Investigation of lectin activity in Theileria annulata piroplasms

Adhesion to target cells is an essential step in the pathogenesis of many protozoal infections. Some protozoa have been reported to have a lectin activity involved in their attachment to the cell surface. The ligand-receptor interaction involved in Theileria annulata infection is unclear at present, in spite of the fact that some aspects of the process have been investigated. To this end, T. annulata piroplasms have been screened for lectin activity. Blood taken from infected cattle was first depleted of leukocytes and then subjected to ammonium chloride lysis in order to isolate the piroplasms. The piroplasms were homogenised and a crude membrane extract was prepared by centrifugation. To investigate lectin activity in piroplasm proteins, a simple screening procedure was employed for analysing piroplasm proteins binding to various lectin ligands. Numerous immobilised lectin ligands (L-fucose-sepharose, N-acetyl-neuraminic acid-sepharose, N-acetyl-D-galactosamine-agarose, N-acetyl-D-glucosamine-agarose, D-mannose-agarose, β-D-glucose-agarose, α-methyl-D-mannoside-agarose) were incubated with T. annulata piroplasm crude membrane extract. The ligand-bound proteins were eluted and separated by a brief centrifugation and determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The present study suggests that a 32 kDa protein of piroplasm binds to D-galactosyl residues of the agarose matrix and that the binding is inhibited by galactose and not by the other monosaccharides tested.

A Simple Screen for Murein Transglycosylase Inhibitors

ABSTRACT A simple assay for detection of compounds that bind to the active site in the transglycosylation domain of the essential bifunctional transglycosylase and transpeptidase penicillin-binding proteins (PBPs) is reported. The method is based on a competition with the specific transglycosylase inhibitor moenomycin. With moenomycin coupled to Affi-Gel beads, a simple filtration procedure allows the amount of labeled PBPs that bind to moenomycin beads in the presence of test substances to be determined. The PBPs can easily be labeled by the covalent binding of penicillin derivatives. Crude membrane extracts can be used as a source for the PBPs, and different kinds of labels for the penicillin-PBP complexes can be used. The assay can be adapted to high-throughput screens.

Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.