Extracellular mobilization of Group IIA 14-kD phospholipase A 2 (PLA 2 ) in glycogen-induced rabbit inflammatory peritoneal exudates is responsible for the potent bactericidal activity of the inflammatory fluid toward Staphylococcus aureus (1996. J. Clin. Invest. 97:250-257). Because similar levels of PLA 2 are induced in plasma during systemic inflammation, we have tested whether this gives rise to plasma bactericidal activity not present in resting animals. Baboons were injected intravenously (i.v.) with a lethal dose of Escherichia coli and plasma or serum was collected before and at hourly intervals after injection. After infusion of bacteria, PLA 2 levels in plasma and serum rose Ͼ 100-fold over 24 h to ‫ف‬ 1 g PLA 2 /ml. Serum collected at 24 h possessed potent bactericidal activity toward S. aureus, Streptococcus pyogenes, and encapsulated E. coli not exhibited by serum collected from unchallenged animals. Bactericidal activity toward S. aureus and S. pyogenes was nearly completely blocked by a monoclonal antibody to human Group IIA PLA 2 and addition of purified human Group IIA PLA 2 to prechallenge serum conferred potent antistaphylococcal and antistreptococcal activity equal to that of the 24 h postchallenge serum. PLA 2 -dependent bactericidal activity was enhanced ‫ف‬ 10 ϫ by factor(s) present constitutively in serum or plasma. Bactericidal activity toward encapsulated E. coli was accompanied by extensive bacterial phospholipid degradation mediated, at least in part, by the mobilized Group IIA PLA 2 but depended on the action of other bactericidal factors in the 24-h serum. These findings further demonstrate the contribution of Group IIA PLA 2 to the antibacterial potency of biological fluids and suggest that mobilization of this enzyme during inflammation may play an important role in host defense against invading bacteria.( J. Clin. Invest. 1998. 102:633-638.)

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Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template.Nonaqueous enzymology has emerged in the last decade as a promising research field (for reviews, see, for example, refs. 1-3), not only because of a large number of widely recognized advantages (2) but especially as a result of the realization that enzymes placed in nearly anhydrous environments can exhibit exciting features, such as an increased conformational rigidity, which confers on the protein greatly enhanced (thermo)stability (4), or a profoundly altered substrate specificity (5). In particular, one interesting facet of the exploitation of the drastically lowered flexibility of a protein in very low-water media is the so-called ligand-induced enzyme "memory" (or simply ligand memory) (6), presumably based on inducing, upon ligand binding, an enzyme conformational change that (after freeze-drying of the solution) will be preserved in the lyophilized sample when exposed to anhydrous solvents. This strategy, also referred to as molecular (bio)imprinting (7,8), has been extended to nonenzymic proteins and other macromolecules (9, 10) and shares a conceptual analogy with the previously known molecular imprinting of polymers (for a review, see ref. 11). In the case of enzyme (bio)imprinting, only scarce efforts have been reported so far, which have focused on a very few related proteases and made use of competitive inhibitors (amino acid derivatives) as print molecules (6-8). Although these preliminary results seem indeed encouraging in terms of an activity enhancement or even an apparent (stereo)selectivity alteration in the organic milieu, the nature of the presumptive conformational changes induced has not been characterized, and the molecular reasons for the experimental observations are still open to debate (12).Paradoxically, in this context, no effort aimed at assessing the possibility of imprinting lipolytic enzymes has been reThe publication costs of th...

The role of the membrane lipid composition and the individual Trp residues in the conformational rearrangement of gramicidin A along the folding pathway to its channel conformation has been examined in phospholipid bilayers by means of previously described size-exclusion high-performance liquid chromatography HPLC-based strategy (Bañó et al. (1991) Biochemistry 30, 886). It has been demonstrated that the chemical composition of the membrane influences the transition rate of the peptide rearrangement from double-stranded dimers to beta-helical monomers. The chemical modification of Trp residues, or its substitution by the more hydrophobic residues phenylalanine or naphthylalanine, stabilized the double-stranded dimer conformation in model membranes. This effect was more notable as the number of Trp-substituted residues increased (tetra > tri > di > mono), and it was also influenced by the specific position of the substituted amino acid residue in the sequence, in the order Trp-9 approximately Trp-13 > Trp-11 > Trp-15. Moreover, it was verified that nearly a full contingent of indoles (Trp-13, -11, and -9) is necessary to induce a quantitative conversion from double-stranded dimers to single-stranded monomers, although Trp-9 and Trp-13 seemed to be key residues for the stabilization of the beta-helical monomeric conformation of gramicidin A. The conformation adopted for monomeric Trp --> Phe substitution analogues in lipid vesicles resulted in CD spectra similar to the typical single-stranded beta6.3-helical conformation of gramicidin A. However, the Trp --> Phe substitution analogues showed decreased antibiotic activity as the number of Trp decreased.