~ Pergamon 0965-1748(95)00067-4 Insect Biochem. Molec. Biol. Vol. 26, No. 1, pp. 95-100, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/96 $15.00 + 0.00 Purification and Characterization of a Digestive Cathepsin D Proteinase Isolated from Tribolium castaneum Larvae (Herbst) A. BLANCO-LABRA,*t N. A. MARTINEZ-GALLARDO,* L. SANDOVAL-CARDOSO,* J. DELANO-FRIER* Received 7 March 1995; revised and accepted 2 August 1995 A digestive proteinase was isolated from larval extracts of Tribolium castaneum. The enzyme was partially purified using gel-filtration and ion-exchange chromatography. It is an acidic proteinase with a maximal activity at pH 3. Considering its inhibition by Pepstatin A, plus its selectivity to hydrolyze hemoglobin but not bovine serum albumin, it was classified as Cathepsin D proteinase. Its relative molecular weight is 22 kDa and it shows a high sensitivity to temperature. Unlike other cathepsin D found in animals, this enzyme is free of carbo- hydrate, and its activity is not affected by the presence of different anions which are known to affect the activity of plant aspartic proteinases. Digestive proteinase Tribolium castaneum Cathepsin D Aspartic acid proteinase Protease Insect enzymes INTRODUCTION Proteinases which cleave internal peptide bonds, are grouped into four categories according to their essential catalytic residues at their active sites: serine (EC 3.4.21), cysteine (EC 3.4.22), aspartic (EC 3.4.23) and metallo proteinases (EC 3.4.24). The type to which a specific pro- teinase belongs is also determined by the pH range over which it is active, by its similarity to well characterized proteinases and by its sensitivity to various inhibitors (North, 1982). Different types of proteinases have been identified in the insect digestive system. They are, in general, similar in their catalytic classes to the ones found in vertebrates. However, some of their main characteristics, such as location, optimal pH activity, thermostability, kinetic constants, etc. may be different. Their relative import- ance might also be of interest, since their presence could be the result of an evolutionary response for adaptation to certain type of food sources. Such is the case in the Coleoptera, whose high cysteine catheptic proteinases have been proposed to be a response to the need to feed on certain types of food, for instance, seeds that contain high levels of trypsin inhibitors (Murdock et al., 1987). Serine proteinases, which include the well known *Department of Biotechnology and Biochemistry, Centro de Investiga- ci6n y de Estudios Avanzados del IPN, Unidad Irapuato, Mexico. tAuthor for correspondence, at: Apdo. Postal 629, C.P. 36500 Irapu- ato, Gto. Mexico. 95 extracellular enzymes trypsin and chymotrypsin, have an essential serine and histidine amino acid residue in their active site and they are the best understood insect diges- tive proteinases (Applebaum, 1985). Aspartic proteinases contain two aspartic residues at their active centers involved in catalysis. It is thought that general acid-base catalysis, rather than the formation of covalent enzyme- substrate intermediates, is operative in the mechanism of these enzymes. Among these are included the extracellu- lar pepsin and cathepsin D, most often found as an intra- cellular enzyme. Cathepsin D is a major proteolytic enzyme of lysosomes (Barret, 1969, 1977), and like most of the lysosomal enzymes, it is usually found as a glyco- protein. Metalloproteinases contain metal ions (usually zinc) at the active center, which are an integral part of their structures. Cysteine proteinases contained an essen- tial cysteine residue involved in a covalent intermediate complex with substrates. Some proteinases of unknown catalytic mechanism have been assigned to a new, tem- porary subclass (EC 3.4.99). This subclass includes enzymes that either have not been sufficiently purified to allow assignation to one of the mechanistic classes, or clearly do not fit one of the four classical groups (Bond and Butler, 1987). This paper reports the purification and partial charac- terization of the most abundant acid proteinase present in the gut of Tribolium castaneum larvae.