Receptor chemical purification

Use of radioligands in the chemical purification of receptors. Here, the bound radioligand allows the receptors to be tracked through the various purification steps — for example, in the fractions eluting from separation columns. In such experiments, it is important for the radioligand to be irreversibly bound to the receptor. 

Simon EJ, Gioannini TL. Opioid receptor multiplicity isolation, purification and chemical characterization of binding sites. In Opioids I. Handbook of Experimental Pharmacology. Vol. 104 (Herz A, ed). Springer, Heidelberg, 1993 3-26. 

Biosensor devices must operate in liquids as they measure effects at a liquid-solid interface. Then, the immobilization of the receptor molecule on the sensor surface is a key step for the efficient performance of the sensor. When the complementary analytes are flowing over the surface, they can be directly recognized by the receptor through a change in the physico-chemical properties of the sensor. In this way, the interacting components do not need to be labeled and complex samples can be analyzed without purification. 

The insulin receptor is present in trace amounts in the plasma membrane of liver cells. Using radiolabeled arylazido derivatives of insulin, which on irradiation yield reactive nitrenes, components of the receptor with molecular weights of 135,000 and 90,000 daltons have been identified (Jacobs etal., 1979 Yip etal., 1980 Wisher etal., 1980). The identity of the two polypeptides has been confirmed by chemical crosslinking to radiolabeled insulin and by purification of the receptor by affinity chromatography. 

Stein M, Meyer HE, Hasilik A, von Eigura K. 46-kDa mannose 6-phosphate-specific receptor purification, subunit composition, chemical modification. Biol. Chem. Hoppe-Seyler 1987 368 927-936. 

Antibodies can have distinct advantages over the natural receptor molecules that are used in some competitive binding assays. They are stable, soluble proteins with known chemical and physical properties. - Procedures for their purification are available, " but usually diluted antisera is used in fluid phase radioimmunoassays. It has been estimated that an individual animal has the potential to produce antibodies specific for approximately 10 diverse immunodominant moieties. These molecules may possess binding constants for individual antigens on the order of... 

To identify the cell-surface TGF(B receptors, investigators first reacted the purified growth factor with the radioisotope iodine-125 under conditions such that the radioisotope covalently binds to exposed tyrosine residues. The labeled TGF(B protein was Incubated with cultured cells, and the incubation mixture then was treated with a chemical agent that covalently cross-linked the labeled TGFp to Its receptors on the cell surface. Purification of the labeled receptors revealed three different polypeptides with apparent molecular weights of 55, 85, and 280 kDa, referred to as types RI, RII, and RIII TGFp receptors, respectively. 

---