Paul Towner

Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta.

Manduca sexta is a nicotine‐insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose α subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]α‐Bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nm and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [125I]α‐bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 μm and 2 μm for larvae and adults, respectively) competed with an affinity similar to that found for nicotine‐sensitive insects. No α‐bungarotoxin‐insensitive binding sites labelled by [3H]epibatidine could be detected. Using the α‐like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other α‐subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an α‐like subunit capable of binding α‐bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.

Transport of d-fructose and its analogues by Trypanosoma brucei

Kinetic parameters for entry of D-fructose into Trypanosoma brucei brucei have been determined. The net uptake of D-fructose was found to be rapid and occurred at a rate which was comparable with that observed for uptake of D-glucose. The Km and Vmax were 3.91 +/- 1.58 mM and 69.1 +/- 7.2 nmol min-1 (mg protein)-1. D-Fructose was metabolized to pyruvate under aerobic conditions and to pyruvate and glycerol under anaerobic conditions in a manner similar to D-glucose. Comparisons of the kinetic parameters for D-fructose transport and metabolism indicated that uptake was rate limiting. Inhibition constants (Ki) for inhibition of 6-deoxy-D-glucose by D-fructose and D-fructose transport by 6-deoxy-D-glucose were consistent with the Km values for these two substrates. These interactions indicate that D-fructose and 6-deoxy-D-glucose share a single common transporter. 1,5-Anhydro-D-glucitol and 1,5 anhydro-D-mannitol (the fused pyranose ring analogues of D-glucose and D-mannose) have been found to interact well with the transporter, while L-sorbose (a D-fructose analogue with a pyranose ring) had only low affinity. However, 2,5-anhydro-D-mannitol (a fused furanose ring analogue of D-fructose) inhibited both 6-deoxy-D-glucose and D-fructose transport with a Ki of approx. 0.8 mM. The high affinity for 2,5-anhydro-D-mannitol (2-deoxy-D-fructofuranose) indicates that D-fructose is transported in the furanose ring form.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and purification of plasmid‐encoded Thermoplasma acidophilum citrate synthase from Escherichia coli

The citrate synthase gene from the thermophilic archaebacterium. Thermoplasma acidophilum was expressed in Escherichia coli, yielding an active product of the expected molecular weight. Manipulation of the citrate synthase gene in a series of pUC19 constructs showed that the presumed Thermoplasma ribosome binding site is recognized by the E. coli ribosome. A rapid purification of the expression of the expression product to homogeneity was achieved, based on the thermostability of Thermoplasma citrate synthase.

Crystallization and preliminary crystallographic study of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum.

Single crystals of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group P6122 or P6522, with unit cell dimensions a = b = 121.9 angstrom, c = 229.6 angstrom and with two molecules in the asymmetric unit.