Masters Theses

Date of Award

6-1982

Degree Type

Thesis

Degree Name

Master of Science

Major

Zoology

Major Professor

John H. Abel

Committee Members

Thomas T. Chen, Leaf Huang, Lawrence Etkin

Abstract

Gonadotropin receptors on the surface of lutein cells once bound to LH or hCG are slowly drawn into patches and internalized by endocytosis. New receptors replace the lost receptors and the process is repeated many times for the life of the cell. The source of new "hidden" receptors however has not been identified. Corpora lutea from pigs in early pregnancy were collected from a local slaughter house, homogenized in 25 mM Hepes, 5 mM NaHCO3, 0.28 M sucrose and 1 mM EDTA at pH 7.4 and 4°C, and the homogenate exposed to differential centrifugation. Pellets 3 and 5 were analyzed for their content of the following marker enzymes; acid phosphatase (Lysosomes), Na+-K+ATPase and alkaline phosphatase (plasma membrane PM), thiamine pyrophosphatase (Golgi), NADH cytochrome C reductase (ER), and succinic dehydrogenase (mitochondria). Pellet 4 was highly enriched in PM and ER. Pellet 3 required a much greater purification and was first placed on a Percoll density gradient (18-26%) and Fractions 1, 2 and 3 were resuspended spun at 10,000 × g for 30 min. and layered on either a sucrose (20-40%) or Ficoll density gradient (7.5-30%) and spun at 100,000 × g for 8 hrs. Fractions significantly enriched in PM&Golgi, PM&ER, mitochondria, lysosomes and secretory granules were obtained. Each was used in a competitive binding assay 125 for gonadotropin-receptors using 125I-hCG. The results of the binding assay indicate that the Golgi fraction contains by far the highest relative binding for hCG with ER second and PM third. The fractions enriched with mitochondria, lysosomes and secretory granules possessed very little binding activity. These data indicate that the secretory granule membrane does not appear to possess receptors and therefore probably does not participate in the insertion of new receptors in the cell surface. The Golgi apparatus, however, apparently possesses abundant receptors and we are currently attempting to determine if and how they are transported to the plasma membrane.

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