The aim of the work in my thesis was to establish the extent to which the pathways of carbohydrate oxidation, namely, glycolysis and the oxidative pentose phosphate pathway, are compartmented in non-photosynthetic cells, with particular reference to the extent and organisation of these pathways in leucoplasts and amyloplasts. I pursued this aim by developing a technique for the isolation of both intact leucoplasts and amyloplasts from three-day-old soybean suspension cultures. This technique involved the enzymic preparation of protoplasts, gentle mechanical lysis of the protoplasts to release intact organelles, the layering of the organelles onto a 16-60% (w/w) linear sucrose density gradient, followed by the centrifugation of the gradient to allow separation of the organelles according to their densities. I then fractionated the gradient and measured marker enzymes in each fraction to show firstly, that I had concentrated the different plastids in different locations on the gradient, and secondly that the separate plastid fractions were not significantly contaminated by other cell components. I then measured the activities of all the enzymes of glycolysis and the oxidative pentose phosphate pathway in the isolated plastids. The results indicated that the leucoplasts contained all the enzymes of glycolysis, with the possible exception of enolase, and all the enzymes of the oxidative pentose phosphate pathway. The amyloplasts contained all the enzymes of both pathways. I confirmed these results with data from latency and protection experiments. I used my results to estimate the relative activities of the enzymes of carbohydrate oxidation in the plastids and the cytosol of soybean suspension cultures. In order to complement the results of the enzyme distributions, I developed a second technique, involving the use of a 0-40% (w/w) linear Nycodenz density gradient in place of the sucrose gradient, for the preparation of pure, intact, functional leucoplasts. I fed specifically labelled [<SUP>14</SUP>C]glucose 6-phosphate to these leucoplasts. The pattern of specifically labelled [<SUP>14</SUP>C] released as CO<SUB>2</SUB> suggested that the oxidative pentose phosphate pathway was operating in the leucoplasts, with recycling of both hexose and triose phosphates, and that glycolysis was operating down to 3-phosphoglycerate.

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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

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The H$ sp+$-translocating inorganic pyrophosphatase from vacuolar membranes of red beet storage roots (Beta vulgaris L.) was purified after solubilization in Triton X-100 through a combination of anion-exchange and size exclusion chromatographies. SDS-PAGE showed strong correlation between a 67 kDa polypeptide and pyrophosphatase activity. Radiation-inactivation studies of the H$ sp+$-PPase indicate a functional size of 91 kDa for hydrolysis and 320 kDa for H$ sp+$ translocation, suggesting an oligomeric structure for the holoenzyme. Affinity purified antibodies were used to screen cDNA libraries of Arabidopsis thaliana yielding clones which contained sequences matching amino acid sequences obtained from tryptic fragments of the 67 kDa hydrolytic subunit. The predicted protein is highly hydrophobic with a molecular size of 81 kDa. Southern analyses show a single copy for the H$ sp+$-PPase in Arabidopsis. The lack of sequence identities between the H$ sp+$-PPase and other known proteins implies a novel class of ion translocases.

Cette recherche comprend l'etude de la bioacatalyse de la chlorophyllase partiellement purifiee a partir d'algue (Phaeodactylum tricornutum) dans differents milieux organiques incluant les solvants miscibles et non miscibles a l'eau et les systemes micellaires tertiaires. Les quantites optimales d'eau et de solvant, choisi en fonction de son degre d'hydrophobicite et incluant l'acetone, l'ethanol, le propanol, le butanol, le pentanol, l'hexanol, le toluene, le pentane, l'hexane, l'heptane et l'octane, necessaire a la catalyse de l'effet hydrolytique de la chlorophyllase, ont ete mesurees. Les valeurs d $V sb{ rm max}$ obtenues mesurant l'activite hydrolytique de la chlorophyllase sont plus elevees chez les solvants miscibles a l'eau (log P 0.8). L'activite hydrolytique de la chlorophyllase decroit d'approximativement 12 fois lorsque le nombre de carbone pour les alcools, passent de 2 a 5. Certains resultats a partir du FTIR ("Fourier transform infrared spectroscopy") tendent a montrer que le phytol pourrait agir comme un donneur d'electron, ainsi, si un compose nucleophilique approprie est ajoute a un syteme biphasique, la solubilite de la chlorophylle dans l'hexane augmentera et l'activite hydrolytique de la chlorophyllase augmentera. En milieu biphasique (hexane/eau), l'addition de solvants polaires tels que l'acetone, l'ethanol, et le propanol augmente l'activite de la chlorophyllase et ce seulement a certaines concentrations. La mesure optimale de l'activite hydrolytique de la chlorophyllase en milieu micellaire tertiaire (tampon/hexane/surfactant) utilisant les polysorbates et les Spans ainsi que differentes chai nes hydrophobiques comme surfactant indique que les concentrations necessaires de surfactants sont dependantes a la fois de la chai ne hydrophobique et du groupement polaire. Toutefois, l'utilisation du Span 85 s'est avere plus approprie et les valeurs de $V sb{ rm max}$ obtenues mesurant l'activite enzymatique de la chlorophyllase sont 288 fois

Biomass production of chlorophyllase of the marine alga (Phaeodactylum tricornutum) at the exponential and stationary stages was performed. The results demonstrated that the biomass yield at the stationary stage was three times that of the exponential stage. A procedure for the extraction and purification of chlorophylls from fresh spinach leaves, used as substrate, was also developed. Chlorophyllase was extracted from photosynthetic membranes of the disrupted cells and partially purified. The purification procedure resulted into 70-fold increase in enzyme activity. Further purification of the partially purified enzyme was performed, using preparative isoelectric focusing on Rotofor-Cell System. Three enzyme fractions, FI$ sp prime$, FII$ sp prime$, and FIII$ sp prime$ were separated, however, most of enzyme activity (84%) was located in fraction FII$ sp prime$. The partially purified chlorophyllase was further purified by native preparative gel electrophoresis on Prep-Cell System, which resulted into a single active fraction. The purified enzyme fraction was then subjected to further purification, using automated Fast Protein Liquid Chromatography (FPLC) System, on ion-exchange Mono Q HR 5/5 column. The purification procedure resulted into two well separated isozymes, FI$ sp prime$ and FII$ sp prime$. Enzyme fraction (FI$ sp prime$) showed the highest enzymatic activity compared to FII$ sp prime$. The homogeneity of each fraction was demonstrated by a single protein band on SDS-PAGE. The molecular weights of these fractions FI$ sp prime$ and FII$ sp prime$ were 67 kD and 66kD, respectively. The optimum pH for chlorophyllase activity fractions FI$ sp prime$, and FII$ sp prime$ were 8.0 and 8.3, respectively. The enzymatic fraction FI$ sp prime$ showed higher activity towards commercial purified chlorophyll b when it was compared to that with the crude chlorophyll, partially purified chlorophyll and commercial purified chlorophyll a. However, enzymatic fraction FI