The glutathione S-transferases (ligandins) are a ubiquitous system of xenobiotic metabolising enzymes. In the rat liver they comprise up to 10% of soluble hepatic protein. Studies in the rat suggested that ligandin was an accurate and sensitive marker of hepatocellular necrosis. and of renal tubular necrosis. The first part of this thesis examines the release of ligandin from liver and kidney in human liver and renal disease in an attempt to determine whether the measurement of ligandin is clinically useful. Ligandin was purified from human liver cytosol using a combination of anion exchange chromatography and gel filtration. The purified protein had similar physicochemical characteristics to ligandin purified by others. The protein was used to raise a monospecific antibody. Ligandin was iodinated by the Chloramine-T method. which yielded a labelled protein of high specific activity. A sensitive and specific radioimmunoassay for human ligandin was developed which had a low intra- and interassay variation. The assay was applied to the study of human liver disease. In acute hepatitis ligandin is released from the liver into serum early in the illness. High serum ligandin levels are seen in the first week of acute hepatitis. The rapid return to normal suggests that ligandin may provide an early indication of recovery. In chronic hepatitis ligandin levels correlated significantly with histological severity of disease. whereas SGOT showed no such correlation. Ligandin may be a better index of severity of disease and for treatment than SGOT. Ligandin was released from the kidney in severe renal ischaemia and in acute tubular necrosis, but was not a reliable predictor or indicator of acute tubular necrosis. Part two examines the distribution of GSH-T activity in organs and in hepatocellular carcinoma. Ligandin was shown to be immunologically similar in all tissues studied. Isoelectric focusing of cytosol separated the three groups of GSH-T activity. Considerable variety in the distribution and activity of GSH-T's was shown in different organs from a single donor, and in the same organs from different donors. Anionic transferase activity was shown to contribute a significant proportion of activity in organs other than the liver. and to be the major source of activity in ovary and lung. In hepatocellular carcinoma cationic GSH-T activity was present in amounts varying from near normal to absent. The anionic and neutral GSH-T's were present in amounts similar to that seen in normal liver. Immunohistochemical studies using a peroxidase-antiperoxidase method showed a rough correlation between tumour differentiation and the amount of ligandin in the tumour.

No description available.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

Cyanobacteria produce a diverse array of natural products with significant potential in many biotechnological, clinical and commercial applications. These include pharmaceuticals, such as antitumour products, antibiotics, immunosuppressants, anticholesterolemics and anti-parasitic agents, as well as veterinary therapies and agrochemicals. These compounds are synthesised by complex secondary metabolism pathways involving polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), both of which require an essential phosphopantetheinyl transferase (PPT) for their activity. PPTs activate the acyl, aryl and peptidyl carrier proteins within various biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. Phylogenetic analysis of the large superfamily of PPTs has revealed two separate families based on substrate specificity, which have been designated AcpS and Sfp-like. The AcpS PPT family activate acyl carrier proteins of fatty acid synthesis, while the Sfp-like PPT family, typified by the Bacillus subtilis PPT Sfp, has diverse roles in primary and secondary metabolism. The majority of cyanobacterial PPTs are of the Sfp-like PPT family and occur in genomes lacking an AcpS PPT. Phylogenetic analysis uncovered a distinct clade of cyanobacterial PPTs involved in heterocyst differentiation. Heterologous expression and functional analysis of NsPPT, the heterocyst-associated PPT in Nodularia spumigena NSOR10, represented the first characterisation of a cyanobacterial PPT. PCR-based screening was utilised to identify NsPPT and Southern hybridisation suggested this was the only PPT encoded by the N.spumigena NSOR10 genome. Enzymatic analyses demonstrated the ability of NsPPT to phosphopantetheinylate PKS and NRPS carrier proteins from a range of metabolism pathways and cyanobacterial species. Nostoc punctiforme ATCC 29133 encodes three PPTs. One of these PPTs, NgcS, is also a heterocyst-associated PPT and is homologous to NsPPT of N.spumigena NSOR10. Expression and enzymatic analysis of NgcS from N. punctiforme ATCC 29133, revealed contrasting phosphopantetheinylation activity to that seen for NsPPT, and indicated that NgcS may have evolved to have a strict specificity for the glycolipid biosynthesis pathway. Although the Sfp-like family of PPTs are normally associated with secondary metabolite biosynthesis, Synechocystis sp. PCC 6803 harbours a unique Sfp-like PPT (Sppt) but does not produce NRPS or PKS compounds. Genetic disruption of Sppt was attempted and expression of Sppt allowed the characterisation of its enzyme kinetics. Sppt displayed the ability to activate non-cognate cyanobacterial carrier proteins from NRPS and PKS biosynthetic pathways, although only at a low level of activity. This suggested that wild-type Synechocystis sp. PCC6803 would not be suitable for heterologous expression of cyanobacterial secondary metabolites. These results have important implications regarding the expression and manipulation of cyanobacterial bioactive compounds in heterologous hosts. Applications of this research may provide a biotechnological platform for the sustainable production of cyanobacterial natural products.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 127-153).

xxi, 98 leaves : ill., tables, graphs ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1978) from the Dept. of Biochemistry, University of Adelaide

xxi, 126 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1978

Thesis (Ph.D. 1978) from the Department of Biochemistry, University of Adelaide.

Thesis (Ph.D.) -- University of Adelaide, Department of Biochemistry, 1978.