Interaction of cytoplasmic l-glycerol-3-phosphate dehydrogenase with liposomes

NAD-linked l-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Interaction of dopamine-beta-monooxygenase with chromaffin granule membrane lipids.

The interaction between bovine adrenal medullary dopamine-beta-monooxygenase and liposomes from chromaffin granule membrane lipids as a function of pH, lipid and salt concentration was studied by ultracentrifugation. Efficient adsorption of dopamine-beta-monooxygenase to liposomes occurs in the pH range 5.0-6.5 and at low ionic strength. The adsorption was not detected in the case of apoenzyme. The membrane dopamine-beta-monooxygenase forms a complex with liposomes more effective than soluble does. The data obtained lead to certain conclusions about the specificity of complex between the enzyme and liposomes.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Role of GPI-anchored Enzyme in Liposome Detergent-Resistance

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.     

艾氏乳腺癌腹水细胞质膜NADH-铁氰化钾氧化还原酶质子泵活性诱导细胞与脂质体融合

在本文中,我们用荧光能量共振转移分析和荧光显微技术证明,小鼠艾氏乳腺癌腹水细胞质膜NADH-铁氰化钾氧化还原反应的电子传递所偶联的质子泵活性能诱导细胞与人工脂质体融合。糖酵解代谢的抑制剂碘乙酸能抑制融合,同时融合过程是吸取质子的。近几年来,我们实验室已报道了多种生物膜质子泵均具有诱导膜融合的功能。因此,质子泵诱导膜融合可能具有比较广泛的生理意义。并为细胞中存在有受能量代谢控制的驱动膜融合的生理机制提供了实验证据。     

Various properties of urease encapsulated into liposomes

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.     

Enzymatically activated microencapsulated liposomes can provide pulsatile drug release

A system for the delayed or pulsed release of biologically active substances was achieved by encapsulating liposomes containing the substance of interest inside microcapsules. The microcapsules retain the liposomes but allow controlled diffusion of the active substance when it is released from the liposomes. Furthermore, by coating the liposomes with phospholipase A2 (an enzyme that removes an acyl group from the 2 position of phospholipids) before placing them within the microcapsule, a pulsatile release pattern was achieved both in vitro and in vivo. The time of onset of the pulse as well as the release rate can be controlled by the amount of phospholipase A2, the molecular weight of the poly(L-lysine) that is used to coat the microencapsulated liposomes, and the composition of the phospholipid bilayer membrane. Even at 37 degrees C the system would protect a model enzyme (horseradish peroxidase). When not placed inside the microencapsulated liposomes, the enzyme lost its activity in solution at 37 degrees C in a few days, whereas it retained 40% of the initial activity after 30 days of incubation at 37 degrees C inside the microencapsulated liposomes.     

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.     

Interaction of d-β-hydroxybutyrate dehydrogenase with lecithin vesicles

Rat liver mitochondrial d-β-hydroxybutyrate dehydrogenase has an absolute requirement for lecithin. The nature of the interaction between the enzyme and phospholipid has been investigated. Single bilayer lecithin liposomes of shell-like structure bring about maximal enzyme activation, whereas the interaction with larger vesicles leads to enzyme inactivation. The strong binding of the enzyme to lecithin confers great stability to the enzyme activity as compared with the nonlipid-activated enzyme, and permits the isolation of a lipoprotein complex by chromatography on Sephadex G-200. Only 20% of the proteins solubilized with d-β-hydroxybutyrate dehydrogenase from mitochondrial membranes bind to lecithin liposomes, thus a 5-fold purification of the enzyme is achieved. The liposome-bound proteins had a significantly lower polarity than the remaining 80% of solubilized mitochondrial membrane proteins.     

Reconstitution in liposomes of the electron-transport chain catalyzing fumarate reduction by formate

Fumarate reduction by formate in Vibrio succinogenes is catalyzed by a membrane-bound electron-transport chain, and is coupled with the phosphorylation of ADP. The electron-transport chain was reconstituted in liposomes from the isolated components. The formate dehydrogenase complex (three different peptides), the fumarate reductase complex (three different peptides) and vitamin K-1 were required for the electron transport. The pathway of the electrons from formate to fumarate in the reconstituted chain was identical with that in the bacterial membrane. Each of the active enzyme complexes in the liposomes participated in the electron transport. This was valid for proteoliposomes with ratios of the contents of the two enzyme complexes ranging between 0.1 and 10. This indicates that vitamin K-1 forms a diffusible pool within the liposomal membrane that allows every quinone molecule to react with each molecule of the two enzyme complexes.     

Characterization of a glucosyltransferase activity in liver plasma membrane: modulation by cations and lipidic effectors

1. Glucosyltransferase activity incorporating [14C]glucose from UDP-[14C]glucose onto endogenous lipidic acceptors was localized primarily in the plasma membrane of liver. 2. Incubation of plasma membrane by phosphatidyl-choline liposomes loaded with dolichyl-phosphate stimulated the enzymatic activity. 3. This enzyme required Mg2+ for maximal catalitic activity. Ca2+ could substitute Mg2+. 4. Mn2+ acted as a partial non-competitive inhibitor of the Mg2+-activated glucosyltransferase. 5. This enzyme can be modulated by neutral and acidic phospholipids; the most efficient were phosphatidyl-serine and phosphatidyl-inositol. 6. The enzymatic activity was not significantly changed by cholesterol alone but it is greatly enhanced by liposomes loaded with dolichyl-phosphate and cholesterol.     

Diffusion and Catalysis Processes in Unilamellar Liposomes

Abstract

This research concerns the study of enzyme loaded unilamellar vesicles as bioreactors. The plant enzyme Ascorbic Acid Oxidase (AAO) was entrapped in unilamellar liposomes.

Kinetic study shows that the enzyme activity is largely criptic and that the enzyme in liposomes is partially protected against proteolysis. In order to describe the reactivity of such a system, a kinetic model was developed which accounts both for the enzyme location and for the substrate diffusion across the lipid membrane. For the building of the kinetic model we have used information derived from previous studies of freeze-fracture and label fracture microscopy, which have shown that the protein is located both inside the aqueous core and across the lipid membrane of the vesicles.     

DMPC单分子层膜及细胞色素C氧化酶重组脂质体表面的扫描隧道显微镜观察

本文报导了扫描隧道显微镜对固相磷脂单分子层膜以及重组了细胞色素C氧化酶后的脂质体表面结构的观察.对DMPC单分子层著,得到了有关磷脂头部形态,大小及排态状况等结构信息,达到分子水平分辨;对重组酶后的脂质体表面,也得到了氧化酶分子在膜上的结构信息.     

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.     

Introduction of purified hexosaminidase A into Tay-Sachs leukocytes by means of immunoglobulin-coated liposomes.

To determine whether ligand-receptor interactions could engender the selective uptake by deficient cells of enzyme-laden liposomes, aggregated human IgG was used to coat liposomes which had previously trapped purified hexosaminidase A (Hex A). By a new, high-yield procedure, Hex A was purified 7000-fold from human placenta: the homogeneous protein had a pI of 5.4, permitting nonelectrostatic trapping in the aqueous interstices of anionic multilamellar liposomes (molar ratios of phosphatidyl-choline-dicetyl phosphate-cholesterol, 7:2:1). Trapped Hex A was separated from free enzyme by means of Sephadex G-200 chromatography: 1.3 +/- 0.3 mUnits of Hex A/mumol of phospholipid became associated with liposomes and trapped glucose, utilized as a marker of the aqueous compartment. Once sequestered, the enzyme remained latent until lamellae were disrupted by Triton X-100. Presence of enzyme in aqueous compartments was proved by the demonstration of increased trapping (0.02-1.33 mUnits/mumol of phospholipid) with increments in like-sign repulsion of the bilayers produced by increasing molar ratios of anionic dicetyl phosphate (5-20%). To provide for ligand-receptor interaction with surface Fc receptors of human polymorphonuclear leukocytes (PMN's), liposomes were coated by heat-aggregated (62 degrees C, 10 min) human IgG. PMN's from Tay-Sachs patients genetically deficient in Hex A activity readily incorporated exogenous Hex A provided in this fashion. PMN's exposed to enzyme-laden liposomes coated with aggregated IgG incorporated significantly more Hex A than when the enzyme was presented in uncoated liposomes or in liposomes coated with native IgG, which engages Fc receptors with less avidity. Free enzyme was not endocytized. Acquisition of specific Hex A isozyme activity by cells (determined by DEAE-cellulose chromatography) was not due to surface adsorption since cytochalasin B, which prevents phagocytosis but not surface adherence; blocked uptake. Incorporation of the isozyme by deficient cells was also demonstrated by starch gel electrophoresis, and ultrastructural studies showed that the immunoglobulin-coated, Hex A-containing liposomes were taken up into PMN lysosomes after membrane fusion. The studies indicate that liposomes coated with surface ligands may be used to introduce enzyme or other materials into deficient cells possessing appropriate surface receptors.     

Structural and functional aspects of bacterial membranes and liposomes interactions

Effect of exogenous lipids on the morphology and enzymatic activity of Bacillus cereus B4368 membrane has been studied. Specific character of different lipids action on the membrane enzyme activity was found. This peculiarity had been taken into account by using liposomes in biological systems.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Immobilization of γ-glutamyl transpeptidase,a membrane enzyme,in gel beads via liposome entrapment

Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallstén et al. (Biochim. Biophys. Acta, 982, 47–52, 1989). The gel beads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl transpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme-phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase, γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.