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Protein L11 was isolated from the 5 0 4 subunit of Esclzericlzia coli ribosomes, using two salt extractions and two chromatographic separations on CM-cellulose. The unusual behavior of the protein when run on sodium dodecyl sulfate electrophoresis showed multiple bands.The complete primary structure of protein L11 is presented in detail. Its sequence was derived from peptides obtained by digesting the protein with trypsin, chymotrypsin, thermolysin, Stuplzylococcus aureus protease and, after modification, with trypsin. Chemical cleavage was performed with cyanogen bromide. Sequencing of the various peptides was achieved by manual micro-dansyl-Edman degradations and automatic methods. The N-terminal residue of the protein is blocked and was not degradable in the liquid-phase sequenator by the Edman method. It was identified by a combination of enzymatic cleavage and mass spectrometry.Protein L11 contains three methylated amino acid residues, a N"-trimethylalanine, and two residues of N"-trimethyllysine. Their behaviour and influence in the sequence elucidation are described. The protein contains 141 amino acid residues and has a molecular weight of 14874. Secondary structure predictions of the protein are given, and its sequence is compared with those of other E. coli ribosomal proteins.

The complete amino acid sequence of the murine t2a heavy chain CBPC-101 Fc region of allotype IgIb was determined by automated and manual Edman degradation procedures. Both chemical and enzymatic cleavages were used to obtain peptides which were purified by gel filtration followed by high-pressure liquid chromatography.The sequence was in good agreement with that predicted from the nucleotide sequence of a y2a gene of allotype IgIb except for two positions. Comparison with published data on MOPC 173 y2a heavy chain of allotype Igla revealed 8 amino acid substitutions in the CH2 domain (7% differences) and 28 substitutions (27%) in the CH3 domain. Many of the observed interchanges occur at positions at which murine heavy chains of other classes also differ from the y2a chains. Our data suggest that the divergence of the y2a Igla gene found in BALB/ c mice and of the y2a IgIb gene found in CB 20 mice must have occured long ago and that the CH2 domain was much more conserved than the CH3 domain.Mouse immunoglobulin heavy chains display antigenic differences which are detected by alloantisera. These polymorphic determinants (allotypes) have been found on six of the eight known immunoglobulin isotypes (1, 2). Unlike the rabbit or human allotypes which have been extensively characterized both at the serological and the structural level, the mouse allotypes have been mainly defined by alloantisera specificities. Most recently, the nature of the antigenic determinants corresponding to the Igla and the Igib allotypes found on the -y2a heavy chains has been reexamined by the use ofa panel ofmonoclonal antibodies (3). The serological studies suggest that most of the determinants are confined to the hinge and COOH-terminal half (Fc) of the y2a polypeptide chain. The precise structural basis of the mouse allotypes, however remains poorly defined.We suggested in a preliminary report (4) that the structural differences between the Fc regions from Igla and Iglb y2a chains are probably extensive. We found 3 substitutions in the first 25 positions of the Fc region. In the present study, the complete sequence of the Fc region of the Iglb y2a chain of protein CBPC 101 is compared to the previously published corresponding sequences of protein MOPC 173, a -y2a chain of allotype Igla (5), of protein MPC 11, a y2b chain (6), and of protein MOPC 21, a yl chain (7). Both the protein and the nucleic acid sequences, when available, were used for the comparison.When comparing the two y2a chains which differ only by their allotype, we found a total of36 differences in 217 positions examined in the Fc region; 8 (7%) of these substitutions are located in the CH2 and 28 (27%) are located in the CH3 domain. Preparation and Purification of the CBPC 101 Immunoglobulin. The CBPC 101 immunoglobulin was purified essentially as described (4). In short, the myeloma protein was precipitated from ascites fluid with ammonium sulfate at 45% saturation. The resuspended pellet was dialyzed against 0.01 M KH2PO4/NaOH pH 8.0 buffer and centrifuged at ...

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Three methylated amino acid residues, one residue of TV-trimethylalanine and two of 7V e ,jV e^/ V e -trimethyllysine residues, are present in protein LI 1. The methods used for the identification and location of these unusual amino acids in the sequence of protein LI 1 are described. Temperature and pH modifications to the eluting buffers enabled the detection of the methylated derivatives of lysine and arginine with a Durrum analyser using routine 90 min amino acid analyses. The presence of 7V e ,7V e -dimethyllysine in the hydrolysate of proteins, was revealed by ascending chromatography on thin-layer cellulose plates. The blocked TV-terminal amino acid of protein LI l^^^V-trimethylalanine, although non-volatile, was identified by field desorption mass spectrometry. The identification was confirmed by comparing the jV-terminal dipeptide of protein LI 1 with the synthesised dipeptide Me 3 Ala-Lys. The behaviour of these methylated amino acids during sequence analysis is described. Identifizierung von methylierten Aminosäuren bei der Sequenzanalyse: Anwendung auf das Protein Lll aus Escherichia-coli-RibosomenZusammenfassung: Drei methylierte Aminosäu-rereste, ein Rest W,AfN-Trimethylalanin und zwei Reste AV^>N e -Trirnethyllysin, sind im Protein L11 enthalten. Die Methoden, die zur Identifizierung und Lokalisierung dieser ungewöhnlichen Aminosäuren in der Sequenz des Proteins Ll l geführt haben, werden beschrieben. Der Nachweis der methylierten Derivate von Lysin und Arginin erfolgte in einem Aminosäu-renanalyser der Fa. Durrum durch Variation von Temperatur und pH der Elutionspuffer in einer 90 min dauernden Auftrennung. Der Nachweis von A re^Ve -Dimethyllysin im Proteinhydrolysat wurde durch aufsteigende Dünnschichtchromato-graphie erreicht. Die blockierte TV-terminale Aminosäure von Protein Ll l, TV^TV-Trimethylalanin, wurde mittels Felddesorptionsmassenspektrometrie identifiziert, da diese Aminosäure nicht flüchtig ist. Als weiterer Nachweis für A^A^TV-Trimethylalanin wurde das ./V-terminale Dipeptid von Protein Lll mit synthetisch hergestellter Substanz, Me 3 Ala-Lys, verglichen. Die Eigenschaften dieser methylierten Aminosäuren werden beschrieben.