N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells.

In plants, N-linked glycans are processed in the Golgi apparatus to complex-type N-glycans of limited size containing a beta(1,2)-xylose and/or an alpha(1,3)-fucose residue. Larger mono- and bi-antennary N-linked complex glycans have not often been described. This study has re-examined the structure of such plant N-linked glycans, and, through both immunological and structural data, it is shown that the antennae are composed of Lewis a (Le(a)) antigens, comprising the carbohydrate sequence Gal beta 1-3[Fuc alpha 1-4]GlcNAc. Furthermore, a fucosyltransferase activity involved in the biosynthesis of this antigen was detected in sycamore cells. This is the first characterization in plants of a Lewis antigen that is usually found on cell-surface glycoconjugates in mammals and involved in recognition and adhesion processes. Le(a)-containing N-linked glycans are widely distributed in plants and highly expressed at the cell surface, which may suggest a putative function in cell/cell communication.

Characterization, purification and properties of the yeast mitochondrial dicarboxylate carrier (Saccharomyces cerevisiae).

The dicarboxylate carrier has been characterized and purified from mitochondria of wild strain Saccharomyces cerevisiae. The mitochondria were solubilized with Triton X-100 and the detergent extract was chromatographed on hydroxylapatite. SDS-PAGE of the hydroxylapatite pass-through showed five protein bands with M(r)s ranging from 28,000 to 35,000, by silver nitrate staining. The n-butylmalonate-sensitive succinate(out)/malate(in) exchange activity of the hydroxylapatite pass-through reconstituted into liposomes, was increased nine-fold with respect to the activity of the Triton X-100 extract. The exchange activity was inhibited by p-chloromercuriphenylsulfonate (PMPS), 4.4'diisothiocyanostilbene-2.2'-disulfonate (DIDS) and pyridoxal-phosphate, suggesting that one or more thiol groups and basic residues are implicated in the binding mechanism. The purification of the carrier was achieved by affinity chromatography on Sepharose-immobilized malate dehydrogenase. The purified protein presented the same properties as the dicarboxylate carrier in native mitochondria and displayed a single protein band with an M(r) of 28,000 as determined by SDS-PAGE. The specific activity of the purified carrier showed a 53-fold increase compared to that of the initial material. The Km for the reconstituted exchange was 2 mM for succinate with a V of 1.5 mumol min-1 mg-1 protein at 22 degrees C. The high purification state achieved for the yeast dicarboxylate carrier should allow the study of its molecular properties.

Purification of the rat-liver mitochondrial dicarboxylate carrier by affinity chromatography on immobilized malate dehydrogenase.

The dicarboxylate carrier of rat-liver mitochondria, extracted by Triton X-100 and partially purified by hydroxylapatite chromatography, was retained by malate dehydrogenase immobilized on Sepharose gel, and eluted with 0.4 M NaCl. SDS-polyacrylamide gel electrophoresis of the eluate showed a predominant peptide band with an M(r) of 28,000. The purified protein, incorporated into liposomes, mediated a butylmalonate sensitive malonate(out)/malate(in) exchange that was inhibited by p-chloromercuriphenylsulfonate. Sulfate, malate and phosphate decreased the rate of exchange. The highly purified protein displayed all the properties of the dicarboxylate carrier. Moreover, the results suggest a possible functional interaction between mitochondrial carrier protein and malate dehydrogenase.

Comparative partial purification of the active dicarboxylate transport system of rat liver, kidney and heart mitochondria.

Hydroxylapatite chromatography of Triton-extracted inner-membrane proteins from rat liver mitochondria allowed a ten-fold purification of the dicarboxylate carrier. The purified system, reconstituted into liposomes, displayed all the properties of the dicarboxylate carrier and mediated malonate-malate and malonate-phosphate exchanges. Six protein bands of Mr ranging from 27,000 to 34,000 could be resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The purification of the dicarboxylate carriers of liver, kidney and heart mitochondria were carried out by this method and their properties were compared with respect to transport activity and electrophoresis patterns. Our results demonstrate that the dicarboxylate carrier of rat mitochondria can be obtained in an advanced state of purification and with a high specific activity.

Reconstitution of the dicarboxylate exchange activity by incorporation into liposomes of a Triton-extract of mitochondrial rat-liver inner membranes.

The exchange between external [14C] malonate and internal malate or phosphate was reconstituted in liposomes prepared by incorporation of a Triton-extract of mitochondrial rat-liver inner membranes. The conditions of transport were investigated and the kinetic parameters of malonate-malate and malonate-phosphate exchanges were determined. The exchange was sensitive to butylmalonate and to organomercurials. Sulfate and phosphate decreased the rate of malonate-malate exchange and phosphate inhibition was found to be competitive. This report demonstrates the possibility to reconstitute a functional dicarboxylate transport and to use the system for further purification and for studies at the molecular level.

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria.

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.

Influence of adenine nucleotides on oligomycin inhibition of energy-transducing reactions in intact rat-liver mitochondria.

The inhibitory effect of oligomycin was investigated in intact mitochondria through oxidative phosphorylation and uncoupler induced ATPase activity. Results show that oligomycin inhibition curves can be either sigmoidal or hyperbolic depending on experimental conditions and chiefly on the metabolic state of mitochondria with regard to the distribution of mitochondrial endogenous adenine-nucleotides. Active respiration and uncoupler-induced ATPse activity produce sigmoidal titration curves for a high initial ATP : ADP ratio and hyperbolic curves for a low ATP : ADP ratio. Time-dependent inhibitions are observed for the two reactions. The maximal inhibitory action for low concentrations of the inhibitor is delayed by the initial presence of ATP or the possibility of generating from inorganic phosphate before adding oligomycin. Results presented here show that the initial adenine-nucleotide distribution is important for oligomycin sensitivity of energy-linked reactions. Although a limited conformational change of the oligomycin-sensitivity to the inhibitor, it is more likely that a gross structural change of the inner membrane induced by adenine-nucleotides modifies membrane permeability to oligomycin.