Development and application of a novel acridinium ester for use as a chemiluminescent emitter in nucleic acid hybridisation assays using chemiluminescence quenching.

Chemiluminescent acridinium esters (AEs) permit the development of high sensitivity ligand binding assays due to a combination of high intensity light emission and very low backgrounds. Here these advantages are exploited for use in homogeneous nucleic acid hybridisation assays using quenched chemiluminescence. AE chemiluminescence is conventionally initiated at highly alkaline pH. Novel "active" AEs were designed that permit initiation under conditions compatible with maintenance of nucleic acid hybrids (i.e. pH less than 9). Methyl red was found to be a dark quencher species capable of functioning at this pH. Practical application of the chemiluminescence quenching assay system has been demonstrated using two model nucleic acid hybridisation assays based on intra- and intermolecular emitter/quencher pairs.

Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Delta(10)-nonadecenoate showed that the enzyme is an n -6 desaturase rather than a Delta(12)-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b (5) as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b (5)) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n -6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n -6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms.

Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme.

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.