Thyrotropin-thyrotropin receptor complexes Disulphide-linked subunit (SDS-polyacrylamide gel electrophoresis)

The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino-hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration.

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Studies of the TSH receptor using affinity labelling with photoactive derivatives of TSH and analysis by SDS-PAGE have shown that the receptor contains 2 subunits (A and B), linked by a disulphide bridge. Similar results are obtained with TSH receptors from human, porcine and guinea pig thyroid tissue and from guinea pig fat. Analysis of affinity labelled receptors under non-denaturing conditions suggest that subunits additional to the A and B subunits are not present.Hydrodynamic measurements indicate that the receptor A subunit has an approximately spherical structure (Stokes' radius 70\ l =A%o\ )and when this interacts with TSH (an elongated structure with Stokes' radius 56\ l =A%o\ ) a very elongated complex (Stokes' radius 104\l =A%o\)is formed.Isoelectric focusing studies of the TSH receptor A subunit, TSH and TSH receptor antibodies indicate that charge-charge interactions are of considerable importance in the binding of hormone and antibody to the receptor.The TSH receptor is an integral membrane glyco¬ protein which forms a binding site for TSH on the outside surface of thyroid follicular cells. The binding of TSH to the receptor causes the hor¬ mone-receptor complex to interact with the regu¬ latory subunits of adenylate cyclase. This interac¬ tion takes place in the lipid bilayer or at the cytoplasmic surface of the cell membrane and results in production of increased amounts of cyclic AMP which mediate most, if not all, of the actions of the hormone. In Graves' disease, a relatively common autoimmune disorder, auto¬ antibodies to the TSH receptor are formed. They bind to the receptor in such a way as to mimic the effects of TSH and this results in the development of hyperthyroidism (Rees Smith 1981;Rees Smith et al. 1985).This short review considers the properties of the receptor and the nature of its interaction with TSH and TSH receptor antibodies. In addition, the relationship of the TSH receptor-receptor antibody system to other autoantibody-autoantigen system will be considered. Photoaffinity labelling of the TSH receptorThe TSH receptor is present on the cell surface in very small amounts (in the region of 103 sites per cell; Rees Smith et al. 1985), and consequently special techniques are often required to study the receptor. One of the most powerful techniques currently available for studying trace amounts of cell surface receptor is affinity labelling (Ji 1979). In the case of the TSH receptor, we have found TSH coupled to photoactive cross-linking rea¬ gents to be particularly useful (Buckland et al. 1986). These studies have indicated that both subunits of TSH form part of the hormone's receptor binding site (Buckland et al. 1986). Analysis by SDS-PAGE of porcine TSH recep¬ tors cross-linked to 125I-labelled TSH have shown that the receptor contains 2 subunits linked by a disulphide bridge. One of the subunits (A; mol wt 50 K) is water soluble and forms the binding site for TSH on the outside surface of the cell mem¬ brane. The other (B) subunit (mol wt 30 ) penetrates the lipid bilayer (Ka...

Ornithine aminotransferase is shown to bind 1 mol of amino[14C]hexynoate per mol of coenzyme in the ‘suicide’ inactivation process. At the same time the coenzyme pyridoxal phosphate becomes irreversibly bound to the enzyme protein. Apart from the inactivation, the labelled enzyme is indistinguishable from native ornithine aminotransferase by several separation techniques. Because the rate of degradation of the labelled enzyme is the same as that of the normal enzyme it is concluded that loss of coenzyme does not initiate turnover. Free aminohexynoate is rapidly eliminated from the liver, and 70% of the compound is excreted unchanged in 7.5 h. Inactivated ornithine aminotransferase accounts for 11% of the total labelled liver protein and significant amounts of label are found in aspartate aminotransferase which is also extensively inactivated. The rate of return of enzyme activity is determined and found to be more rapid than expected for a process in which the enzyme is synthesized at a constant rate and degraded in a single, first-order process.