Separation of jack bean (Canavalia ensiformis) urease isoforms by immobilized metal affinity chromatography and characterization of insecticidal properties unrelated to ureolytic activity

In this work we described the separation of two isoforms of urease from jack bean seeds, the "classical" jack bean urease (JBU) and canatoxin (CNTX), using immobilized metal affinity chromatography (IMAC). Jack bean urease isoforms presented differential behavior on a cobalt-loaded iminodiacetic acid (IDA)-Sepharose column and IMAC is proposed as an efficient method to isolate the isoenzymes. The metal content of the urease isoforms was determined by particle induced X-ray emission (PIXE). CNTX displays ca. 1 eq. of nickel per monomer, contrasting with 2 eq. of nickel found per monomer for JBU. Beside nickel, CNTX contains I eq. of zinc per monomer, while no zinc is found in JBU. The insecticidal property of these ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Both ureases were lethal to the insects, being CNTX more potent than JBU. This property was not affected by treatment with p-hydroxymercurybenzoate (pHMB), an irreversible inhibitor of ureolytic activity. Altogether the data show that IMAC is a suitable method for separating jack bean urease isoforms and that the isoenzymes display entomotoxic effects, independent of their ureolytic activity, suggestive of a role in plant defense against insect predators. (C) 2004 Elsevier Ireland Ltd. All rights reserved.