Insect Biochem. Vol. 21, No. 5, pp. 457~165, 1991 0020~1790/91 $3.00+ 0.00 Printed in Great Britain.All rights reserved Copyright© 1991 PergamonPress plc PROPERTIES AND INTRACELLULAR DISTRIBUTION OF A CATHEPSIN D-LIKE PROTEINASE ACTIVE AT THE ACID REGION OF MUSCA DOMESTICA MIDGUT FRANCISCOJ. A. LEMOSand WALTERR. TERRA* Departamento de Bioqulmica, Instituto de Quimica, Universidade de S~o Paulo, C.P. 20780, S~o Paulo 01498, Brasil (Received 22 October 1990; revised and accepted 22 March 1991) Abstract--Musca domestica larval midgut display in cells and luminal contents a proteolytic activity with a pH optimum of 3.0-3.5. This activity is abolished by pepstatin and is insensitive to soybean trypsin inhibitor and to sulfhydryl proteinase inhibitors. The acid proteinase occurs in multiple forms with M~ values in the range 40,000-80,000 and with pI values of about 5.5. The proteinase inactivates at 60°C according to apparent first-order kinetics and Lineweaver-Burk plots of its activity against albumin concentration are rectilinear, suggesting that the multiple forms have similar properties. The proteinase reacts slowly with diazoacetylnorleucine plus CuSO4, is stable in alkaline media, is inhibited by dithiothreitol, hydrolyses hemoglobin better than albumin and is virtually not active upon synthetic substrates for pepsin. These properties are similar to those of cathepsin D. The specific activity of the acid proteinase determined by titration with pepstatin is 680 units/rag of proteinase and the KD of the pepstatin-proteinase complex is 1.5 nM at 30°C. The acid proteinase occurs mainly in midgut subcellular fractions characterized by a high specific activity of molybdate-inhibited acid phosphatase and a large number of secretory-like vesicles. It is proposed that the M. domestica midgut acid proteinase is a cathepsin D-like proteinase evolved to function in luminal contents. The lack of ATP activation of the midgut enzyme supports this hypothesis, since ATP is thought to regulate cathepsin D-proteolysis inside lysosomes. Key Word Index: cathepsin-like; pepsin-like; luminal cathepsin D; midgut cathepsin D; cathepsin D titration; cathepsin D properties INTRODUCTION Working with homogenates of whole bodies of larvae, pupae and adults of Musca domestica, Greenberg and Paretsky (1955) found a strong proteolytic ac- tivity at pH 2.5-3.0 and at pH 8-9. They interpreted the activity at low and high pH as due to a pepsin-like and a trypsin-like enzyme, respectively. Their results were confirmed by Labremont et al. (1959), which showed that the acid proteinase is restricted to the midguts, known to possess in flies a region displaying a low pH. Similar results were also found with other cyclorrhaphous Diptera (Pendola and Greenberg, 1975). The functional significance of the acid proteinase in cyclorrhaphous Diptera became more clear after detailed studies on the compartmentalization of the *Author for correspondence. Abbreviations used: APheI2Tyr, N-acetyl-L-phenylalanyl-3,5 diiodo-tyrosine; BAPA, ~-N-benzoyl-DL-arginine-p- nitroanilide; BSA, bovine serum albumin; DAN, diazo- aeetylnorleucine; DTT, dithiothreitol; E64, transepoxy- succinyl-L-leucyl-amido (4-guanidino) butane; EDTA, ethylenediamine-tetracetic acid; LpNA, L-leucine-p-ni- troanilide; M, relative molecular weight; NPP, p-nitro- phenyl phosphate; pHMB, p-hydroxymereuribenzoate; SBTI, soybean trypsin inhibitor; Tris, tris-hydroxymetyl aminomethane; ZGluTyr, N-carbobenzoxy-L-glutamyl- L-tyrosine; ZHispNPhe2, N-earbobenzoxy-histidyl-p- nitro-L-phenylalanyl-L-phenylalanine methyl ester. digestive process in M. domestica larvae (Espinoza- Fuentes and Terra, 1987; Espinoza-Fuentes et al., 1987; Terra et al., 1988b). According to these studies, cyclorrhaphous maggots living in highly infected food ingest large amounts of bacteria, which are their main food. Bacteria, once ingested, are killed in the middle midgut by the combined action of low pH, lysozyme and the acid proteinase. The nutrients released by the bacteria then pass to the posterior midgut, where the majority of the digestion takes place. In spite of the functional importance of the acid proteinase in cyclorrhaphous midguts, its characteriz- ation was attempted only by Pendola and Greenberg (1975). They prepared homogenates from Caliphora vicina larva midguts and assayed them in several conditions. They found that the acid activity over casein was inhibited by diphenyldiazomethane, and that the homogenates hydrolyzed APheI2Tyr and ZgluTyr; the first substrate was hydrolyzed better at pH 7.0 than at pH 2.5, the contrary being true for the second substrate. Since the good pepsin substrate (APheI2Tyr, see Fruton, 1976) is poorly hydrolyzed at acid pH, and diphenyldiazomethane inhibits several different acid proteinases (Fruton, 1976), the results of Pendola and Greenberg (1975) remain inconclusive, despite they favored the hypothesis that cyclorrhaphous Diptera possess a true pepsin. In this paper we showed, by several critheria, that the acid proteinase from Musca domestica larval midguts is cathepsin D-like, and that it occurs particle In 21/5---A 457