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Abstract. Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli. This method shows 21 proteins in the 30S, and 34 proteins in the 50S, subunit.Waller' showed that the ribosomes of Escherichia coli contain a large number of protein components. Determination of the exact number of ribosomal proteins is important for precise knowledge of the structure and function of the ribosomes. Combinations of two or more conventional methods, such as disk electrophoresis on acrylamide gels under different conditions, ion exchange, and molecular sieve chromatography, indicate that the 30S and 50S ribosomal subunits contain 20 and 30-35 proteins, respectively.2-6 We have recently developed a two-dimensional (2D) polyacrylamide gel electrophoresis procedure' by means of which all of the proteins of the ribosomal subunits can be rapidly and reproducibly separated in one operation. This paper describes the results of this procedure for the separation of proteins derived from 30S and 50S subunits, as well as 70S ribosomes, of E. coli. We find that the 30S subunits contain 21 proteins, and the 50S subunit as many as 34 proteins. A numbering system is proposed based on the position of the protein spots on the 2D electropherogram.Materials and Methods. Electrophoresis: Two-dimensional electrophoresis was performed as described.7 The acrylamide concentration in the first dimension was 8%, except in a few cases where it was 4%. Tris-EDTA-boric acid buffer, pH 8.6, and less frequently, pH 9.6, was used. The electrophoretic conditions in the second dimension were always 18% acrylamide gel in acetate buffer, pH 4.6.Preparation of the proteins: The 70S ribosomes obtained by differential centrifugation were washed with 0.5 M NH4Cl, 1 mM Mg++. Subunits were separated by sucrose gradient centrifugation in a zonal rotor, B XV. Proteins were extracted from ribosomes by treatment with 67% acetic acid in the presence of 30 mM Mg++ (ref. 4). The RNA precipitate was washed with extracting solution for more quantitative extraction of the proteins. No further proteins were detected in the RNA precipitate after washing with 67% acetic acid in the presence of higher salt concentrations. The proteins were lyophilized and dissolved in the components of the sample gel. The solubility of the proteins improved when the extracted proteins, instead of being lyophilized, were directly dialyzed against the gel components. When mentioned in the text, the proteins were oxidized with performic acid or reduced and alkylated with iodoacetamide.8Results and Discussion. The protein mixtures extracted from a large number of ribosome preparations were compared by two-dimensional electrophoresis.

Abstract. M\lolecular weights of the isolated proteins from the 30S and 50S ribosomal subunits of Escherichia coli were determined by two independent methods: polyacrylamide gel electrophoresis with sodium dodecylsulphate and equilibrium sedimentation. The values for the molecular weights determined by gel electrophoresis range from 10,900 to 65,000 for the proteins of the 30S subunit and from 9,600 to 31,500 for those of the 50S subunit, with number averages of 19,000 and 16,300, respectively, in agreement with those obtained by equilibrium sedimentation.The ribosomes of Escherichia coli consist of a number of different proteins,' some of which have been isolated and characterized.2-8 According to recent studies there are about 55 individual proteins in the 70S ribosomes of E. coli.9 l0 The molecular weights of some 20 proteins isolated from the 30S subunit have been determined by equilibrium sedimentation.8 Furthermore, molecular weights have been determined, by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels, for partially fractionated ribosomal proteins.9 The present paper describes the determination of the molecular weights of the individual ribosomal proteins11-'3 by two independent methods. Materials and Methods. The ribosomal proteins of E. coli K12, strain A19, were isolated as described."-'3 All were homogeneous as judged by one-and two-dimensional polyacrylamide gel electrophoresis. These proteins were also found to be homogeneous (unpublished data) when tested by cellulose acetate electrophoresis.14 SDS-polyacrylamide gel electrophoresis was conducted essentially by the procedure of Weber and Osborn."5 However, the gels were not polymerized in columns but in slabs. Samples and a bromophenol-blue marker were applied to small slits in the gel. The bromophenol blue mobility was taken as a standard; all protein migrations were normalized to this value. Using the two-dimensional apparatus of Kaltschmidt and Wittmann,'6 it was possible to simultaneously run five gel-slabs (a total of 75 samples); thus, identical conditions were created for all of the ribosomal proteins in one run. Electrophoresis was usually for 17 hr at 1.5 V/cm. Each gel slab was then removed from its individual frame and the distance the bromophenol blue band had traveled was marked. Proteins were stained with Coomassie brilliant blue.

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 950/, as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps.Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity.The protein moiety of ribosomes is very complex [I]. 30 S ribosomal subunits of Escherichia coli contain 21 proteins which were isolated and studied in various laboratories [ 2 -141. The number of ribosomal proteins in E . coli 50s subunits is about 50°/, higher than in 30 S subunits [8,10,15]. Because of the large number of 50 S proteins and their relatively similar chemical and physical properties, separation and isolation are very difficult and only possible by a combination of various methods which separate according to different criteria, e.g. protein charge and size as well as interaction of ribosomal proteins with each other and/or with ribosomal RNA in the particle.The present paper describes the isolation of 50s proteins in relatively large quantities (6-90 mg). The chemical, physical and immunological properties of the ribosomal proteins isolated by these methods are described elsewhere [I1 -14,271. MATERIAL AND METHODSIsolation of ribosomes and subunits from E . coli K12 (strain A19) and separation of proteins by CMcellulose and Sephadex G-I00 were described earlier [16]. Treatment of 50s subunits with LiCl followed previous procedures [17-221 with the following modifications.12g of 50s subunits were brought with 0.05M Tris-HC1 pH 7.8, 0.01 M Mg2+ to a concenbration of 10 mg/ml and dialyzed a t 4 "C for 4 h against 2 x 5 1 of 0.05 Tris-HC1 pH 7.8, 0.01 M Mg2+ and then for I2 h against 3 x 5 1 of the same buffer with 0.1 M Mg2+.One volume (= 1200ml) of 50s and one volume of cold 4 M LiC1, 0.1 M magnesium acetate, were mixed and stirred for 8 h in ice water. After centrifugation for 16 h a t 25000 rev./min in Spinco 42 rotors the supernatant (= UI) was dialyzed for 18 h against 4 x 5 1 100/, acetic acid plus 0.1 O/, mercaptoethanol.The sediment was resuspended in 1200 ml of cold 0.01 M Tris-HC1 pH 7.8, 0.1 M Mg2+, and dialyzed a t 4 "C for 4 h against 5 1 of this buffer and then for I2 h against 3 x 5 1 0.05 M Tris pH 7.8, 0.1 M Mg2+. The suspension was diluted 1 : 1 with cold 7 M LiC1, 0.1 M Mg2+, stirred in ice water for 8 h and centrifuged as described. The supernatant (= UII) was dialyzed as described for UI.The sediment was resuspended in 600ml cold distilled water. The...

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