Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads.
In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.
D004800:Enzymes, Immobilized / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D011134:Polysaccharides / Q000138:chemical synthesis*; D011549:Pseudomonas / Q000201:enzymology*; D012685:Sepharose / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology; D013251:Steroid Isomerases / Q000378:metabolism*
C038153:CMESA-sepharose; D004800:Enzymes, Immobilized; C038152:O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone; D011134:Polysaccharides; D012685:Sepharose; D007535:Isomerases; D013251:Steroid Isomerases; C022660:steroid delta-isomerase