Abstract
Acyloxyacyl hydrolase, a leukocyte enzyme previously has been shown to catalyze the hydrolysis of secondary (acyloxyacyl-linked) fatty acyl chains from the nonreducing glucosamine of the lipid A region of rough Salmonella typhimurium lipopolysaccharide (LPS). We describe here the activity of this enzyme toward smooth S. typhimurium LPS and LPS from Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Acyloxyacyl hydrolase released the secondary acyl chains from all of these lipopolysaccharides, regardless of the location of the acyloxyacyl linkage on the diglucosamine backbone or the structure of the acyl chains. The two acyloxyacyl linkages present in each LPS molecule apparently were hydrolyzed separately, so that free fatty acids released from the different sites accumulated at different rates. The purified enzyme also removed greater than 90% of the secondary acyl chains in each LPS, indicating that the enzyme acts not only on intact LPS but also on LPS molecules that have only one secondary acyl chain. The enzyme did not release the glucosamine-linked 3-hydroxyacyl chains. The specificity and versatility of the enzyme for cleaving acyloxyacyl linkages suggest that it may be a useful reagent for studying the structure and bioactivities of lipopolysaccharides with diverse carbohydrate and lipid A structures.
Highlights
Medical Center, Acyloxyacyl hydrolase, a leukocyte enzyme, previously has been shown to catalyze the hydrolysis of secondary fatty acyl chains from the nonreducing glucosamine of the lipid A region of rough Salmonella typhimurium lipopolysaccharide (LPS)
Analysis of free fatty acids recovered at various times during incubation of the LPS from bacteria grown at 13 “C with acyloxyacyl hydrolase showed that palmitoleate was released more slowly than myristate or laurate (Fig. 4B)
In each LPS at least two of the 3-hydroxyacyl chains are substituted by secondary fatty acyl of Lipopolysaccharides chains of 12 to 16 carbons to form acyloxyacyl groups; in some cases the secondary acyl chains are hydroxylated or unsaturated
Summary
The following broth media were used: for E. coli and S. typhimurium, proteose peptone-beef extract [15]; for H. influenzae, brain heart infusion (Difco) supplemented with 10 fig/ml hemin and nicotinamide adenine dinucleotide; for P. aeruginosa and for N. meningitidis, tryptic soy (Difco); for N. gonorrhoeae, Morse medium [16]. Elson-Morgan reaction [22]; the specific activity of the glucosamine in the LPS was calculated Release of 3H-labeled fatty acids was calculated by correcting for intact LPS taken up with the chloroform < 2.5% of added LPS) and for spontaneous release of fatty acids (determined in control tubes, containing the same amount of substrate and reaction mix, but no enzyme; ~0.7% of ‘H in the LPS). 3-OH-120 3-0(12:0)-14~0 allowed determination of LPS fatty acid specific activities Equation for the curve the estimated were compared yloxyacyl myristate, bonds that link the secondary acyl chains, laurate and to the primary (glucosamine-linked)
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