PURIFICATION AND CHARACTERIZATION OF MULTIPLE-CHARGED FORMS OF PERMETHRIN CARBOXYLESTERASE(S) FROM THE HEMOLYMPH OF RESISTANT COLORADO POTATO BEETLE

Permethrin carboxylesterases (e.g., pI 4.2-4.8 CbEs) have been purifie d from the hemolymph of a permethrin-resistant strain of Colorado pota to beetle, Leptinotarsa decemlineata, through several chromatographic procedures in order to determine those proteins most responsible for t he DEF-synergized aspect of resistance. The pI 4.8 CbE is a 46- to 48- kDa monomeric protein whereas the pI 4.5 CbE appears to be a 57- to 59 -kDa dimeric protein. Both the pI 4.8 CbE and the pI 4.5 CbE are, in f act composed of groups of multiple-charged forms. All pI 4.5-4.8 CbEs are glycoproteins bur the charge heterogeneity is not associated with N-glycan moieties. A consistent tendency of increasing affinities for the pI 4.2-4.8 CbEs as the hydrophobicity of naphthyl substrate increa sed implicates that the catalytic sites of all the pI CbEs have hydrop hobic properties. The hydrophobic catalytic site, however, is separate entity from the domain determining overall surface hydrophobicity of CbE as determined by hydrophobic interaction:chromatography of the pur ified pI 4.5-4.8 CbEs. Overall, pI 4.8 CbE possessed the highest affin ity and catalytic efficiency for all three naphthyl substrates tested and for permethrin. The hydrolysis rates for permethrin, however, were extremely low compared to those of the naphthyl substrates. Neverthel ess, the over production and the high affinity toward hydrophobic subs trates of the permethrin CbE in the hemolymph in the permethrin-resist ant strain of Colorado potato beetle is likely to result in efficient hydrolysis of permethrin. In general, the PI 4.8 and 4.5 CbEs share a number of similarities in their biochemical properties;ind functional role in permethrin resistance despite their distinct molecular propert ies. The availability of highly purified and characterized forms of pe rmethrin CbE will allow the development of efficient immunochemical an d DNA-based diagnostics for the monitoring of permethrin resistance in field populations of beetles. (C) 1998 Academic Press.