High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin

Abstract

The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.

Fluorescence studies on the interaction of adenine with ricin A‐chain

“…Binding of adenine to RTA, however, has not been detected by equilibrium dialysis [12]. Here, we report that adenine-binding induces a large enhance- …”

A systemic antiviral resistance‐inducing protein isolated from Clerodendrum inerme Gaertn. is a polynucleotide:adenosine glycosidase (ribosome‐inactivating protein)

“…Unless otherwise stated, polynucleotide:adenosine glycosidase activity was determined by measuring adenine released from various substrates by HPLC [10] essentially following the procedure of [11] as described by [12]. Reactions were run for 40 rain at 30°C in a final volume of 50 I.tl containing 50 mM Na-acetate, pH 4.0, 100 mM KCI and RIP and substrate as indicated in the legends to the appropriate figures.…”

The N-Glycosidase Activity of the Ribosome-inactivating Protein ME1 Targets Single-stranded Regions of Nucleic Acids Independent of Sequence or Structural Motifs

“…23SSO-A12 contains only one adenine residue, and the activity was measured using a slight modification of a fluorometric method described previously by Zamboni et al (33). Using a fixed preincubation temperature and reaction time (45°C/20 min), assays containing 10 nM 23SSO-A12 yielded an apparent K D value of 105.9 nM (Fig.…”