Peptide studies on two acidic proteins, A, and A,, from 50-S ribosomes of Escherichia coli I. Amino acid compositions of the tryptic peptides of the two proteins are identical, with 2. The N-terminal amino acid sequence of A,-protein is Ser-Ile-Thr-Lys, while that of 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent 4. I n 500/, of the polypeptide chains, in both A,-or A,-protein, it specific lysine residue is indicate a closely related primary structure. exception of the amino terminal peptide.A,-protein is N-acetyl-Ser-Ile-Thr-Lys.with the physical molecular weight estimate.replaced by s-N-monomethyl-lysine.As reported in the preceding paper and their biological activity [S] which seems to be related to the phosphate energy liberation in the translocase step.The main objective of this study has been the precise determination of the difference in chemical structure between the two proteins. Thorough analysis of the tryptic peptides of the protein revealed that both have probably an identical primary structure and that the difference between the two is only confined to an acetyl group on the amino terminal end of the A,-protein, which is absent in A,-protein.From the peptide compositions the chemical molecular weight of the A-proteins have been estimated.
MATERIALS AND METHODS
Preparation of Tryptic PeptidesThe purification of A-protein, isolation and amino acid composition of A,-and A,-protein have been described elsewhere [i].Unusual Abbreviation. Dansyl, 1-dimethyl-aminonaphthalcne-5-sulphonyl.2 mg protein, determined by the microbiuret assay [l], was dissolved in 1 ml lo/, (NH,),CO, (pH 8.85) to which 20 p1 of a I mg/ml solution of trypsin treated with ~-l-tosyl-amido-2-phenylethylchloromethylketone, (Serva, Heidelberg, Germany) in 0.001 N HC1 was added to start the reaction at 37 "C. A second aliquot of enzyme (20 pg) was added after 1 h, the total digestion time being 4 h, after which the digest was freeze-dried, dissolved in 1 .O ml water and freeze-dried again. This residue was used directly for peptide mapping. Tryptic peptides for column chromatography were prepared by a 6-h digestion in dilute ammonia on a Radiometer pH-stat a t p H 8.0,37 "C. The enzyme to substrate weight ratio was also 1 to 50. The mixture was then frozen and lyophilized once.
Separation of the Tryptic PeptidesPeptide maps were made essentially according to Clegg et al. [9]. High-voltage paper electrophoresis on Whatmann 3-MM paper was performed in a "Varso1"-cooled tank (Savant Instruments, Hicksville, U.S.A.) at 30 V/cm for 3 h with a buffer (pH 4.7) of pyridine-acetic acid-water (5:5: 390, by vol.). The solvent for paper chromatography was n-butanol-acetic acid-water-pyridine (15:3: 12: 10, by vol.) (Hills solvent). Peptides were detected by staining the paper with a 0.050/, ninhydrin-acetone solution a t 40 "C for 30 min. The coloured spots were subsequently eluted from the paper with ZOO/, pyridine in water.
