A rapid and sensitive method to measure the enzymatic activity of ribosome-inactivating proteins.
A method is described in which the adenosine- N -glycosidase activity of ribosome-inactivating proteins (RIPs) is measured using as substrate a 2251 bp [3H]DNA obtained by PCR amplification of the 731-2981 region of the pBR322 plasmid. The DNA, labelled in the purine ring of adenine, proved a good substrate for all three RIPs tested (PAP-S, ricin and shiga-like toxin I). The method, which measures directly the [3H]adenine released, is highly specific, extremely rapid and quantitative in a wide range of RIP concentrations.
Brigotti
M
M
Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy.
Barbieri
L
L
Valbonesi
P
P
Stirpe
F
F
Montanaro
L
L
Sperti
S
S
D000225:Adenine / Q000032:analysis; D001427:Bacterial Toxins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D007700:Kinetics; D009699:N-Glycosyl Hydrolases; D037121:Plant Lectins; D010940:Plant Proteins / Q000378:metabolism*; D010947:Plants, Toxic; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011865:Radioisotope Dilution Technique; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000378:metabolism*; D012278:Ricinus; D012639:Seeds; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1; D014316:Tritium
D001427:Bacterial Toxins; D017931:DNA Primers; D037121:Plant Lectins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D022622:Shiga Toxin 1; D014316:Tritium; D004247:DNA; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein; D000225:Adenine