title: Luciferase-based assay for adenosine: application to S-adenosyl-L-homocysteine hydrolase.
abstract: S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, United States.
authors.forename: Emmanuel S
authors.initials: ES
authors.lastname: Burgos
authors.forename: Shivali A
authors.initials: SA
authors.lastname: Gulab
authors.forename: María B
authors.initials: MB
authors.lastname: Cassera
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000241:Adenosine / Q000032:analysis* / Q000737:chemistry; D001681:Biological Assay; D057230:Limit of Detection; D008156:Luciferases / Q000737:chemistry*; D012435:S-Adenosylhomocysteine / Q000737:chemistry*
chemical_list: D012435:S-Adenosylhomocysteine; D008156:Luciferases; D000241:Adenosine