Student Theses and Dissertations
Date of Award
1969
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
George Palade
Keywords
Golgi complex, hepatocyte, very low density lipoprotein (VLDL), ethanol intoxication, Golgi-derived vesicles, enzyme activity
Abstract
The elements of the Golgi complex of the hepatocyte are characterized by their content of 30-80m osmiophilic particles, which have been identified by others as very low density lipoprotein. The vast majority of such particles in the cell are in the elements of the Golgi complex. Moreover, the number in the complex can be increased still further by acute ethanol intoxication. This light lipoprotein content afforded the opportunity to isolate a purified fraction of Golgi-derived vesicles from the liver cell, since the content itself would make these vesicles lighter than vesicles derived from other intracellular sources (which do not contain lipoprotein particles) and since the clusters of particles could serve as a unique marker for Golgi-derived material. Accordingly, a fraction of Golgi-derived vesicles has been isolated from microsomes obtained from the livers of ethanol-intoxicated rats, by centrifugation on a discontinuous sucrose gradient under conditions designed to favor purity at the expense of yield. The fraction has been examined in the electron microscope and found to consist almost exclusively of vesicles containing clusters of electron opaque 30-80m particles. There are no recognizable mitochondria or rough surfaced microsomes in the fraction and only a small number of smooth microsomes not containing dense particles. About 1% of the vesicles in the fraction have an appearance suggestive of lysosomes. On purely morphological grounds, we estimate that at least 90-95% of the vesicles in the fraction are derived from the Golgi comples. The vesicles have been broken open by suspension in alkaline hypotonic medium followed by shearing in a steep pressure gradient (French press). After this treatment, a fraction of membranes largely free of lipoprotein particles could be isolated by centrifugation. This fraction is assumed to represent the membranes which bounded the Golgi vesicles in the cell. The total Golgi fraction (membrane and contents) and the isolated Golgi membranes have been examined with respect to gross chemistry and to a series of enzymatic activities, and compared to a total microsomal fraction (and in some cases to lysosomal and mitochondrial fractions). The Golgi membranes have the same phospholipid-to-protein ratio as microsomal membranes, and contain no detectable RNA. They have no detectable glucose-6-phosphatase, NADPH-cytochrome c reductase, or cytochrome P-450 activity, all enzymes associated with microsomal fractions, and their specific activity for NADH-cytochrome c reductase and cytochrome b5, enzymes associated with both microsomal and plasma membranes, is only about 15% of that of microsomal fractions and about equal to that reported for plasma membranes. The Golgi fractions have an AMPase activity about equal to that of microsomal fractions and about 10% that of plasma membrane fractions. We believe this represents, in part at least, activity in the Golgi membranes themselves, not merely plasma membrane contamination of the fraction. The Golgi fractions also have high thiamine pyrophosphatase activity. The Golgi fraction contains less than 1% of the cytochrome oxidase specific activity of purified mitochondrial fractions, consistent with our failure to observe mitochondrial contaminants morphologically. The acid phosphatase activity of the Golgi fractions is about 3% that of purified lysosomal fractions. This may reflect the lysosomal contamination seen in the fraction with the electron microscope, but it may also come from acid phosphatase activity in the Golgi vesicles themselves. The biochemical evidence supports the conclusion drawn from morphology that the fraction consists at least 90-95% of Golgi-derived material. The enzyme activities are discussed with respect to the interrelationships which they imply among the Golgi complex, the rest of the endoplasmic reticulum, and the plasma membrane.
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Recommended Citation
Ehrenreich, John Herman, "The Golgi Complex of the Hepatocyte: Isolation and Partial Enzomological Characterization" (1969). Student Theses and Dissertations. 560.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/560
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy