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 These proteins were also found to be homogeneous (unpublished data) when tested by cellulose acetate electrophoresis.14 SDS-polyacrylamide gel electrophoresis was conducted essentially by the procedure of Weber and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.

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Mouse brain proteins were separated by two-dimensional electrophoresis (2-DE). The proteins of a section of the 2-DE pattern were blotted onto hydrophobic membranes and 43 of them were excised and hydrolyzed by liquid-phase hydrolysis. The amino acid composition of these proteins was determined by orthophthaldialdehyde precolumn derivatization and compared with the compositions of known proteins stored in the NBRF sequence database. An identification program named ASA was developed for this purpose. The ASA program includes correction and weighting factors, data reduction by molecular weight windows, and exclusion or inclusion of certain organisms as desired. As a control, eight test proteins and five well-known proteins from mouse brain, all separated by 2-DE, were correctly identified by the program. Out of the 43 brain proteins selected, 19 were identified with high confidence.

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