Filter binding assay for the geldanamycin-heat shock protein 90 interaction.

Article Details

Citation

Copy to clipboard

Carreras CW, Schirmer A, Zhong Z, Santi DV

Filter binding assay for the geldanamycin-heat shock protein 90 interaction.

Anal Biochem. 2003 Jun 1;317(1):40-6.

PubMed ID

12729599 [ View in PubMed

]

Abstract

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Geldanamycin	Endoplasmin	Protein	Humans	Unknown	Inhibitor	Details