[3H]Triamcinolone acetonide was used to tag covalently specific glucocorticoid receptors by photoaffinity labelling at lambda greater than or equal to 320 nm. Receptors of wild‐type mouse lymphoma cells and two glucocorticoid resistant mutants of “nuclear transfer deficient” (nt‐) and “increased nuclear transfer” (nti) phenotypes, respectively, were used. Wild‐type and nt‐ receptors yielded radiolabelled polypeptide bands of mol. wt. 98 000 as revealed by gel electrophoresis under denaturing conditions and fluorography. In contrast, the nti receptor had a mol. wt. of 42 000. Partial proteolysis of the wild‐type receptor with alpha‐chymotrypsin resulted in a fragment of mol. wt. 39 000 which still contained the steroid binding site but had increased affinity for DNA indistinguishable from that of the nti receptor. Chymotrypsin thus removed a domain from the wild‐type receptor polypeptide which is involved in modulating DNA binding. The same domain is missing from the nti receptor.

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Introdu~ionThe purple membrane ofHalobacterium halobium contains a single protein known as bacteriorhodopsin, which is composed of 1 mol retinal]mol bacterio-opsin [ 1 ]. The organization of the protein within each purple membrane sheet is that of an exact 2-dimensional crystal. X-ray and electron diffraction data have revealed that the bacteriorhodopsin in purple membrane is arranged as trimeric units in a hexagonal lattice [2]. The average distance between bacteriorhodopsin molecules within a trimeric unit is significantly shorter than the spacing between bacteriorhodopsin molecules from adjacent trimers.One could predict that crosslinking of purple membrane with bifunctional reagents would lead to the selective formation of crosslinked trimers. We demonstrate here that this is indeed the case.The biosynthetic precursor of purple membrane is the brown membrane, which also contains bacterioopsin [3 ]. When this membrane is treated with crosslinking reagents, polymeric protein molecules are formed exclusively.Crosslinked purple membrane exhibits the same photointermediates as the native membrane, although the cycle of events is slowed down. Materials and methods ChemicalsDimethyl-3,3'-dithiobispropionimidate-2 HC1 (DTBP) and dimethylsuberimidate.2 HCI (DMS) were obtained from Pierce, Rotterdam. Tartryldihydrazide for the preparation of tartryldiazide (TDA) was obtained from Fluka, Neu-Ulm, and L-[3SS]methionine (500 Ci/mmol)from Amersham Buchler, Braunschweig. Crosslinking of bacteriorhodopsin DTBPPurple membrane containing of 6 nmol bacteriorhodopsin was suspended in 200 #1 0.2 M N-methylmorpholine acetate pH 8.0, 2.5 mg DTBP.2 HCI (dissolved in 100 #10.1 M N-methylmorpholine) were added and the mixture incubated at 37°C for 2 h. DMSPurple membrane containing 6 nmol bacteriorhodopsin was suspended in 500/zl 0.1 M NaHCOs and 1 mg DMS.2 HC1 (dissolved in 25 gl water) added and the mixture incubated at 37°C for 2 h. TDATDA was prepared from tartryldihydrazide as in [4]. Purple membrane containing 6 nmol bacteriorhodopsin was suspended in 100/.d 1 M N-methylmorpholine acetate, pH 8.0 and 100 gl TDA solution (15 gmol) added and the mixture neutralized with 2 M KOH then incubated at 56°C for 1 h.After the crosslinking reaction the purple membrane was collected by centrifugation and the precipitate washed 3 times with water. Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis and fluorography Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis was performed using slab gels con- Elsevier/North-Holland Biomedical Press123