Glucosinolates are a diverse group of molecules found in plants of the order Capparales and the genus Drypetes. Hydrolysis of glucosinolates is catalysed by a thioglucosidase, myrosinase. Myrosinase has not only been detected in almost all glucosinolate-containing plants but also in insect and microbial species. Phylogenetic analysis of glucosinolate-containing plants found that all were clustered together with the exception of the outlier genus Drypetes. The important question is whether myrosinase in Drypetes arose from the same ancestral gene as that in the Capparales, or whether it arose from a different source. Myrosinaselike activity was detected in D. natalensis. A candidate molecule for the observed activity was isolated and found to be a 50 kDa heterodimer with subunits of approximately 30 kDa and 20 kDa. This contrasts with Capparales myrosinases which are typically 130-150 kDa homodimers. A 1047 bp partial sequence corresponding to the larger subunit was obtained. Analysis of the nucleic acid and amino acid sequence showed that it was similar to the cupin superfamily of proteins which include the ubiquitous seed storage proteins. Phylogenetic analysis showed that the isolated protein was closely related to seed storage proteins. The results obtained here are suggestive of an independent origin of myrosinase activity in Drypetes. With the degree of functional plasticity observed in the cupin superfamily it is proposed that the isolated factor could have acquired myrosinaselike activity. However, purification and further characterisation of the enzyme responsible for the observed activity is still required to confirm the results

A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy, Johannesburg, 1996 / The glutathione S-transferases (GST) are a supergene family of h0111o-or heterodimeric Phase II detoxification enzymes which catalyse the S-conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions; a highly specific Gssite for binding the physiological substrate glutathione and a nonspecific If-site for binding nonpolar electrophilic substrates. Unfolding of porcine class Pi isoenzyme (pGSTPl~l) was monitored under equilibrium conditions using different physicochemical parameters. The coincidence of unfolding curves obtained with functional and structural probes, the absence of thermodynamically stable intermediates such as a folded monomer, and the dependence of pGSTPl··l stability upon protein concentration, indicate a cooperative and concerted two-state unfolding transition between native dimeric pGSTPl-l and unfolded monomeric enzyme. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were preformed to study the effect of ligand binding to the G-site on the unfolding and stability of the porcine class pi glutathione S-transferase against urea. The presence of glutathione caused a shift in the equilibrium-unfolding curves towards lower urea concentrations and enhanced the first-order rate constant for unfolding suggesting a destabilisation of the pGSTPl-l structure against urea. The presence of either glutathione sulphonate or S-hexylglutathione, however, produced the opposite effect in that their binding to the G-site appeared to exert a stabilising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway. / MT2017

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Master of science Johannesburg. 1996 / An attempt has been made to purify and isolate a starch endo-hydrolase enzyme produced by Streptococcus sanguis, an organism that may be implicated in dental caries. In order to isolate the enzyme by affinity binding,& chemically modified amylopectin Was prepared, similar to the preparation of chromogenic substrate for the assay of a-arnylase, but without dye. The amylopectin was treated with 10 percent ammonium sulphate at 55°C for 45 minutes. A crude enzyme extract was prepared from concentrated culture medium and by precipitation with 60 percent ammonium sulphate. The concentrated culture medium was added to the modified amylopectin substrate and the mixture was incubate for 30 minutes at 37'C and centrifuged. The precipitate was resuspended in 20 mM bepes buffer pH 6.5 which contained 0.02 M KCI to release the enzyme from the enzyme-substrate complex. The suspension was tested for enzyme activity and the presence of proteins, More than 50 percent of the yield of the enzyme was achieved by this process, after three assays, from both crude enzyme extracts and enzyme serum samples. A 5.2 fold purification was obtained from the extraction process of the crude enzyme extract and a protective enzyme activity effect was noticed in the presence of ammonium sulphate. The analytical methods selected for the activity assay were mainly used for the activity evaluation of a-amylases and carbohydrate hydrolysing enzymes. The result showed carbohydrate interference. The isolation method proved sensitive and highly specific for the isolation of a starch metabolizing enzyme produced from Streptococcus sanguis 1MC 204. The purification of the enzyme by gel filtration and its characterization by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that this enzyme was a glycoprotein . SDS-PAGE was performed with mucin and heparin in the. presence of other. proteins as markers, and stained with the periodic acid- aldan prestained silver method. This gave one transparent-band around the phosphorylase b marker and four other more slowly running clear bands. Further, the comparison of scans of several proteins and glycoproteins with the scan of the eluted sample of the amylopectin extracted enzyme showed a similarity with the UV scan of mucin and confirmed that the enzyme was a glycoprotein. It may be further characterized by selecting methods that take into account the ambohydrate content of the protein and by eliminating the carbohydrate interference in the assays. 3

Coenzimas piridínicos desempenham papel fundamental relacionado à suas propriedades de óxido-redução. Assim, participam da cadeia respiratória, onde se situam, devido ao valor do potencial de óxido-redução do par, logo no início, sendo os primeiros intermediários entre certos substratos e oxigênio. Constituem-se também no primeiro local de síntese de ATP; muito importante, presumivelmente em conexão com a função respiratória, é de fato de os coenzimas piridínicos intervirem nos sistemas de algumas transhidrogenases. / Not available

Thesis (M.S.)--Chinese University of Hong Kong. / Includes bibliographical references.

Thesis (M. Phil.)--Chinese University of Hong Kong, 1976. / Bibliography: leaves 82-91.

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