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Title:Two-Dimensional Gel Electrophoretic Analysis of Thylakoid Membrane Polypeptides in Developing Chloroplasts of Euglena Gracilis
Author(s):Gilbert, Carl Walter
Department / Program:Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, General
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.
Issue Date:1983
Description:242 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.
Other Identifier(s):(UMI)AAI8309944
Date Available in IDEALS:2015-05-14
Date Deposited:1983

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