Two-Dimensional Gel Electrophoretic Analysis of Thylakoid Membrane Polypeptides in Developing Chloroplasts of Euglena Gracilis
Gilbert, Carl Walter
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/77635
Description
Title
Two-Dimensional Gel Electrophoretic Analysis of Thylakoid Membrane Polypeptides in Developing Chloroplasts of Euglena Gracilis
Author(s)
Gilbert, Carl Walter
Issue Date
1983
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
Two-dimensional (2D) gel electrophoresis is used for the first time to analyze the polypeptide composition of thylakoid membranes during chloroplast development in Euglena gracilis. The 2D-gel technique of O'Farrell {(1975) J. Biol. Chem. 250, 4007} was modified for maximal resolution of thylakoid membrane polypeptides. The modifications include delipidation of thylakoid membranes with acetone/ether, solubilization of the polypeptides with Triton X-100, and sonication before electrophoresis in the first dimension.
The number of polypeptide spots resolved in the 2D-gels is 3-4 times the number of bands previously resolved on one-dimensional gels. Groups of multiple polypeptides with the same molecular weights, but differing isoelectric points, are detected throughout thylakoid membrane development. The pattern of polypeptide synthesis and insertion into the forming thylakoid membrane of dark-grown Euglena exposed to continuous light conforms with previous biochemical measurements on the assembly of the electron transport chain located in these membranes.
Continuously- and pulse-labeled cells show basic, high molecular weight polypeptides (> 100 kd) detectable during early greening. Further development is marked by the appearance of increasing numbers of acidic, lower molecular weight polypeptides (< 67 kd) in the membranes. Additionally, analyses of the thylakoid membrane polypeptides from pulse-labeled cells show that they are synthesized in two waves during greening. The total complement of thylakoid membranes appear to be synthesized and assembled in two portions with the first portion essentially completed before the second portion is started. The data suggest that thylakoid membrane development is highly regulated temporally.
Thylakoid membrane polypeptides labeled in the presence of protein synthesis inhibitors show that (TURN)8% of the polypeptides are synthesized on ct ribosomes, 86% are synthesized on cytoplasmic ribosomes, and 6% depend on both cytoplasmic and ct ribosomes being functional in protein synthesis before they are synthesized.
Experiments with a specific thylakoid membrane polypeptide, i.e., the DCCD-binding CF(,o)-polypeptide, show that individual polypeptide spots resolved in 2D-gels can be identified and that the pattern of appearance of individual polypeptides in the forming thylakoid membrane can be determined.
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
