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SynopsisPeptide agonists covalently attached to tobacco mosaic virus exhibit such unusual properties as superpotency, superaffinity, enhanced resistance towards enzymic degradation, and prolonged action at the target cell. These properties can be exploited for the isolation by density-gradient centrifugation of membrane vesicles bearing specific receptors for the peptides and for radioactive and fluorescent labeling of cell-surface receptors. Our observations can be explained by cooperative-affinity phenomena caused by the deployment in space of the agonist molecules. DEPLOYMENT OF AGONIST MOLECULES AS A FACTOR IN BIOLOGICAL ACTIVITYSince early work on the conformation of cyclic peptides and on the organization of information in linear peptide agonists (for reviews, see Refs. 1-3), it has become increasingly clear that there exist causal relationships between the amino acid sequences of peptides, their conformational possibilities, their interactions with receptors, and their biological activity. In this review, we would like to point out the importance of another factor governing receptor interactions and biological activity, namely the deployment in space of groups of individual agonist molecules. Cooperative AffinityThe strong affinity of antibodies for membrane-bound antigens has been explained by the simultaneous binding of the two recognition sites on the antibody molecule to two antigen molecules connected through the membrane.4.5 Cooperative affinity effects of this type should prevail in any situation in which ligand and acceptor each possess more than one mutual recognition or binding site, provided that these sites are deployed in such a manner that simultaneous interaction is stereochemically feasible. A classical example of almost perfect complementarity is the DNA double helix, in which one single strand can be regarded as the ligand and the other as the acceptor molecule, both containing favorably deployed binding sites: the purine and pyrimidine bases.

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SummaryThe use of tobacco mosaic virus (TMV) covalently loaded with hormones or other small molecules for various purposes including receptor detection and isolation is proposed. The basic principle is that of cooperative affinity interactions involving (large) numbers of artificially introduced sites (e.g. hormones) of the modified virus on the one hand and membrane-bound sites (e.g. receptors) of a cell or of a cellmembrane particle on the other. In order to test the feasibility of such TMV/hormone conjugates, TMV carrying about 500 molecules of a biologically active a -melanotropin analogue was synthesized, and characterized by its aspect under the electron microscope, by its infectivity, its melanophore-stimulating activity, and its reaction with antisera against a -melanotropin. The observed hormonal activity is in accordance with the idea of cooperative affinity interactions.The chemical nature of membrane-bound receptors for drugs and hormones is still an unsolved problem of molecular biology. This paper is the first of a series investigating the use of tobacco mosaic virus (TMV) covalently attached to hormones, drugs, and other comparatively small molecules for the study of receptors. First, some pertinent chemical properties of TMV and of polypeptide-hormone receptors are reviewed and the concept of cooperative affinity labelling is introduced. It is then demonstrated that some 500 molecules of an a-melanotropin (a-MSH) analogue can be covalently attached to the virus particle without destroying the essential chemical and biological properties characteristic of the virus and the hormone. I )This work was supported by the Swiss National Science Foundation. Abbreviations: TMV= tobacco mosaic virus; EM. = electron microscopy; symbols for amino acids and peptides according to the IUPAC-IUB rules [ 11.

Adrenocorticotropin-(1-24)-tetracosapeptide was covalently attached to tobacco mosaic virus in two different manners: (i) through a handle near the C-terminus on tyrosine-(23) and (ii) through a handle at the N-terminus on serine-(1). Compounds of type (i) with their N-terminal message sequence freely exposed on the virion surface were considerably more potent for stimulating steroidogenesis in isolated adrenocortical cells than those of type (ii) with a more congested message. Conjugates with 50 or less hormone molecules per virion were less potent per peptide unit than the "free" handle-substituted hormones, whereas conjugates with 150 ACTH units exhibited superpotency effects. Superpotency disappeared when the substituted virions were disaggregated into (substituted) capsomers, suggesting influences of hormone clustering and virion geometry on biological activity. Superpotent stimulation was irreversible under conditions that immediately inhibited steroidogenesis by ACTH (dilution, addition of a peptide antagonist). Thus, superpotency might be caused by superaffinity arising from a slow rate of dissociation of the conjugates from the target cell receptors. The reason for the slow dissociation rate is still unclear: possible explanations include cooperative affinity, rapid internalization of the conjugate-receptor complexes, or decreased rates of peptide degradation at the receptor site.