Vol. 62, No. 1, 1975 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS BINDING OF INSULIN AND OTHER HORMONES TO NON-RECEPTORMATERIALS: SATURABILITY~ SPECIFICITY AND APPARENT '~EGATIVE COOPERATIVITY" Pedro Cuatreeasas and Morley D. Hollenberg Departments of Pharmacology and Experimental Therapeutics~ and Medicine~ The Johns Hopkins University School of Medicine 3 Baltimore~ Maryland 21205 Received November 15, 1974 SUMMARY. Studies of interactions of 1251-insulin with non-tissue materials (tal% sil- ica~ protein-agarose derivatives~ glass test tubes) are presented as examples of non- receptor ("nonspecific") interactions which share~ at least superficially~ the criteria (saturability~ specifieity~ high affinity and reversibility) commonly attributed to "specific" hormone-receptor interactions. Such "specific" nonreceptor interactions~ which can potentially complicate interpretations of receptor binding studies~ also ex- hibit characteristics analogous to those observed in liver membranes where the data have been interpreted (3) to indicate "negative eooperativity" between receptors. Similar data are presented here for insulin and epidermal growth factor binding to placenta membranes~ and the effects of porcine insulin are compared to those of guinea pig insulin~ a derivative which dimerizes very poorly. The "negative cooperativity" observed in the nonreceptor systems is most likely attributable to insulin-insulin interactions. Wherever ligand (e.g.~ hormone) self-association or isomerization oc- cur% the binding data (e.g. Scatchard plot) may yield nonlinear relationships which can falsely be interpreted as indicating cooperative interactions between receptors~ or the presence of "additional groups" of binding sites. There are by now numerous studies of the binding of a variety of hormones to puta- tive tissue receptors (reviewed in refs. 1-2). The binding is generally believed to reflect a "specific" (i.e.~ receptor) interaction if it satisfies certain criteria such as chemical and tissue specificity~ saturability~ high affinity and reversibility. Although these are necessary criteria for receptor identification~ it is often over- looked that nonreeeptor ("nonspecific") interactions may also display certain of these properties. For examplej it is now almost standard practice in receptor binding assays to assume as "specific" that portion of the binding which can be "displaced" (by com- petition) by very high concentrations of the unlabelled hormon% since this is indica- tive of a "saturable" process. In the present report we describe examples in which the binding of insulin to sub- stances in the absence of tissue can be shown to demonstrate saturability~ reversibil- ity 3 relatively high affinity and some specificity. The awareness that nonspecifi% nonreceptor interactions can exhibit certain of the properties expected for receptors should prove cautionary. Furthermor% the availability of simple systems for measuring such nonspecific interactions should prove useful for testing certain properties of the binding which are observed in complex biological systems and which are believed to re- flect special properties of receptors. For example~ certain features of insulin bind- ing to cell membranes which have been interpreted as "negatively cooperative" inter- actions between receptors (3) can be reproduced in the nonreceptor systems to be Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. 3 1