The Amino Acids of the Cell Wall of Mycobacterium tuberculosis var. bovis, Strain BCG

A detailed investigation of the amino acids of cell wall preparations obtained by various methods from the BCG strain is reported. 1 “Non-peptidoglycan” amino acids represent about 15% of the weight of crude, delipidated cell walls (Table 1); trypsin-chymotrypsin treated, delipidated cell walls contain mainly the peptidoglycan amino acid alanine, glutamic acid and meso-2,2′-diaminopimelic acid, the ratio Ala/A2pm being 1.5 and the ratio Glu/A2pm being 2.3, as compared with the expected values of 2 and 1 for a classical peptidoglycan; there are, however, equal molar amounts of d-glutamic acid and diaminopimelic acid and the excess of glutamic acid is the l-form. 2 After enzymatic solubilization of the peptidoglycan by lysozyme or by Myxobacter AL1 enzyme 90% of the peptidoglycan amino acids are found in the soluble part; this has been fractionated on Sephadex G-50 and in most of the fractions obtained the ratio Glu/A2pm is 1, as expect ed, but the ratio Ala/A2pm is closer to 1 than to 2, indicating some other, not yet defined cross link to diaminopimelic acid. 3 The insoluble residue obtained after lysozyme or Myxobacter AL1 enzyme digestion contains a poly (l-glutamic acid); partial acid hydrolysis allows the solubilization of various l-glutamic acid oligopeptides, which have been studied by mass spectrometry, after N-acetylation and per-methylation; Fig. 3 shows a partial mass spectrum obtained from a peptide containing 6 or 7 glutamic acid residues, proving the sequence Glu-Glu-Glu-Glu; pyroglutamic acid is observed in all spectra, indicating that at least the N-terminal glutamic acid residue is in α-linkage. The intact polymer could not be isolated, but the largest fragment obtained has an average chain length of eleven glutamic acid residues. 4 The presence of l-glutamic acid polymers seems to be restricted to human and bovine strains of Mycobacteria and to Mycobacterium kansasii.