Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Interactions of tocopherols and ubiquinones with monolayers of phospholipids.

1. The penetration of alpha-tocopherol and seven of its derivatives, and five compounds in the ubiquinone series, having differing chain lengths, into monolayers at the air/water interface of 11 different synthetic phospholipids and cholesterol was investigated; the properties of mixed monolayers of the tocopherols and of ubiquinones with phospholipids were also studied. 2. Penetration of alpha-tocopherol into diarachidonylglycerylphosphorycholine was approximately constant for molar ratios of tocopherol/phospholipid ranging from 0.4:1.0 to 2.0:1.0. 3. Tocopherols with shorter or longer side chains than alpha-tocopherol had a lesser ability to penetrate monolayers of phospholipid molecules with 16 or more carbon atoms in their acyl chains. 4. All the tocopherols penetrated more readily as unsaturation in the phospholipids was increased, and their penetration into mixed monolayers of phospholipids was greatly facilitated by the presence of relatively small quantities of unsaturated phospholipid molecules. 5. There was relatively little interaction between the tocopherols and cholesterol, or between the ubiquinones and phospholipids. 6. The possible significance of the observed interactions between alpha-tocopherol and polyunsaturated phospholipids is discussed in relation to the biochemical actions of alpha-tocopherol in vivo. 7. It is suggested that fluidity of the lipid bilayer in membranes containing polyunsaturated phospholipids may allow alpha-tocopherol to interact in a dynamic manner with a number of phospholipid molecules.     

Neurotransmitter movements in nerve endings. Influence of substances that modify the interfacial potential

Polysialogangliosides, sulphatides, glycerylmonooleate, unsaturated fatty acids, myelin basic protein and sucrose inhibit the Na+-coupled uptake and induce a Ca2+-dependent release of dopamine from nerve endings. Substances chemically related to those referred to above, such as monosialogangliosides, neutral glycosphingolipids, glycerylmonostearate, saturated fatty acids and albumin, do not show these effects. Mixtures of polysialogangliosides or sulphatides with myelin basic protein or albumin inhibit, to different degrees, the effects of the individual components. The decreased uptake induced by sucrose reverted to control levels upon reduction of the concentration of the perturbing agent. The restoration of the uptake was probably mediated by the Na+-pump reconstituting the transmembrane Na+-gradient necessary for the Na+-coupled cotransport of dopamine. It is suggested that the effects of uptake inhibitor or release inducer agents derive from their ability to decrease the surface potential and modify the molecular organization of phospholipid interfaces which can result in changes of the membrane ionic permeability.     

Cytoplasmatic domain of Na,K-ATPase α-subunit is responsible for the aggregation of the enzyme in proteoliposomes

We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes.     

Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids.

1. The surface potentials of mixed monolayers of synthetic phospholipids with lipids that are fusogenic for hen erythrocytes were investigated. 2. At pH 5.6 and 10, but not at pH2, mixed monolayers of the fusogenic lipid, glycerol mono-oleate, with phosphatidylcholine exhibited negative deviations from the ideality rule in surface potential per molecule which were accompanied by negative deviations in mean molecular area. 3. Interactions of this type were not seen with chemically related but non-fusogenic lipids, nor were they found in mixed monolayers of any of the lipids with phosphatidylethanolamine. 4. Experiments with dihexadecyl phosphate and hexadecyltrimethyl-ammonium indicated that the complete head group of phosphatidylcholine is required for its observed behaviour with fusogenic lipids. 5. Bivalent cations (Ca2+, UO2(2+) or Zn2+) in the subphase at pH 5.6 significantly modified the behaviour of mixed monolayers of fusogenic lipids with phospholipids; there was a parallel perturbing effect of fusogenic lipids on interactions between monolayers of phospholipids and bivalent cations. 6. Possible molecular interactions of fusogenic lipids with membrane phospholipids, and the role of Ca2+, are discussed which may be relevant to cell fusion in erythrocytes induced by low-melting lipids in the presence of Ca2+.     

Large quantities of recombinant PR-5 proteins from the extracellular matrix of tobacco: Rapid production of microbial-recalcitrant proteins

A procedure for the extraction of large quantities of PR-5 proteins that have been recalcitrant to microbial-based expression systems is described. Targeting of the recombinant proteins to the extracellular matrix allowed efficient protein extraction by a vacuum infiltration/centrifugation system. Approximately 1 kg of fresh leaves from transgenic tobacco plants overexpressing either truncated osmotin (Liu et al., 1996) or A9 fromAtriplex nummularia L. (Casas et al., 1991) yielded between 3 and 5 mg of purified proteins that fully retained their antifungal activity. The entire system of overexpression, extraction, and purification could be easily scaled up for the production of several grams of protein.     

Equivalent Aqueous Phase Modulation of Domain Segregation in Myelin Monolayers and Bilayer Vesicles

Purified myelin can be spread as monomolecular films at the air/aqueous interface. These films were visualized by fluorescence and Brewster angle microscopy, showing phase coexistence at low and medium surface pressures (<20-30 mN/m). Beyond this threshold, the film becomes homogeneous or not, depending on the aqueous subphase composition. Pure water as well as sucrose, glycerol, dimethylsulfoxide, and dimethylformamide solutions (20% in water) produced monolayers that become homogeneous at high surface pressures; on the other hand, the presence of salts (NaCl, CaCl₂) in Ringer's and physiological solution leads to phase domain microheterogeneity over the whole compression isotherm. These results show that surface heterogeneity is favored by the ionic milieu. The modulation of the phase-mixing behavior in monolayers is paralleled by the behavior of multilamellar vesicles as determined by small-angle and wide-angle x-ray scattering. The correspondence of the behavior of monolayers and multilayers is achieved only at high surface pressures near the equilibrium adsorption surface pressure; at lower surface pressures, the correspondence breaks down. The equilibrium surface tension on all subphases corresponds to that of the air/alkane interface (27 mN/m), independently on the surface tension of the clean subphase.     

Thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) prevents internalization of TSH receptor

The thyroid-stimulating hormone (TSH) receptor (TSHr) was made specifically fluorescent by insertion of a tetracysteine motif (TSHr-FlAsH) into the C-terminal end and transiently transfected into COS-7 and HeLa cells. The observation that TSH administration caused the intracellular level of cAMP to increase in both TSHr-FlAsH-transfected cell types indicated that the FlAsH binding motif did not alter normal TSHr functioning. When transfected into HeLa cells and stimulated with TSH, the TSHr-FlAsH receptor exhibited a pronounced perinuclear labelling pattern, whereas labelling remained on the cell surface following pre-incubation with 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). Chinese hamster ovary (CHO)-TSHr cells probed with anti-TSHr antibodies were fluorescent mainly in the proximity of the plasma membrane, with fluorescence being primarily restricted to a juxta-nuclear position when exposed to 10 mU/ml TSH for 1 or 5 min. However, in the presence of DDT, the anti-TSHr fluorescence maintained a peripheral location along the cell plasma membrane, even if CHO-TSHr cells were stimulated with TSH for 1 and 5 min. To verify that DDT acted specifically on the TSHr, CHO cells transfected with the A₂a receptor were used as controls. Following a 1-min stimulation with 5’-(N-ethyl-carboxamido)-adenosine, A₂a receptors were gradually internalized regardless of the presence of DDT in the culture medium. Finally, immunoelectron microscopy of CHO-TSHr cells showed that a 1-min exposure to TSH sufficed to displace anti-TSHr antibodies tagged with 10-nm gold particles into coated pits and vesicles but that their superficial location was retained along the plasma membrane in the presence of DDT.     

Surface behavior,microheterogeneity and adsorption equilibrium of myelin at the air-water interface

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.     

Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline

The first step of proline biosynthesis is catalyzed by gamma-glutamyl kinase (GK). To better understand the feedback inhibition properties of GK, we randomly mutagenized a plasmid carrying tomato tomPRO1 cDNA, which encodes proline-sensitive GK. A pool of mutagenized plasmids was transformed into an Escherichia coli GK mutant, and proline-overproducing derivatives were selected on minimal medium containing the toxic proline analog 3,4-dehydro-dl-proline. Thirty-two mutations that conferred 3,4-dehydro-dl-proline resistance were obtained. Thirteen different single amino acid substitutions were identified at nine different residues. The residues were distributed throughout the N-terminal two-thirds of the polypeptide, but 9 mutations affecting 6 residues were in a cluster of 16 residues. GK assays revealed that these amino acid substitutions caused varying degrees of diminished sensitivity to proline feedback inhibition and also resulted in a range of increased proline accumulation in vivo. GK belongs to a family of amino acid kinases, and a predicted three-dimensional model of this enzyme was constructed on the basis of the crystal structures of three related kinases. In the model, residues that were identified as important for allosteric control were located close to each other, suggesting that they may contribute to the structure of a proline binding site. The putative allosteric binding site partially overlaps the dimerization and substrate binding domains, suggesting that the allosteric regulation of GK may involve a direct structural interaction between the proline binding site and the dimerization and catalytic domains.