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A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

For elucidation of a relationship between chemical structure and biologic activity of lipid A, syntheses of 13 derivatives of glucosamine beta (1-6) disaccharide, which were acylated at 3, 4, and 6'-hydroxyl groups and/or phosphorylated at positions 1 and 4', were carried out. The results showed the importance of the presence of the phosphate group at either position 1 or 4' and of the beta-hydroxyl group in acyl function, particularly in N-linked fatty acids, for the exhibition of biologic activities characteristic of lipid A. New evidence on the positions of fatty acids in natural lipid A of Escherichia coli species obtained by means of 2D-H nuclear magnetic resonance was also demonstrated; the findings indicate that the 3'-hydroxyl group is acylated and the 6'-hydroxyl group in the molecule must be free.

The specificity of monoclonal antibodies directed against the Salmonella minnesota R595 lipid A and the structural requirements of lipid A epitopes were studied with chemically synthesized lipid A analogues by enzyme-linked immunosorbent assay and its inhibition test. The results suggest that lipid A has specific and common epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.

Structural principles of the three regions of the lipopolysaccharide molecule are briefly discussed. One of the recently available, chemically synthesized lipid A analogues has been tested for biologic activities. The preparation was found to be toxic, pyrogenic, and mitogenic. The amounts, however, needed for expression of effects similar to those of lipid A were about 500 times higher. These investigations are aimed at verifying the proposed structure of lipid A and at evaluating structure-activity relationships.