title: Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.
abstract: A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.
authors.affiliation: School of Pharmacy, University of Wisconsin, Madison 53706.
authors.forename: M L
authors.initials: ML
authors.lastname: Moss
authors.forename: R E
authors.initials: RE
authors.lastname: Palmer
authors.forename: P
authors.initials: P
authors.lastname: Kuzmic
authors.forename: B E
authors.initials: BE
authors.lastname: Dunlap
authors.forename: W
authors.initials: W
authors.lastname: Henzel
authors.forename: J L
authors.initials: JL
authors.lastname: Kofron
authors.forename: W S
authors.initials: WS
authors.lastname: Mellon
authors.forename: C A
authors.initials: CA
authors.lastname: Royer
authors.forename: D H
authors.initials: DH
authors.lastname: Rich
mesh_terms: D000199:Actins / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D016572:Cyclosporine / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D000345:Affinity Labels; D006360:Heat-Shock Proteins; D016572:Cyclosporine