Leucyl-phenylalanine

The biological action of several X-Phe-D-Leu-Phe-D-Leu-Z (X=3',5'-dimethylphenyl-ureido; Z=Phe, Lys, Glu, Tyr) analogues was analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-D-Leu-Phe-D-Leu-Phe analogues obtained as C-terminal olo or amido derivatives and T-Phe-D-Leu-Phe-D-Leu-Phe analogues (T=thiazolyl-ureido) were also analysed. The activities of pentapeptide derivatives were compared with those of X-Phe-D-Leu-Phe-D-Leu-Phe chosen as reference antagonist. Our results demonstrate that X-Phe-D-Leu-Phe-D-Leu-Phe-olo, X-Phe-D-Leu-Phe-D-Leu-Glu and X-Phe-D-Leu-Phe-D-Leu-Tyr are more active antagonists than X-Phe-D-Leu-Phe-D-Leu-Phe. The presence of Lys (X-Phe-D-Leu-Phe-D-Leu-Lys) seems, instead, to inhibit the formyl-peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor antagonist properties of T-Phe-D-Leu-Phe-D-Leu-Phe-OH.

A mild synthetic method for N-formyl-Met-Leu-Phe-OH (1) is described. After Fmoc solid phase peptide synthesis, on-bead formylation and HPLC purification, more than 30 mg of the fully (13)C/(15)N-labelled tripeptide 1 could be isolated in a typical batch. This peptide can be easily crystallised and is therefore well suited as a standard sample for setting up solid-state NMR experiments.

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier.

Designed octapeptides Boc-Leu-Val-Val-Aib-(D)Xxx-Leu-Val-Val-OMe ((D)Xxx = (D)Ala, 3a;(D)Val, 3c and (D)Pro, 5a) and Boc-Leu-Phe-Val-Aib-(D)Ala-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I' beta-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-(D)Pro segment, is shown to adopt a stable type I' beta-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a beta-hairpin conformation. Replacement of (D)Pro5 by (D)Ala/(D)Val (3a-c) results in sequences that form beta-hairpins in hydrogen bonding solvents like CD(3)OH and DMSO-d(6). However, in CDCl(3) evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl(3) and a significant population of both helices and hairpins in CD(3)OH and DMSO-d(6).