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The present study was undertaken to establish the mechanism(s) involved in the endocytic pathway, in particular, early endosome processing and delivery to the lysosomes. Two models for endosome processing have previously been proposed in the literature, namely the maturation and vesicular traffic models. The general consensus has been an early phase of intermingling of the endocytic contents markers within early endosomes that mature to form non-fusogenic late endosomes (maturation model). This maturation phase is followed by a segregation phase where intermingling of contents between vesicles no longer takes place. To establish the mechanism(s) involved in early endosome processing and delivery to lysosomes, a kinetic analysis was made using results from cellular fluid-phase uptake assays. This unique approach offers an alternative view to previous studies on the mechanisms in operation during endocytic processing. The results and conclusions made could thus confirm or disprove previously proposed mechanisms.

The present thesis looks at the behaviour of internalised cell surface-derived membrane marker in comparison with the behaviour of endocytosed HRP (horse-radish peroxidase) as a fluid-phase contents marker. The pooling and/or segregation in the endosome was measured by determining co-localization with HRP. Colocalization of the two markers in the endosome is studied by using the ability of HRP to catalyse the crosslinking of membrane marker in endosomes with DAB (3,3'-diaminobenzidine), rendering the membrane marker detergent insoluble. To study the kinetic behaviour of membrane marker, radioactive galactose was covalently bound to cell-surface glycoconjugates on mouse macrophage-cells, P388D₁, as catalysed by galactosyltransferase. This provided a general membrane marker. After endocytosis-derived redistribution of membrane marker between the cell surface and endosomal membrane, a steady state was established with about 16% of the label on internal membranes. The bulk of the label on the cell surface was removable by subsequent treatment with β-galactosidase.

This dissertation represents an attempt to understand the mechanism of the action of pesticides derived from chlorinated phenols (pentachlorophenol, pentachlorobenzenethiol, and 2,4,5-trichlorophenol) in lipid membranes; specifically, the kinetics of pesticide induced hydrogen ion transfer in lecithin-cholesterol membranes and its relationship to uncoupling activity in energy transducing membranes. Information on pesticide induced charge transport in membranes was obtained from the measurements of steady state and transient membrane conductance and membrane potentials as a function of the composition of the aqueous phase surrounding the membrane and of membrane composition. In addition, a systematic theoretical study of a series of membrane transport models was performed in order to elucidate the various aspects of membrane transport kinetics and to make predictions relevant to the interpretation of experimental results. Based on the theoretical results, two kinetic schemes of membrane transport were proposed to explain the experimental results; one for pentachlorophenol and 2,4,5-trichlorophenol, and another for pentachlorobenzenthiol. The schemes differ in the proposed mechanism of charge transfer across membrane water interface. Some conclusions regarding the rate limiting step in the charge transport process are drawn. Measurements of membrane surface potentials based on the microelectrophoretic method reveal that the density of ionized pentachlorophenol at the membrane surface can be predicted from the Langmuir adsorption model, provided that electrostatic repulsion between pentachlorophenol ions adsorbed at the membrane and free ions in the aqueous phase is taken into account. The fact that the adsorption of positively charged tetraphenylarsonium ions and negatively charged salicylate ions modify the membrane charge transport induced by 2,4,5-trichlorophenol, can be explained by electrostatic arguments. The 2,4,5-trichlorophenol, can be explained by electrostatic arguments. The ability of pentachlorobenzenethiol and of chlorinated phenols to induce proton transport in membranes is regarded as the origin for both the membrane electrical conductivity and their toxic action in energy transducing membranes. We have found similarities between the pH dependence of pentachlorobenzenethiol induced membrane conductance and the pH dependence of the pentachlorobenzenethiol uncoupling activity found in mitochondria, as reported in the literature. We have also found that the level of pentachlorophenol concentration at which the membrane conductance increases above the background level corresponds to the onset of reduction of carbon uptake by algae.

Early studies of the permeability of the animal cell membrane were difficult to compare, because of the variety of cell types involved. In the late nineteenth century, workers chose to use erythrocytes due to their advantages over using a variety of cell types. With erythrocytes a comparison could be developed between strictly homologous cells of many different species. Erythrocytes are easily obtainable and mature cells are separate from all other cells of the body as well as from each other. Of even greater importance is the fact that the mature erythrocyte’s major function is the uptake of the greatest possible amount of gases and solutes in the shortest possible time and elimination of these materials without loss or change. The erythrocyte eliminates the complications arising from metabolism of the penetrant being studied. This is attributed to the fact that the erythrocyte is metabolically almost inactive. TO illustrate this point, the human erythrocyte has a rate of glucose utilization of 0.019 ug/hr/million cells while the human leucocyte’s glucose utilization rate is 7.24 ug/hr/million cells or nearly 380 times as great (Guest et al., 1953). The importance of a nearly inactive cell is the lack of complications due to interpretation of data as a function of metabolic processes rather than membrane transport.

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