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

Abstract

Eugkna chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrel (1975 J Biol Chem 250 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as weli as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Eugkna chloroplast polypeptides are resolved on one-dimensional sodium dodecyl-sulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the ftrst dimension, sodium dode-cylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Eugkna chloroplasts with acetone-ether and subsequent solubilization of polypep-tides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when 35S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, auto-radiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. 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 ' point; RuP2Case, ribulose-1,5-bisphosphate carboxylase. sensitive resolution. 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). 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. For that study (18), we used an IEF gel for the first dimension and a 10%o polyacrylamide slab gel for the second dimension. We present here additional studies which: (a) further improve the resolution of Euglena chloroplast polypeptides with 2D-gel electrophoresis; and (b) compare the degrees of resolution provided by ID-and 2D-gels when either 10%o or linear gradient polyacrylamide gels are used. MATERIALS AND METHODS Culture Conditions. Euglena gracilis strain Z (obtained from J. A. Gross) was grown to the late exponential phase of growth at 25 C in continuous light (0.0674,uE m 2s-' = 5,500 lux) on 200 ml of a defined sulfur-limited medium (12) supplemented with 22.15 ,Ag Na2SO4/ml (equivalent to 5 ,tg "S"/ml) and 0.1 M ethanol (13) as a carbon source. Some cultures were supplemented after 96 h of growth with 20 uCi [35S]Na2SO4/ml (New England Nuclear, MEX-041, carrier-free) in the late exponential phase and incu-bated in the light for 12 h more. Preparation of Chloroplasts. Cells were harvested by centrifu-gation, resuspended in 25 ml cold (4 C) KTM5 buffer (25 mm KCI, 50 mm Tris-HCl, pH 7.6, and 5 mM MgCl2) containing 0.25 M sucrose (3), washed three times in 25 ml of cold buffer by centrifugation at l,000g each time, and then resuspended in 5 ml cold buffer and ruptured in a French pressure cell (American Instrument Co.) at 6,000 to 8,000 p.s.i. All subsequent steps were done at 0 to 4 C. Lysates were homogenized in a Dounce homog-enizer (5 strokes with a B pestle, A. H. Thomas Co.) and centrifuged at 500g for 2 min. The supernatant fraction was saved. Two ml ofcold buffer was added to the pellet which was rehomogenized (5 strokes with a TenBroeck homogenizer) and then centrifuged at 500g for 2 min. The supernatant fraction was added to the previous one and centrifuged at l,500g for 10 min (20). The resultant crude chloroplast pellet was washed three times by resuspension in 5 ml cold buffer and centrifugation at 2,000g for 15 min each time. The final washed chloroplast pellet was resus-623 GILBERT AND BUETOW pended in 2 ml cold buffer, extracted once with acetone (80% final concentration) and centrifuged at 5,000g for 5 min. The resultant pellet then was extracted twice by resuspension in 5 ml of anhy-drous ethyl ether followed by centrifugation at 5,000g for 5 min each time (19). The extracted chloroplast pellets were stored at-80 C until used.