ISO FORMS OF LEUCYL-AMINOPEPTIDASE OF CERAMBYX CERDO ( COLEOPTERA , CERAMBYCIDAE ) LARVAE

ISO FORMS OF LEUCYL-AMINOPEPTIDASE OF CERAMBYX CERDO (COLEOPTERA, CERAMBYCIDAE) LARVAE. N. Bozie', Z. Vujcic 2, V. Nenadovie' and' J. Ivanovle', Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, Studentski trg 12-16, 11000 Belgrade; Department of Biochemistry, Faculty ofChemistry, University ofBelgrade, Studentskitrg 12-16, 11000 Belgrade; "Sinisa Stankovic " Institute ofBiological Research, Department of Insect Physiology and Biochemistry, 29 Novembar 142, 11000 Belgrade, Serbia and Montenegro

The activity of leucyl-aminopeptidase was determined using specific the chromogenic substrate leucine-p-nitroanilide (Lee andAnstee, 1995).Reaction mixtures contained 5 III of crude midgut extracts and 20 III of the fraction in 0.5 ml of 50 mM Tris-HCl buffer with pH 8.0, which has been shown to be the pH optimum for LAP in this species (Bozic et al., 2001), and 1 mM of substrate dissolved in N,N-dimetilformamide.The reaction run for 5 min at 30°C.After that, enzymatic reactions were terminated by adding 0.1 ml 30% acetic acid.The concentration of the resulting p-nitroaniline was estimated by measuring absorbance at 410 nm (Erlanger et aI., 1961).
Native slab 10 % polyacrylamide gel electrophoresis (Davis, 1964) was used for detection ofLAP isoforms, Activity of LAP was detected by zymogram analysis based on the formation ofazo-eolor (Bozic et al., 2003).The semipreparative gel contained 100 III of crude midgut extract (2,5 Vlml) (lane B) and 300 IIIofpartially purified LAP (0.8 Vlml) (lane A) (Fig. 2).Two bands of LAP activity in the crude extract as well as in the .partiallypurified fraction were visible in the zymogram resolved by native pag.
There are a very few literature data about C. cerdo proteolytic enzymes (Ivano vi c and Mi lan ov i c 1970, Nenado vi c et aI., 1982;1994;1999); among a report them is concerning leucyl-aminopeptidase (LAP) (Bozic ef al., 2001) which has been shown to be the most abundant enzyme in the midgut of C. cerdo larvae.It is also the most abundant enzyme in the midgut of the species Morimus funereus, likewise a member of the family Cerambycidae (B oz ic et al., 2003).In the latter species, there are four isoforms, that differ in efficiency and specificity.This could be of great influence in maintenance of the high polyphagy of M funereus, which attacks a number of deciduous and coniferous trees, because isoenzymes provide increased capability of the organism to adapt to different sources of food and to overcome the activities of plant proteinase inhibitors (Wagner et al., 2002).
Cerambux cerdo favors all species of the genus Quercus, but can also be found on beech, ash, and walnut trees in Russia (Neriado v ic ef al.,1999), thus having a much narrower host range than M.funereus.From that point of view, it was interesting to find out how many isoforms of leucyl-aminopeptidase are present in the midgut of C. cerdo larvae and clarify whether there is any connection between this enzyme and polyphagy in some members of the family Cerambycidae.
Larvae collected outdoors in December on the mountain of Fruska Gora, were used in the present work.Midguts were dissected out, weighed, and homogenized with a pre-chilled mortar and pestle in 4 vol.(glml) ofice-eold 50 mM Tris buffer, pH 7.5, with the addition of quartz sand.After centrifugation, the resulting supernatants were treated with an equal volume of carbon tetrachloride for lipid removal and centrifuged again (Bozic et al., 2003).Protein concentration was determined in the extract (B radford, 1976).
A measured volume of midgut extract of C. cerdo larvae was.loaded onto a Sephadex G-100 column (1,6 x 60 em, Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCl in 20 mM acetate buffer, pH 6.0, and calibrated with molecular weight markers.Fractions of2 ml were collected andA 2So values monitored.Fractions containing LAP activity were pooled, and protein concentration and LAP activity were determined.The results are summarized in Fig. 1.Since enzyme activities were

B
Leucyl-aminopeptidase in the midgut of C. cerdo larvae exists in two forms.In view of the possible role of isoenzymes in polyphagy of M.funereus larvae and the wider host range of the species M.funereus, these results were expected.They enable us to assume that LAP is a potential marker for cerambycid beetles, not only through its existence, but also through the presence of different forms of it.