Showing papers by "John L. Harwood published in 1999"

Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus.

Abstract: A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Lipoxygenase activity in olive (Olea europaea) fruit

Abstract: The present work was designed to characterize lipoxygenase activity in olive fruit pulp, in order to determine its significance in the biosynthesis of virgin olive oil aroma. Lipoxygenase activity has been detected in particulate fractions of enzyme extracts from olive pulp subjected to differential centrifugation. The activity in different membrane fractions showed similar properties, with optimal pH in the range of 5.0–5.5 and a clear specificity for linolenic acid, which was oxidized at a rate double that of linoleic acid under the same reaction conditions. The enzyme preparations displayed very low activity with dilinoleoyl phosphatidylcholine, suggesting that olive lipoxygenase acts on nonesterified fatty acids. The enzyme showed regiospecificity for the Δ-13-position of both linoleic and linolenic acid, yielding 75–90% of Δ-13-fatty acid hydroperoxides. Olives showed the highest lipoxygenase activity about 15 wk after anthesis, with a steady decrease during the developmental and ripening periods. Olive lipoxygenase displayed properties that support its involvement in the biogenesis of six-carbon volatile aldehydes, which are major constituents of the aroma of virgin olive oil, during the process of oil extraction.

Re-evaluation of plant sulpholipid labelling from UDP-[14C]glucose in pea chloroplasts.

Abstract: The sulphoquinovosyldiacylglycerol (sulpholipid) zone obtained by TLC of pea chloroplast lipids yields, on hydrolysis, not only sulphoquinovose but also galactose and glucose. Following incorporation from UDP-[(14)C]glucose, the percentages of the total radioactivity in these three sugars were typically 1, 5 and 85%, respectively. The occurrence of the glucolipid causes difficulties in elucidating further the biosynthetic pathway for sulphoquinovose.

Identification and characterisation of metallothioneins from environmental indicator species as potential biomonitors

Abstract: This study has given the first indication that two commonly used environmental indicator species contain members of the metallothionein superfamily. This demonstrates that these species use similar proteins to higher life forms to adapt to heavy metal conditions. Future studies will investigate if the MT proteins are present in the specialised compartments containing high levels of sulphur and copper/cadmium or if other cysteine-rich proteins such as the phytochelatin family are involved. Previously reported functions of the MT proteins in metal homeostasis suggests that MT expression may be up-regulated as a consequence of the high metal concentration of the environment from which these organisms were collected. The use of the MT expression profiles, as sensitive indicators of the metal concentration of the environment, will be investigated.