Tobacco mosaic virus as a carrier for small molecules: Artificial receptor antibodies and superhormones

Abstract

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.

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

“…Pretreatment The present results are in complete agreement with our earlier results on fluorescence labeling of these cells using a water-soluble class of fluorescent melanotropic macromolecular reagents (6, 7) as well as with those of Schwyzer and coworkers, who developed conjugates of MSH with tobacco mosaic virus (8)(9)(10)(11)(12). Using the melanotropin fluorescein conjugate, we demonstrated that both melanotic as well as amelanotic human melanoma cells expressed membrane melanocortin receptors (6,7).…”

“…In this respect, these results are similar to electron microscopic visualization of internalized receptor-ligand complex for a glucagon-serum albumin preparation labeled with 17 nanometer gold particles (23). These results are consistent with those obtained earlier using fluorescent tobacco mosaic virus-MSH conjugates (8)(9)(10)(11)(12) and fluorescent polyvinyl alcohol-MSH conjugates (6, 7).…”

“…This binding undoubtedly resulted from the multiplicity of the peptide ligand on these solid-phase probes, such that a single assembly of the bead-peptide conjugate is most likely able to interact simultaneously with more than one receptor on the cell membrane. This behavior has earlier been reported on interaction of tobacco mosaic virus-MSH conjugates with mouse melanoma cells (8)(9)(10)(11)(12). Similarly, certain classes of polyvalent antibodies that also are capable of establishing multiple interactions exhibit much higher binding affinities (22).…”

“…These reagents complement our previously reported fluorescent polyvinyl alcohol-conjugated (6, 7) MSH (8)(9)(10)(11)(12) constructs for use in receptor identification. Most importantly, every cell of every melanoma cell line possessed melanotropin receptors as visualized by these reagents.…”

“…These methods provide simple alternatives to the use of fluorescence techniques previously described by Schwyzer and coworkers (8)(9)(10)(11)(12) and recently by us (6,7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established.…”