Detecting Ricin: Sensitive Luminescent Assay for Ricin A-Chain Ribosome Depurination Kinetics

Abstract

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic ribosomal RNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of elongation factor binding resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Sub-picomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 × 10 8 M −1 s −1 , a diffusion limited reaction indicating catalytic perfection even with large reactants.

Identification of a Novel Functional Domain of Ricin Responsible for Its Potent Toxicity

“…Previous studies have demonstrated that the catalysis of the 80 S ribosome by RTA approached the diffusion rate limit for enzymatic reactions with a catalytic efficiency of 2.6 ϫ 10 8 M Ϫ1 s Ϫ1 for the discontinuous assay conditions (15,30). Among these RIPs, ricin has evolved to become a near perfect catalyst for mammalian ribosomes.…”

“…The relative binding avidity of each mAb was assessed by ELISA using RTA protein as the target antigen (14). The inhibition of the enzymatic activity of ricin by RTA mAbs was measured in a cell-free in vitro translation assay using rabbit reticulocyte lysates (Promega) as both the source of mRNA and ribosomes (14,15). Then, a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay was performed to evaluate the neutralization of ricin-mediated cytotoxicity.…”

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway

“…Reactions were initiated by the addition of CT263 to final concentrations of 100 -200 nM. A luciferase-coupled assay converted product adenine to ATP in 100 mM Tris-HCl (pH 7.7) (22). Briefly, luminescence was converted to picomoles of adenine (correcting for background and fitted to a standard curve).…”

Rapid Detection of Ricin in Serum Based on Cu-Chelated Magnetic Beads Using Mass Spectrometry

“…R icin is a naturally occurring toxin produced by the seeds of the castor bean plant [1,2]. It is among the deadliest known poisons with an LD50 in humans of 5-10 μg/kg through inhalation or 350-700 μg for a 70 kg person [3,4].…”

“…The ricin toxin A chain (RTA) is a ribosome inactivating protein (RIP) that inhibits protein synthesis in mammalian cells. The ricin toxin B chain (RTB) is a lectin that binds to galactose residues on the surface of cells [1].…”