Abstract
The polypeptide hormone insulin and the binding portion of ricin toxin, the B chain, were linked via a disulfide bond. This insulin-ricin B chain conjugate bound to insulin receptors with a potency one-twentieth that of native insulin. Rat HTC hepatoma cells, a cultured cell line that has relatively few insulin receptors, bound the conjugate to a much greater degree than insulin. Binding occurred predominantly via the ricin B chain portion of the conjugate since binding was not inhibited by insulin but was inhibited by galactose, a known inhibitor of the interaction of ricin B chain to its receptor. In HTC cells, the insulin-ricin B chain conjugate at 330 nM stimulated amino acid uptake to 225% of controls, a value higher than that for insulin which stimulated uptake to only 167% of controls. The conjugate also stimulated tyrosine aminotransferase activity in HTC cells with a potency value approximately one-half that of insulin. Both of these activities of the insulin-ricin B chain conjugate in HTC cells were inhibited by 100 mM galactose (90% and 80%, respectively), whereas the ability of insulin to stimulate these activities was not inhibited significantly by this sugar. The results suggest, therefore, that one can construct hybrid molecules consisting of binding proteins and polypeptide hormones and that these hybrid molecules can have binding and biological activities which are different from the parent hormone molecule.
Highlights
Thepolypeptidehormoneinsulinandthebinding can be constructedfrom the A chain of a particulartoxin and portion of ricin toxin, the B chain, were linked via a the binding portion of another molecule so that theresulting disulfidebond,Thisinsulin-ricinBchainconjugate hybrid molecule exerts toxicity through a new cell surface bound to insulin receptors with a potency one-twen- receptor
The results suggest, that onecan construct hybrid molecules consisting of binding proteins and polypeptide hormones and that these hybrid molecules can have bindingandbiological activities whicharedifferent from the parent hormone molecule
These lattertwo molecules couldthen be eluted with 200 rnM galactose (Fig. 1A).Third, after thereducing agent 2mercaptoethanol was added to this conjugate preparation, the larger molecular weight band disappeared, and o d y one band migrating with ricin B chain was present on staining the gel (Fig. l A ), and another band migrating with insulin and its chains appeared by autoradiography (Fig. 1B)
Summary
The binding molecule can be a toxin B chain [6], tieth that of native insulin.Rat HTC hepatoma cells, a plant lectin [7,8],antibody [9], or even a polypeptide hormone cultured cell line thathas relatively few insulin recep- [10, 11].For instance,the A chain of ricin has been coupled to tors, boundtheconjugate to a much greater degree human chorionic gonadotropin andthe hybrid molecule trniathioscIaktnirnnnHeionciTnBwehCspinubtcoliihcitrnneea..dhlilnBisb,binitpythdooeiriornnftignistoushonulecinlcioiunnfbr-wtrruetiehatrcdaisencipntcBrihooeincdnbohjoiufatmegrindiaicntbcieanoynnBgstjliauynclghcaveaacitatienobtsaihentteo,dinphs( h1groeAo0rsmw)lwe.tnnohatnotsouet hsghehat ovAietshtcuoahrxfsaaiiccniebotyferetieonncxecidepnUetsomstrowos nicctsehatlrnlrsae,tbceiteedphutatoshsreasndtoftoathrsbetehaeibnsvihernehodpiricmnolgreotentodoef 330 nM stimulated amino acid uptake to 226% of con- whether the B chainof toxins can be used to present hormones trols, a value higher than that for insulin which sttiomcuel-ls. HTC cells with a potency value approximately one-haclofrdingly, we have prepared a hybrid molecule consisting of that of insulin. Both of these activities of the insulin- the Bchain of ricin coupled to the polypeptide hormone ricin B chain conjugate iHnTC cells were inhibited by insulin via a disulfide bond.
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