The uptake of ganglioside analogues by a permanent mouse fibroblast cell line has been studied by radio-tracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogues of GM1, GM2, and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9-, or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and was shown to consist of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogues at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile, and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogues, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analogue taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogues.
Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GDt,. Micelles of ['H]ganglioside G D l a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside G M 1 , is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GDl, indicate that the ganglioside uptake by microsoinal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidasc. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GDla does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5 -7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent K , value and an increase in the V,,, value. The V,,, value was also enhanced by freezing and thawing of the microsomal membranes.General anesthetics as well as the series of primary alcohols stimulate the degradation of exogenous ganglioside GDI, by membrane-bound sialidase [l-41. For this system and for the hydrolysis of exogenous sphingomyelin by membrane-bound neutral sphingomyelinase [5] it was demonstrated that the stimulation of the overall enzymic reaction was accompanied by an increase of 'fluidity of the hydrophobic membrane phase' as measured by fluorescence depolarization. Kinetic studies [3,4] showed that the degradation of exogenous ganglioside Gol a by membrane-bound sialidase occurred at the surface of the membrane. However, two important questions remained open. Firstly does membrane-bound sialidase react with Go a micelles adsorbed to the membrane surface or only with GD1, molecules inserted into the membranes or with both of them? Secondly, which of these processes (adsorption, insertion, enzymic degradation) is stimulated by general anesthetics and membraneperturbing agents like n-hexanol?We have therefore studied under various conditions the enzymic degradation o...
Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane-bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20-fold. Melittin in concentrations as high as 100 microM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxyl derivative of SPM, respectively. In the concentration range 100--1000 microM, melittin was observed to rigidify SYM. The incorporation of SPM derivatives into the lipid bilayer of SYM was demonstrated by ESR measurements. Melittin enhanced the uptake of SMP-derivatives into SYM.
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