Thesis

Ultrastructural localization of enzymes in Polytomella agilis

The combination of histochemical methods and electron microscopy provides useful technique for the ultrastructural localization of enzyme activities. Cells from log and stationary phase cultures of the flagellate polytomella agilis were fixed in glutaraldehyde and then incubated in Gomori type lead stains for the enzymes acid phosphatase and g1ucose-6-phosphatase. Following incubation the cells were fixed in osmium and processed for electron microscopy. The cells were viewed for the presence of lead deposits which are the indicators of the enzyme activities. Lead deposits were observed in the proplastid network of a relatively small number of mature trophs incubated for acid phosphatase activity. This was interpreted as a possible indication of acid phosphatase activity in this organelle. The proplastid undergoes considerable differentiation during life cycle changes. It is proposed that the proplastid network may be a site of hydrolytic digestion. No evidence of lysosomes was observed. A significant number of mature trophs and a few pre-cysts incubated for glucose- 6-phosphatase activity contained heavy lead deposits along the membrane limits of storage vesicles and the proplastid network. Some cells also contained deposits along the nuclear membrane. No deposits were observed in the endoplasmic reticulum. These deposits were interpreted as a strong possibility of glucose-6-phosphatase activity in these areas. These storage vesicles and the proplastid are believed to be involved in the manufacture and storage of starch. Glucose-6-phosphate is an integral enzyme in carbohydrate metabolism. It is proposed that the enzyme may also be involved in the intracellular transport of glucose. Various factors concerning the validity of the use of lead stains for enz;yme localizations were reviewed.

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