Student Theses and Dissertations

Andrea Leskes

Date of Award

1969

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid cell and membrane differentiation was studied by electron microscopic histochemistry. Livers from animals 4 days before to 1 day after birth were perfused with 2% glutaraldehyde in the presence of 6 mM glucose-6-phosphate. The short fixation time (3 to 5 minutes) allowed retention of 70-90% of the enzyme activity. Slices 50-100 micra thick were incubated in 1 mM glucose -6-phosphate, 2 mM lead nitrate, and 50 mM cacodylate buffer, pH 6.6. The slices were post-fixed in osmium tetroxide, oriented during embedding, and sectioned perpendicular to their length so that the complete width of a slice could be examined. Observations were confined to the outermost layers of cells where consistent results implied adequate penetration of both lead and substrate. The concentration of lead used did not inhibit glucose-6- phosphatase or cause non-specific hydrolysis of the substrate. At all stages examined the lead phosphate deposit was localized to the endoplasmic reticulum and the nuclear envelope.

At four days before birth» when the enzyme specific activity is only 9% of the adult level, the enzymatic reaction product was present in only a few hepatocytes. In these cells, a light deposit was seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, with a specific activity approximately equal to that of the adult, nearly all cells gave a positive reaction for the enzyme. Again the lead phosphate was evenly distributed throughout the entire endoplasmic reticulum, nuclear envelope included. By 24 hours post-parturition, all of the rough membranes, and in addition the newly-formed smooth membranes 9 contained heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6- phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although non-synchronously in the hepatocyte population as a whole. In addition, the enzyme appears in the smooth endoplasmic reticulum as the membranes form during the first 24 hours after birth.

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