The compartmentation of carbohydrate oxidation in non-photosynthetic cells of higher plants
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 [14C]glucose 6-phosphate to these leucoplasts. The pattern of specifically labelled [14C] released as CO2 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.