Date of Award
1988
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Epidermal cells respond to 20-hydroxyecdysone (20HE) by altering their morphology, rate of proliferation and level of cell-to-cell communication. This response depends on polypeptide synthesis.;To study changes in polypeptide synthesis induced by 20HE, epidermal polypeptides from the midinstar larvae of the mealworm Tenebrio molitor were separated by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). {dollar}\sp{lcub}35{rcub}{dollar}S-methionine-labelled (non-cuticular) polypeptides were extracted from cells incubated either in the absence or presence of 2 ug/ml 20HE (4 uM) for 14-16 h in vitro. Cells incubated with the hormone incorporated 28% more label into polypeptides. Over 250 polypeptides were detected in total cell lysates by this technique. Polypeptides that showed altered rates of synthesis in response to 20HE treatment were identified and characterized. The relative synthetic rate of 54 polypeptides was affected by 20HE; 21 had a raised synthetic rate. The synthetic rate of most "down-regulated" 20HE-sensitive polypeptides began to drop at 10{dollar}\sp{lcub}-3{rcub}{dollar} ug/ml 20HE, whereas that of most "up-regulated" polypeptides increased only at concentrations above 10{dollar}\sp{lcub}-1{rcub}{dollar} ug/ml. An early response to 20HE, involving the synthesis of a 43 kD polypeptide de novo, was seen. It was first detectable after 2 h of exposure to hormone, and peaked after 4 h. The synthesis of this 43 kD polypeptide is selectively enhanced by the addition of 10{dollar}\sp{lcub}-2{rcub}{dollar} ug/ml cycloheximide to medium containing 20HE. The long term effect (12-16 h) of 20HE on polypeptide synthesis in subcellular fractions of the epidermis was also examined. The polypeptide content of the nuclear, mitochondrial/lysosomal, microsomal and soluble fractions all changed in response to 20HE.;Calcium-binding proteins (CaBPs) were isolated by Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent hydrophobic interaction chromatography followed by PAGE. The overall effect of 20HE is to depress CaBP synthesis. Of the 17 newly-synthesized CaBPs detected in total cell lysates, the relative synthetic rate of twelve was depressed while that of only one apparently raised during exposure to 20HE. Most newly-synthesized CaBPs identified were found in the cytosolic fraction of epidermis. The distribution of one CaBP (Mr 32,000) shifted from the cytosolic to the microsomal fraction on adding Ca{dollar}\sp{lcub}2+{rcub}{dollar} (this was inhibited by adding EGTA) to the cell extract before fractionation. The synthesis of this polypeptide was depressed by the presence of 20HE. (Abstract shortened with permission of author.)
Recommended Citation
Ouellette, Yves, "Polypeptide Synthesis In Response To 20-hydroxyecdysone, With Particular Reference To Calcium Binding Proteins, In The Epidermis Of A Larval Insect" (1988). Digitized Theses. 1763.
https://ir.lib.uwo.ca/digitizedtheses/1763