Student Theses and Dissertations
Date of Award
1969
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Philip Siekevitz
Keywords
glucose-6-phosphatase, rat liver development, endoplasmic reticulum, histochemistry, hepatocyte differentiation, enzyme localization
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, 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. To confirm that the pattern of histochemical deposits reflects the actual distribution of enzyme sites, a method to sub-fractionate the rough endoplasmic reticulum was developed, based on the retention of reaction product within fresh microsomes reacted in vitro. Lead phosphate increases the density of glucose-6-phosphatase-containing microsomes and thereby makes possible their separation from enzyme-free vesicles by isopycnic centrifugation on a 2-step density gradient. The procedure was applied to rough microsomes isolated from rats at various stages during hepatocyte differentiation, and the results obtained agree very well with those given by the histochemical experiments in situ. Before birth, when only some of the cells react, only a commensurate proportion of the microsomal fraction can be rendered heavy by the glucose-6-phosphatase reaction. Thus, the distribution of the reaction product in the intact endoplasmic reticulum reflects the actual distribution of enzyme sites in the membranes. The enzyme is evenly distributed in closely-spaced sites throughout the endoplasmic reticulum membranes, and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase at this level of resolution. These findings suggest a mechanism of membrane assembly and differentiation involving molecule-by-molecule insertion into a pre-existing structural framework. The membranes formed are mosaics of old and new components and do not contain regions of entirely "new" membrane, in which all components are newly synthesized or newly assembled.
License and Reuse Information
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Recommended Citation
Leskes, Andrea, "The Localization of Glucose-6 Phosphatase in Developing Rat Liver: A Histochemical and Biochemical Study" (1969). Student Theses and Dissertations. 631.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/631
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy