Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

Abstract

The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized proteins were incorporated in egg yolk phospholipids. The reconstituted proteoliposomes show proton-driven carriermediated uptake of acidic monosaccharides. The reconstituted carrier was compared in several characteristics with the transporter as present in the native lysosomal membrane. Transporter substrate affinity (Kt for glucuronic acid = 0.4 mM) and specificity for acidic monosaccharides are completely retained.The proteoliposomes also demonstrate trans-stimulation properties with both substrates sialic acid and glucuronic acid. The transporter is inhibited, both in its native and in the reconstituted state, by the sulfhydryl-modifying agents p-chloromercuribenzoic acid, N-ethylmaleimide, and phenyl isothiocyanate. In native membrane vesicles, arginine and histidine modifiers phenylglyoxal and diethyl pyrocarbonate inactivated transport in a substrate-protectable manner. In reconstituted proteoliposomes, similar inhibition was observed. However, protection by substrates was achieved only after treatment with phenylglyoxal. These data suggest that arginine or histidine residues or both are present at or near the substrate binding site of the carrier. Possibly, other essential histidines become exposed in the reconstituted state. The successful functional reconstitution of the lysosomal sialic acid carrier represents an important step towards its purification and its detailed molecular characterization.

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes

“…Thus, alcohol particularly increases the uptake of Man, ManNAc and Glc and this effect suggest an alteration of the carbohydrate transporters located in the plasma membrane. In this respect, a lysosomal sialic acid transporter (Mancini et al 1992;Havelaar et al 1998) has been reported in several cell types, but so far this protein has not been identified in astrocytes. In contrast, the effect of alcohol on glucose transport and on glucose transporters in astrocytes and other cell types has been widely studied, although contradictory results have been reported including, e.g.…”

Organic anion transport is the primary function of the SLC17/type I phosphate transporter family

“…The activity saturates with a K m~0 .24 mM, and depends on pH, with higher levels of transport associated with lower extralumenal pH [21,28]. The mechanism has thus been proposed to involve the symport of acidic sugar with H + .…”

Functional reconstitution of the Trypanosoma brucei plasma‐membrane D‐glucose transporter

“…The initial uptake rate for L-glucose was just under half that for D-glucose; liposomes alone accounted for more than 90% of this non-specific L-glucose uptake (Table 1). Background of the L-glucose control in our trypanosomal reconstitution system (about 50%) is notably higher than in the erythrocyte reconstitution system (10-34% ; Kasahara and Hinkle, 1976) but within the range of non-specific uptake in, for example, the lysosomal reconstitution system for acidic monosaccharides (30-50%; Mancini et al, 1992). Nevertheless, specific D-glUCOSe-tranSpOrt activity is well above background and high enough to provide a functional assay for the characterization and purification of the solubilized transporter.…”