This investigation aims at the isolation and chemical and biological characterization of insulin receptors from human placenta. Specifically we plan to develop a procedure that will enable us to prepare homogeneous native receptors on a routine basis in sufficient quantity to permit characterization. To this end we expect to investigate the utility of affinity columns in which N alpha, B1-6 iminobiotinylaminohexyl-insulin is noncovalently attached to AH-Sepharose 4B-immobilized succinoylavidin. The results of preliminary experiments encourage us to develop this system which overcomes the major objections to the use of avidin-biotin affinity columns i.e. the inability to dissociate biotin-containing compounds from avidin columns. The weaker binding iminobiotinyl derivatives can be readily displaced by biotin. We expect to establish whether the placental receptor is identical to that from other sources, we propose to determine the molecular weight and size of the native receptor, the subunit architecture and to ascertain whether or not proteolytic activity is an inherent property of the receptor. In addition, if sufficient material can be prepared, we plan to perform end group determinations and peptide mapping studies. Streptavidin will be crystallized and its molecular architecture will be determined by X-ray crystallographic methods. Techniques drawn from organic synthesis, biochemistry, endocrinology immunology and cell biology will be employed. Insulin without its receptor has no biological activity, thus knowledge of the nature of the receptor will have clinical significance.
| Finn, F M; Ridge, K D; Hofmann, K (1990) Labile disulfide bonds in human placental insulin receptor. Proc Natl Acad Sci U S A 87:419-23 |
| Finn, F M; Hofmann, K (1990) Isolation and characterization of hormone receptors. Methods Enzymol 184:244-74 |
