A Two‐Dimensional Separation of Proteins from Chloroplast Thylakoids and Other Membranes

Abstract

A two‐dimensional, highly reproducible, analytical separation procedure for Triton/urea extractable proteins, using isoelectric focusing in the first dimension and gelelectrophoresis with sodium dodecylsulfate (SDS) in the second, is described. The separation of erythrocyte membrane proteins compares the reliability with that of published procedures. From the thylakoid membranes of the green alga Chlamydomonas reinhardii and of spinach, two‐dimensional maps of the Triton/urea extractable proteins were established, containing 58 and 33 peptide spots, respectively, which were characterized by their molecular weights and apparent isoelectric points. In Chlamydomonas some of the spots could be attributed to the photosystem I and II particles. A few major spots in the maps of the thylakoid proteins from the two non‐related plants were found at the same relative positions. A one‐dimensional electrophoretic comparison of the SDS and the Triton/urea‐extractable proteins showed that in the Triton/urea extract of thylakoid membranes some proteins of the photosystem II region, and in that of erythrocyte membranes, some components of the band 3 and 5 region are reduced or missing.

Intrinsic membrane proteins of the thylakoids of Chlamydomonas reinhardii

“…The conditions for the growth of Chlamydomonas reinhardii Arg-mt + sr3 as well as the isolation of the thylakoids were as described previously [4]. In certain experiments, it was desirable to use cells with radioactively labelled chloroplast-made proteins.…”

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

“…containing 3 S-labeled chloroplast polypeptides were either dried onto Whatman 3 MM paper and autoradiographed at -80 C using Kodak XR-5 x-ray film or prepared for fluorography (8) and fluorographed at -80 C using the same film. (5,6,9,10,14,15,28). Therefore, we resolved Euglena chloroplast polypeptides on a gradient gel (Fig.…”

“…Both 1D3-and 2D-gels have been used, the latter giving the more 'These studies were supported in part by National Institutes of Health (4) and to analyze the protein composition of various chloroplast subfractions from spinach (9,10,23,26,28), Chlamydomonas (9, 10), pea (16) and bean (2).…”

“…Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.Polyacrylamide gel electrophoresis has proven to be a very useful technique for resolving complex mixtures of proteins (22).Both 1D3-and 2D-gels have been used, the latter giving the more 'These studies were supported in part by National Institutes of Health (4) and to analyze the protein composition of various chloroplast subfractions from spinach (9,10,23,26,28), Chlamydomonas (9, 10), pea (16) and bean (2).In the case of Euglena gracilis, chloroplast proteins (5-7, 1 i, 18, 19, 27) have been analyzed on ID-gels. Recently, we (18) adapted the 2D-gel technique of O'Farrell (24) to an analysis of the chloroplast proteins of Euglena and showed that 33% ofthe protein bands resolved on ID-gels are composed of multiple isoelectric species.…”

“…In 1975, O'Farrell (24) introduced a highly sensitive 2D-technique which separates proteins by pI on an IEF gel in the first dimension and then by molecular weight on an SDS-polyacrylamide slab gel in the second dimension. Recently, 2D-techniques, including that of O'Farrell (24), have been used with plants to compare the composition of seed polypeptides in different peanut cultivars (4) and to analyze the protein composition of various chloroplast subfractions from spinach (9,10,23,26,28), Chlamydomonas (9,10), pea (16) and bean (2).…”