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A new, simple procedure is described for the direct gas chromatographic determination of sphingolipid bases as their trimethylsilyl derivatives. This method is used to study the nature of the mixture of bases produced by acidic methanolysis of ceramides, sphingomyelins, free sphingolipid bases, andN‐acetyl derivatives of the latter. Cleavage of sphingolipids with anhydrous methanolic hydrogen chloride results in the production of considerable quantities of secondary products from sphingosine, such as theO‐methyl ethers, which are not formed during methanolysis of free sphingosine. A modified reagent for methanolysis, containing methanol, water and hydrochloric acid, reduces the yield of these by‐products to low levels. Preliminary purification of the sphingolipid bases, before gas chromatography, is achieved by chromatographic procedures. Evidence is presented for the occurrence of C16‐sphingosine in sphingomyelin from human blood plasma.

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The peptidoglycan of bacterial cell walls is synthesized in a reaction mechanism in which a membrane-bound lipid serves as a carrier for the sugars and amino acids that eventually become part of the peptidoglycan.' One intermediate in the reaction cycle is represented as lipid-P-P-disaccharide-pentapeptide. This intermediate is common to a number of different bacteria. However, its disaccharidepentapeptide moiety (GlcNAc-MurNAc-L-ala-D-glu-L-lys-D-ala-D-ala)I becomes modified in various ways in different organisms.3 The modified disaccharidepeptide moiety is then transferred to an endogenous acceptor with introduction of a new unit into a growing peptidoglycan.The purpose of the present paper is to report that lipid-P-P-disaccharide-pentapeptide has been obtained in a highly purified form from Micrococcus lysodeikticus, and that the nature of the lipid moiety of this intermediate has been elucidated.Soon after the discovery of the occurrence of a lipid intermediate in peptidoglycan synthesis, a lipid carrier in the biosynthesis of the lipopolysaccharide of the cell wall of Salmonella was described.4' I The structure of this substance is reported in a paper by Wright et al-'Isolation of Lipid-P-P-Disaccharide-Pentapeptide from M. lysodeikticus.-Largescale preparation of the lipid intermediate was carried out using techniques essentially the same as those described previously.7 Particulate enzyme was prepared after lysozyme lysis of cells of M. lysodeikticus8 and incubated with the uridine nucleotide substrates to generate the lipid intermediate. The incubation mixture contained 1000 ml of particulate enzyme prepared in 0.05 M Tris-HCl buffer, pH 7.4, and 0.01 M MgCl2, 1.7 ml of 12 mM UDP-MurNAc-pentapeptide (containing 8.7 X 106 cpm of C14-lysine),9 8 ml of 10 mM UDP-GlcNAc and 30 ml of 5 per cent deoxycholate. Enzyme prepared after lysozyme lysis of cells of M. lysodeikticus is able to synthesize the lipid intermediates, but is unable to utilize these intermediates for peptidoglycan synthesis. Deoxycholate was included in the incubation mixture in order to minimize the addition of endogenous glycine to the lipid intermediate.8After incubation at 200 for 30 minutes, the mixture was extracted with 1 liter of nbutanol:6 M pyridinium acetate (2:1), pH 4.2, followed by two extractions with the same volume of n-butanol. The combined extracts were back-washed with 2 liters of water and then taken to dryness and redissolved in 50 ml of chloroform: methanol (1:1). The yield was 3 X 106 cpm (34%, based on UDP-MurNAc-,C4-pentapeptide).The extract was placed on a column of O-(diethylaminoethyl) cellulose (DEAEcellulose) (2.5 X 25 cm) and eluted as previously described.7 The radioactive eluate obtained with methanol:6 M pyridinium acetate, pH 4.2 (1: 1), was pooled 1878