reductase and it tightly bound to the reductase protein, most likely the quinone binding proteins.     

Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase

(1) Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. (2) DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c₁ in the reductase. (3) DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. (4) When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. (5) DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. (6) Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. (7) Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.     

Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives

Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.     

Behavior of beef-heart cytochrome c oxidase in reconstituted proteovesicles. A systemtic evaluation of the influence of phospholipid polar headgroup and fatty-acyl side chains

1. Phospholipid-depleted cytochrome c oxidase is incorporated in vesicles, built up of phospholipids of known polar headgroup and fatty-acyl side chains. 2. Maximal reactivation is obtained only when the fatty-acyl side chains provide a fluid environment. 3. Fluid zwitterionic phospholipids are found to be more efficient reactivators than fluid anionic ones. 4. Irrespective of the polar headgroup type, two narrow ranges of activation energies for the enzymatic reaction are calculated from the Arrhenius plots: 81--92 kJ/mol in solid and 51--61 kJ/mol in fluid conditions. 5. Cytochrome c oxidase is also incorporated in a series of vesicles, each built up of an equimolar amount of two phospholipids which differ in their polar headgroup type and/or their fatty-acyl side chain characteristics. From the localization of the enzyme activity profiles, obtained with these mixtures, tentative deductions are made about the preference of cytochrome c oxidase for different phospholipid molecules.     

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

磷脂对琥珀酸-细胞色素c还原酶的作用

琥珀酸细胞色素c还原酶除去90％以上的磷脂后活力丧失约95％。将去脂琥珀酸细胞色素c还原酶与磷脂和辅酶Q_2保温,可恢复其活性。活力恢复程度依赖于磷脂的组成。当磷脂酰胆碱(PC):心磷脂(CL):磷脂酰乙醇胺(PE)=2:2:1时活力恢复最高,比大豆磷脂的效果更为明显,单组分PC,PE或CL恢复活力较差。与酶蛋白紧密结合的CL和PC在活力可逆恢复中有重要作用。     

Activation of isolated NADH:ubiquinone reductase I (complex I) from Escherichia coli by detergent and phospholipids. Recovery of ubiquinone reductase activity and changes in EPR signals of iron-sulfur clusters

NADH:ubiquinone oxidoreductase (NDH-1 or complex I) from Escherichia coli was purified using a combination of anion exchange chromatography and centrifugation in sucrose density gradient. The dependence of enzyme activity on detergent and phospholipids was studied. Artificial hexaammineruthenium reductase activity was not affected by dodecyl maltoside (DDM) and asolectin. Ubiquinone reductase activity had a bell-shape dependence on DDM concentration; 7-10-fold activation could be achieved. Treatment with asolectin subsequently yields additional 2-fold activation with a corresponding increase in the apparent V(max) and without significant changes in apparent K(m). Comparative EPR studies of complex I reduced with NADH, "as prepared" and "activated by asolectin" showed an increase in the signals derived mainly from two [4Fe-4S] clusters in the activated enzyme. One of these signals could be simulated with an axial spectrum with g values of g(xyz)= 1.895, 1.904, 2.05, which corresponds to the parameters reported for the N2 cluster. This data indicates conformational rearrangements of catalytic importance in complex I upon binding of phospholipids.     

Studies on membrane fusion. 1. Interactions of pure phospholipid membranes and the effect of myristic acid,lysolecithin, proteins and dimethylsulfoxide

The interaction and mixing of membrane components in sonicated unilamellar vesicles and also non-sonicated multilamellar vesicles prepared from highly purified phospholipids suspended in NaCl solutions has been examined. Electron microscopy and differential scanning calorimetry were used to characterize the extent and kinetics of mixing of membrane components between different vesicle populations. No appreciable fusion was detected between populations of non-sonicated phospholipid vesicles incubated in aqueous salt (NaCl) solutions. Mixing of vesicle membrane components via diffusion of phospholipid molecules between vesicles was observed in populations of negatively charged phosphatidylglycerol vesicles but similar exchange diffusion was not detected in populations of neutral phosphatidylcholine vesicles. Incubation of sonicated vesicle populations at temperatures close to or above the phospholipid transition temperature resulted in an increase in vesicle size and mixing of vesicle membrane components as determined by a gradual change in the thermotropic properties of the mixed vesicle population. The interaction of purified phospholipid vesicles was also examined in the presence of myristic acid and lysolecithin. Our results indicate that while these agents enhance mixing of vesicle membrane components, in most cases mixing probably proceeds via diffusion of phospholipid molecules rather than by fusion of entire vesicles. Increased mixing of vesicle membrane components was also produced when vesicles were prepared containing a purified hydrophobic protein (myelin proteolipid apoprotein) or were incubated in the presence of dimethylsulfoxide. In these two systems, however, the evidence suggests that mixing of membrane components results from the fusion of entire vesicles.     

Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.     

Lateral diffusion of ubiquinone during electron transfer in phospholipid- and ubiquinone-enriched mitochondrial membranes

After fusion of small unilamellar phospholipid liposomes with mitochondrial inner membranes, the rate of electron transfer between membrane dehydrogenases and cytochrome c decreases as the average distance between integral membrane proteins increases, suggesting that electron transfer is mediated through a diffusional process in the membrane plane (Schneider, H., Lemasters, J. J., H?chli, M., and Hackenbrock, C. R. (1980)., J. Biol. Chem. 255, 3748-3756). The role of ubiquinone in this process was evaluated by fusing liposomes containing ubiquinone-10 or ubiquinone-6, with inner membranes. In control membranes enriched with phospholipid only, ubiquinol-cytochrome c reductase and NADH- and succinate-cytochrome c reductase activities decreased proportionally to the increase in bilayer lipid. These decreases were restored substantially in phospholipid plus ubiquinone-supplemented membranes. The degree to which restoration occurred was dependent upon the length of the isoprenoid side chain of the ubiquinone with the shorter chain length ubiquinone-6, always giving greater restoration than ubiquinone-10. It is concluded that electron transfer between flavin-linked dehydrogenases (Complexes I and II) and cytochrome bc1 (Complex III) occurs by independent, lateral diffusion of ubiquinone as well as independent, lateral diffusion of ubiquinone as well as the protein complexes within the plane of the membrane.     

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.     

Isolation and properties of two classes of low-density vesicles from Saccharomyces cerevisiae.

A mixture of small (0.43-mum diameter) and large (0.62-mum diameter) low-density vesicles from spheroplasts of Saccharomyces cerevisiae was fractionated by rate centrifugation in a gradient of 0 to 8% (wt/vol) Ficoll to yield fractions rich (90 to 95%) in small or large vesicles. The large, but not small, vesicles swelled when diluted into mannitol solutions containing less than 0.4 M mannitol. The pH-electrophoretic mobility curve of the large vesicles showed that they are probably enclosed in a phospholipid-protein membrane. The dyes neutral red and toluidine blue, accumulated into large vesicles by intact cells and spheroplasts, were largely lost from large vesicles when these were separated from stained spheroplasts. Sudan black III stained small and large vesicles, both classes of vesicle retaining the stain on separation. Fractions rich in large vesicles contained proportionately more phospholipid and less free sterols, diacylglycerols, and free fatty acids compared with those enriched in small vesicles. The two classes of vesicles contained about the same proportions of esterified sterols and triacylglycerols. The free fatty acids in both small and large vesicles were free from unsaturated fatty-acyl residues; diacylglycerols and triacylglycerols contained appreciable proportions of unsaturated fatty-acyl residues. Small vesicles were richer in lipase activity, whereas the larger vesicles contained greater beta-glucanase and alpha-mannosidase activities. Phospholipase activity could not be detected in any of the fractions.