The mode of action of a semisynthetic glycopeptide active against vancomycin-resistant bacteria has been investigated. It is shown that the antibiotic, biphenylchloroeremomycin or LY307599, dimerizes strongly and anchors to membranes. It is hypothesized that these two locating devices, previously identified by us when acting separately, might combine to give enhanced binding at a cell surface. This hypothesis is tested experimentally by showing that glycopeptides can bind cell-wall precursor analogues from resistant bacteria (terminating in -D-lactate) in a similar manner to those from susceptible bacteria (terminating in -D-alanine) and by using model cell surfaces where the benefits of dimerization can be expressed and studied. These model systems use vesicles to represent the cell membrane, to which cell wall analogues are anchored Via a docosanoyl chain, so mimicking the arrangement encountered at the cell surface. Using 1 H NMR spectroscopy, we demonstrate enhanced binding due to dimerization and propose that this enhancement will act cooperatively with membrane anchoring in biphenylchloroeremomycin.

Factor VII (FVII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation. A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVII activity when tested in a one- stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVII antigen. Variant FVII purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of normal FVII. Also, the variant FVII was unable to compete with wild-type FVII for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinant human TF to the variant FVII compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus' gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the FVII molecule is important for its function.

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