Phenol-Extracted Plant Proteins Can Be Renatured and Assayedin Gelfor Protein Kinase Activity

Abstract

Extraction of proteins by phenol has been shown to be a rapid and reproducible method for the preparation of samples for two-dimensional gel electrophoresis (1–3). An advantage of this method, which has been commercially exploited (4), is that it yields an aqueous phase containing the extracted nucleic acids and thus allows for the simultaneous analysis of gene expression at RNA and protein levels on the same experimental sample. For protein analysis, a further advantage of phenol extraction is the rapid inactivation of proteases and protein-modifying enzymes. Thus, this method has been successfully used for analyzing the in vivo modulation of protein synthesis (2) and protein phosphorylation (3) in response to environmental stimuli. However, phenol extraction may result in reversible or irreversible inactivation of proteins of interest. In the case of reversible inactivation, one should be able to renature the phenol-extracted enzyme protein and determine its activity. Here, we have tested this possibility by analyzing protein kinase activity of proteins renatured after phenol extraction. We show that phenol extraction is an efficient and cost-effective method for analyzing protein kinase activity in polyacrylamide gels. To demonstrate the relative efficiency of phenol in sample preparation, we compared the in gel renaturable activities of protein kinases extracted by phenol to those extracted by utilizing Triton or sodium dodecyl sulfate (SDS). Three-week-old alfalfa seedlings were used as experimental material. Since cold acclimation (exposure to low but nonfreezing temperatures) is known to alter levels of protein kinase transcripts and activities (5, 6), one set of seedlings was cold acclimated for 6 h by placing them at 4°C. Preparation of protein samples was carried out at 4°C as follows. Extraction with phenol. Extraction was essentially as described elsewhere (1), except for the modifications indicated. Seedlings were ground (3 ml/g fresh wt) in Tris– HCl-buffered phenol, pH 8 (7). Then an equal volume of buffer A (0.05 M Tris, pH 8.0, 0.002 M EDTA, 0.7 M sucrose, 0.5 M NaCl, 0.07 M 2-mercaptoethanol) was added and vigorously mixed with the phenol-containing homogenate. Phases were separated by centrifugation at 10,000g for 5 min. Proteins in the phenol phase (upper) were precipitated with 4 volumes of 0.1 M ammonium acetate in methanol. The protein precipitate was washed three times with 0.1 M ammonium acetate in methanol and once with acetone, dried under vacuum, and resuspended in Laemmli’s sample buffer (8). Extraction with Triton. Seedlings were ground (2 ml/g fresh wt) in buffer B [0.05 M Hepes, pH 7.2, 0.04 M EDTA, 0.001 M EGTA, 0.001 M 4-(2-aminoethyl)benzenesulfonyl fluoride (ICN, Costa Mesa, CA), 0.005 M cysteine, 0.001 M benzamidine, 0.01 M NaF, 0.25 M sucrose, and 0.024 M 2-mercaptoethanol]. Triton X-100 was added to the homogenate to a final concentration of 2% (v/v), and the mixture was stirred for 10 min. The stirred homogenate was cleared by centrifugation at 12,000g for 15 min and the supernatant filtered through glass wool. For SDS–polyacrylamide gel electrophoresis (SDS–PAGE), aliquots of the filtrate were mixed with an equal volume of 2X Laemmli sample buffer (8). Extraction with SDS. Seedlings were ground (2 ml/g fresh weight) in buffer C (0.125 M Tris, pH 6.8, 4% SDS, 1.7 M 2-mercaptoethanol). The homogenate was incubated at 95°C for 5 min (2 min per each milliliter of homogenate) and cleared by centrifugation at 12,000g for 5 min. For SDS– PAGE, aliquots of the supernatant were mixed with an equal volume of 2X Laemmli sample buffer (8). To assess renaturable proteins, protein extracts were fractionated by SDS–PAGE, renatured, and assayed for protein kinase activity in situ. Aliquots of each sample, 25 mg of protein, were separated on minigels containing 10% polyacrylamide and 0.5 mg/ml casein or histone type III-S. Casein or histone was added at the same concentration as above in the electrode buffers to prevent changes in their concentration in the gel during electrophoresis. Protein concentrations were determined by the Coomassie protein assay reagent kit of Pierce (Rockford, IL) and further verified for equal loading by SDS–PAGE (Fig. 1, top). After electrophoresis, washing, denaturation, and renaturation of proteins in the gel were as described (9). Protein kinases were 1 To whom correspondence should be addressed. Fax: (514) 3985069. E-mail: RDHINDSA@BIO1.LAN.MCGILL.CA.