The ribosome inactivating plant proteins (RIPs) ricin and viscumin but not Ricinus communis agglutinin are able induce vesicle-vesicle fusion. A model is suggested in which the toxicity of the RIPs is partially determined by their fusogenicity. Herein, fusion is hypothesized to allow the RIPs to leak across endocytic vesicles to approve their access to cytoplasmic ribosomes.
Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of monocarboxylic acid at one side of BLM were studied by measuring pH changes in the unstirred layers near the BLM surface. The pH changes were assayed by recording protonophore-dependent potentials as well as by direct measurements of pH shifts in he unstirred layers close to the membrane by the pH microelectrode. It was shown that the mechanism of the acid transport changed qualitatively upon the increase of the hydrophobic chain length of the acid. In the case of short-chain acids at pH < pKa, the total transport was limited by diffusion of the anionic form of the acid across the unstirred layers, while at the alkaline pH (pH>>pKa) the transport was limited by diffusion of the neutral form across the membrane. In the alkaline pH range the pH shifts induced by short-chain acids were sensitive to the presence of cholesterol in the BLM as well as to the stirring conditions in the cell. However, in the case of long chain acids (more than 8 carbonic atoms) the transport was limited by diffusion of the anionic form of the acid in the whole range of pH studied. In the latter case, pH changes in the unstirred layers did not depend on the presence of cholesterol in the membrane, and moreover pH shifts were not dependent on the thickness of the unstirred layer. It was proposed that the peculiarities of the long-chain acid-induced proton transport were associated with the formation of micelles of the acid in bathing solutions.
Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of dicarboxylic (DC) acids at one side of BLM are monitored by measuring pH changes in the unstirred layers near the BLM surface via recording protonophore-dependent potentials. Two groups of DC acids are studied: (1) 2-n-alkylmalonic acids with an alkyl chain of different length which carry both carboxylic groups at one terminus of the hydrocarbon chain (alpha,alpha-DC acids); and (2) dicarboxylic acids of different linear chain length having carboxylic groups at the opposite ends of the hydrocarbon chain (alpha,omega-DC acids). It is shown that the pH optimum of hydrogen ion fluxes for the DC acids is shifted considerably to acidic pH values compared to monocarboxylic acids and is located near pH 5. For both types of DC acids at pH&z. Lt;5, the total transport is limited by diffusion of the anionic forms of the acids across the unstirred layers, while at pH&z.Gt;5 the transport is limited by diffusion of the neutral form across the membrane. The fluxes of alpha,alpha-DC acids are similar to those of alpha,omega-DC acids provided that the acids have the similar number of carbon atoms, the fluxes grow with the increase in the chain length of the alkyl radical.
The effects of ribosome-inactivating proteins (RIPs) from Ricinus communis and from Viscum album on the water permeability, Pf, and the surface dielectric constant, epsilon, of model membranes were studied. Pf was calculated from microelectrode measurements of the ion concentration distribution in the immediate vicinity of a planar membrane, and epsilon was obtained from the fluorescence of dansyl phosphatidylethanolamine incorporated into unilamellar vesicles. Pf and epsilon of fully saturated phosphatidylcholine membranes were affected only in the presence of a lectin receptor (monosialoganglioside, GM1) in the bilayer. It is suggested that the membrane area occupied by clustered lectin-receptor complexes is markedly less permeable to water. Protein binding to the receptor was not a prelude for hydrophobic lipid-protein interactions when the membranes were formed from a mixture of natural phospholipids with a high content of unsaturated fatty acids. These membranes, characterized by a high initial water permeability, were found to interact with the RIPs unspecifically. From a decrease of both Pf and epsilon it was concluded that not only water partitioning but also protein adsorption correlates with looser packing of polyunsaturated lipids at the lipid-water interface.
The measurements of pH profiles in the unstirred layers (USLs) near planar bilayer lipid membranes (BLM) were applied for the evaluation of the hydrogen ion fluxes which were induced by nigericin in the presence of potassium ion gradients. It was shown that at high concentrations of KCl the increase in the concentration of citrate buffer caused an anomalous effect, namely, an increase in the local pH shifts in the USLs. The hydrogen ion flux rose 50 times upon the increase in the citrate concentration from 1 mM to 20 mM. Phosphate stimulated the flux 7 times under these conditions. In agreement with our previous results, at low KCl concentrations, when the process is limited by the K + -nigericin interaction, an increase in the buffer concentration led to a reduction of the local pH shifts, under these conditions the usual concentration dependence was observed. The data obtained favor the model implying the existence of the kinetic barrier for proton transfer at the membrane-water interface.z 1998 Federation of European Biochemical Societies.
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