Heterogeneity in the distribution or binding of the membrane phospholipids was demonstrated in the membrane fragments released from Haemophilus parainfluenzae by treatment with ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane (Tris). The membrane fragments released early in the EDTA-Tris treatment contained two-to fivefold higher proportions of cardiolipin and phosphatidylglycerol and less phosphatidylethanolamine as well as phospholipids with threefold lower specific

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... ity of the phospholipid phosphate after a short pulse of 82p than were found in the residue. Heterogeneity was best demonstrated with shorter EDTA-Tris treatments and shorter periods of growth with 32p. EDTA-Tris treatment appeared to progressively strip phospholipids from the cells that were synthesized at progressively later times. The lipid composition of the limiting membrane of gram-negative bacteria is relatively simple (3). In Haemophilus parainfluenzae, 85% of the fatty acids are extractable with solvents and 95% of the extractable fatty acids are found esterified in phosphatidylethanolamine (PE), phosphatidyl glycerol (PG), cardiolipin (CL), phosphatidyl-serine (PS), and phosphatidic acid (PA) (7, 8). The non-extractable fatty acids are associated with the lipopolysaccharide (8). With a relatively simple lipid composition and a large number of membrane-associated activities necessary for metabolism, it might be possible to detect differences in the lipid composition or metabolism that could be correlated with different functions of the membrane. The lipids are closely associated with the electron transport system in H. parainfluenzae (9). Alterations in the composition of the respiratory system coincide with changes in phospholipid metabolism. The slowing of the turnover of the phospholipid phosphate, the change in incorporation of phosphate, the changes in the proportions of PE, PG, and CL, and the increase in the total phospholipid that occur when the electron transport system is modified suggest that lipid metabolism is a necessary part of the modification of the membrane (9). Perhaps these modifications begin at discrete sites in the membrane which could somehow be isolated and studied.