In a clinical isolate of Serratia marcescens different states of low and high resistance to different beta-lactam antibiotics considered to be beta-lactamase-stable, viz. cefotaxime, ceftizoxime, ceftazidime, aztreonam, cefoxitin and imipenem, were found to be connected with the presence of constitutively overproduced, chromosomally encoded beta-lactamase at concentrations in the bacterial periplasm of 0.4 and 0.9 mM, respectively. All the antibiotics were degraded by the beta-lactamase. However, kinetic constants varied widely: k(m) from 92 to 0.012 microM and k(cat) from 3.4 to 2x10(-4)s(-1). The relative contributions to resistance by the functioning of periplasmic beta-lactamase, resynthesis of this enzyme, and limitation of antibiotic penetration by the bacterial outer membrane were analysed by computer simulations according to steady-state and non-steady-state models of interactions in the periplasm. Results for cefotaxime, ceftizomime, ceftazidime, aztreonam and latamoxef revealed overproduced beta-lactamase as the sole cause of the state of low resistance while antibiotic permeability was the same as in non-resistant S. marcescens strains. In contrast, high resistance was due to beta-lactamase action and decreased permeability of antibiotics. For resistance to aztreonam, only, immobilization of the antibiotic as covalent acyl-enzyme by newly synthesized beta-lactamase was essential. For cefoxitin, ampicillin and imipenem the analyses indicated that additional resistance factors may play a role, e.g. induction of beta-lactamase.

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Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ET, a nonselective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-'251]iodotyrosyl ET-1 (*"I-ET-l) binding, with affinities comparable to those of the wild-type receptors (apparent K, = 1-6 X lo-").An average of 10'-106 binding sitedcell was calculated for the wild-type and mutant forms. In competition experiments using 'Z'I-ET-l and unlabeled ET-3, an ET,-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ET,. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ET, chimeras where the symmetrical exchange had been operated. Using 1251-ET-l and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the Nterminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.Cloning and sequencing of the three endothelin(ET)-receptor subtypes ETA, ET, and ET, [l -31 revealed remarkable structural similarities between them and the members of the guanine-nucleotide-binding-regulatory-protein(G-pr0-tein)-coupled, seven-transmembrane-domain receptor superfamily [4, 51. The hydrophobicity profile of the ET receptors is similar to that of bacteriorhodopsin [6], suggesting a common topological organisation. In this model seven hydrophobic domains span the lipid bilayer of the cell plasma membrane; the N-terminal part and three loops linking domains I1 and 111, IV and V, and VI and VII are located on the extracellular side whereas the three loops joining domains I and 11, 111 and IV, and V and VI, as well as the C-terminal tail are located on the cytoplasmic side (Fig. 1). The overall conservation across species is similarly high (= 90% amino acid identity) at least for the ETA and ET, subtypes, where data are available from human, rat and bovine cDNA clones [l, 2, 7-11]. In contrast, only 60% amino acid identity was calculated between ETA and ET, with the best conserved parts being the membrane-spanning domains and the cytoplasmic loops (Table 1).Bi...