Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.     

Effect of temperature on the structure of rat liver microsomes studied by electron spin resonance, fluorescence and activity of enzymes involved in fatty acid biosynthesis

The structural properties of rat liver microsomes were studied by physical and kinetic methods. The microsomes and the lipids extracted from the microsomes were labeled with 16-doxyl-stearic acid- and N-phenyl-1-naphthylamine. The electron spin resonance spectra and the fluorescence intensities were respectively determined at different temperatures from approximately 10 to 40 C. Both methods suggested the absence of a transition temperature indicative of a phase change in the bulk of the lipids of the microsomes in the temperature range studied. The fluidity of the lipid bilayer increased smoothly with the temperature. The Arrhenius plots of the NADH-ferricyanide reductase, NADH-cyt.c reductase, delta 9 desaturase, delta 6 desaturase and palmitic elongation to stearic acid also indicated the absence of a detectable change of phase from crystalline to liquid crystalline in the boundary lipids of these enzymes from 10 C to 40 C. The transference of electrons from the NADH-cyt.b5 reductase to the cyt.b5 is the rate limiting step in the first parts of the electron transport chain. However, the delta 9 desaturase is the rate limiting step of all the series of reactions involved in the delta 9 fatty acid desaturation. Similar conclusions may be extended to the delta 6 desaturation of fatty acids. The physical state of the lipids surrounding the desaturating system would be different from boundary lipids of the cyt.P450 system.     

Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.     

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W.

1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.     

Reduced pyridine nucleotides and cytochrome b5 as electron donors for prostaglandin synthetase reconstituted in dimyristyl phosphatidylcholine vesicles

Prostaglandin synthetase has been reconstituted in dimyristyl phosphatidylcholine vesicles. These vesicles in either 80 or 2 microM flufenamate utilized NADH or NADPH as electron donor in the reductive peroxidative step of prostaglandin H2 formation. Vesicles containing bound cytochrome b5 reductase and cytochrome b5 in order to complete an NADH cytochrome b5 reductase system also utilized reduced cytochrome b5 as the electron donor for this peroxidative step. In systems containing cytochrome and reductase, the rate of NADH oxidation exceeded that of NADPH oxidation, indicating that reduced cytochrome b5 is an effective electron donor for prostaglandin H2 formation, enhancing both the initial rate and the extent of the reaction. The concentrations of reduced pyridine nucleotides and cytochrome b5 employed in these experiments to provide reductants for prostaglandin synthetase are within the concentration ranges that obtain under physiological conditions.     

Investigation of microsomal oxygenases of biosynthetic processes. Stearyl-CoA desaturase of adipose tissue and liver.

Porcine and rat microsomal stearyl-CoA desaturases require reduced pyridine nucleotide and oxygen, are cyanide sensitive, and are insensitive to carbon monoxide. The Km for stearyl-CoA is somewhat larger for liver than for the adipose desaturases, but, in general, assay conditions are quite similar. Adipose tissue microsomes contain cytochromes b5 and P-450, as well as the NADH- and NADPH-specific cytochrome reductases. Compared to liver, the specific contents and activities of electron carriers are much lower in adipose tissue, and activities of 4-methyl sterol oxidase of cholesterol biosynthesis, as well as the cytochrome P-450-dependent aminopyrene demethylase and benzypyrene hydroxylase, are negligible in adipose tissue microsomes. Furthermore, unlike hepatic desaturase, administration of insulin stimulates the adipose desaturase 3-fold without affecting either the amounts or activities of microsomal oxidation-reduction proteins; the changes in desaturase activities produced either by altering dietary fat or by fasting and/or fasting followed by refeeding are, in general, both more extensive and more permanent in adipose compared to liver microsomes. The effects produced by isotopic hydrogen substitution both in stearyl-CoA and in the medium (2H2O) are similar with microsomes from both tissues. The rate-determining step of desaturase appears to be similar in both tissues. The primary isotope effect, k H/Tr, observed with [9,10-3H2]stearyl-CoA is relatively small, 2.88. Since little, if any, primary isotope effect is associated with methyl sterol oxidase, these two mixed function oxidases of biosynthetic processes also appear to share this property in common.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Induction of microsomal stearyl coenzyme A desaturase in the newly hatched chicks.

The development of the stearyl-CoA desaturase system was studied in newly hatched chicks. The desaturation activity was very low in hepatic microsomes from chick embryos, less than 0.05 nmol of oleate formed min^?1 (mg of protein)^?1. After hatching and feeding, the desaturation activity gradually increased to 4–5 nmol of oleate formed min^?1 (mg of protein)^?1 in 6-day-old chicks. This increase could be prevented by administration of cycloheximide or actinomycin D. Measurement of the microsomal electron transfer components throughout the induction period showed no significant changes in the NADH- or NADPH-specific reductases or in the concentrations of cytochromes b₅ and P-450. However, the activity of the terminal component of the desaturase system (the desaturase enzyme) increased in parallel with the desaturation activity. Supplementing the liver microsomes from chick embryos with isolated desaturase enzyme resulted in the formation of an active desaturation system. It is proposed that the induction of the stearyl-CoA desaturase system during development of newly hatched chicks is dependent on the synthesis of the terminal desaturase enzyme.     

Inhibition of the microsomal stearoyl coenyme A desaturation by divalent copper and its chelates

Divalent copper and copper complexes of tyrosine, histidine and lysine inhibited at low concentrations the stearoyl-CoA desaturation reaction in both chicken liver microsomes and in a purified system consisting of chicken liver delta 9 terminal desaturase, cytochrome b5, ascorbate and liposome. Although the copper chelates lowered the steady-state level of ferrocytochrome b5 by 20%, and partially inhibited the NADH-ferricyanide reductase activity, the availability of the ferrocytochrome b5 during the time course of desaturation was not affected, indicating that the site of inhibition of desaturation was at the terminal step, i.e., on the delta 9 terminal desaturase. The presence of chalates during catalysis was essential for the observed inhibition. Based on the observation that O2 is involved in the desaturation and that there is an initial electron reduction of desaturase iron, it is plausible that the copper chelates are inhibiting by acting as superoxide scavengers.     

Regulation of rat hepatic stearoyl coenzyme A desaturase. The roles of insulin and carbohydrate.

The rat hepatic stearoyl-CoA desaturation decreased by 3.7-fold in streptozotocin-induced diabetes. Insulin treatment of diabetic rats increased the enzyme activity by 7-fold. In marked contrast to glucose administration, fructose feeding in diabetic rats resulted in 20-fold stimulation of stearoyl-CoA desaturation, although both carbohydrates stimulated stearoyl-CoA desaturation in normal rats. Measurement of the microsomal electron transfer components showed no significant changes in the NADH-cytochrome b5 reductase activity or in the concentration of cytochrome b5. However, the activity of the terminal desaturase changed in a parallel fashion as the amount of terminal desaturase reflect changes in the overall desaturation. Supplementation of various microsomes with the saturating amount of purified terminal desaturase resulted in the formation of similar amounts of catalytically active complex and increased the stearoyl-CoA desaturation to the same level suggesting that the changes in the amount of terminal desaturase reflect changes in the overall desaturation. The results support the suggestion that both insulin and the intermediates of carbohydrate metabolism are involved in the regulation of terminal desaturase.     

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes.

The involvement of cytochrome b5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or delta-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b5, but those from delta-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b5 and catalyzed the desaturation. The cytochrome b5 content in microsomes from mutant Ole 3 could be varied by changing the delta-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b5 content was increased. The antibody to yeast cytochrome b5, but not the control gamma-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b5 is actually involved in the desaturase system of yeast microsomes. The lack of desaturase activity in mutant Ole 3 grown with Tween 80 plus ergosterol seems to be due to the absence of cytochrome b5 in microsomes, whereas the genetic lesion in mutant E5 appears to be located at ther terminal desaturase.     

Effect of alpha-tocopherol incorporation of glucose permeability and phase transition of lecithin liposomes.

Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.     

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

Sodium ion diffusion through liposome membranes containing cerebroside

Na⁺ efflux from liposomes (small unilamellar vesicles, SUV) of various compositions was studied, using ²²Na⁺ and ³H-labelled stachyose in simultaneous dual isotope measurements, stachyose being used as a measure of liposome disintegration. Dialysis was utilised to separate liposomes from extra-liposomal activity.Liposomes were made from egg lecithin and sphingomyelin and from mixtures of egg lecithin, sphingomyelin, cerebroside, sulphatide and cholesterol. All mixtures produced more leaky and less stable SUVs than pure lecithin and pure sphingomyelin. The incorporation of cerebroside is significantly smaller than that of the phospholipids including sphingomyelin. It was found that membranes containing cerebroside had a significantly higher Na⁺ permeability than membranes without cerebroside.