Browsing by Author "Mtwisha, Linda"

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    ASP 53, a 53 kDa cupin-containing protein from Acacia erioloba seeds that protects proteins against thermal denaturation
    (2004) Mtwisha, Linda; Lindsey, George G; Brandt, Wolf F; Farrant, Jill M
    Includes bibliographical references (leaves 103-111).
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    ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation.
    (CSIRO Publishing, 2007) Mtwisha, Linda; Farrant, Jill M; Brandt, Wolf; Hlongwane, Caswell; Lindsey, George G
    ASP53, a 53 kDa heat soluble protein, was identified as the most abundant protein in the mature seeds of Acacia erioloba E.Mey. Immunocytochemistry showed that ASP53 was present in the vacuoles and cell walls of the axes and cotyledons of mature seeds and disappeared coincident with loss of desiccation tolerance. The sequence of the ASP53 transcript was determined and found to be homologous to the double cupin domain-containing vicilin class of seed storage proteins. Mature seeds survived heating to 60◦C and this may be facilitated by the presence of ASP53. Circular dichroism spectroscopy demonstrated that the protein displayed defined secondary structure, which was maintained even at high temperature. ASP53 was found to inhibit all three stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55◦C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature-dependent aggregation of egg white protein.
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    Isolation and characterisation of a LEA-like protein from yeast (Saccharomyces cerevisiae)
    (1998) Mtwisha, Linda; Brandt, W B; Lindsey, George G
    LEA proteins are plant proteins that are characteristically hydrophilic and soluble at elevated temperature. The consistent correlation between desiccation tolerance in orthodox seed tissue and an accumulation of LEA proteins suggests that these proteins play an important role in protecting cells from desiccation induced damage. Yeast (Saccharomyces cerevisiae) has been known to desiccate as part of its normal growth cycle and to remain viable after long periods in the desiccated state. As a result of these properties this project was designed to investigate the presence of LEA-like proteins in yeast. A protein was isolated from baker’s yeast that fulfils the requirements for being a LEA protein. This protein, with a molecular mass of 11 kDa, was found to be the most prevalent heat soluble protein in the yeast extract. Antibodies raised against LEA group I proteins recognised this 11 kDa yeast protein in the total extract but failed to recognise the protein after heat treatment at 80°C for 10 min. Amino acid analysis showed that the ll kDa protein was highly hydrophilic - a characteristic of LEA proteins. The protein was partially sequenced (10 cycles) after CNBr digestion and the sequence obtained was compared with the sequence of known proteins in the Stanford databank. Only one protein, HSP 12, was identified to be 100 % homologous to the obtained sequence without the introduction of gaps. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was present in a reduced concentration in yeast grown at 37 °C compared with yeast grown at 30 °C. HSP 12 was found to increase in concentration after entry into stationary phase - a time when nutrients are limiting and the yeast is preparing to reduce its water content and sporulate. This might be considered equivalent to plant seed maturation - the stage when LEA proteins are synthesised. Moreover, growth conditions that have been reported to stimulate LEA protein biosynthesis in plants also stimulated HSP 12 synthesis in yeast. Purified HSP 12 was shown to inhibit thermal denaturation of yeast alcohol dehydrogenase (ADH) at elevated temperatures. This is a functional property of the pea seed p11 LEA group I protein. From the above results, it was therefore concluded that HSP 12 should be identified as a LEA-like protein rather than as a heat shock protein.