Identification of cathepsin B, D and H in the larval midgut of colorado potato beetle, Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

Insect Biochem. Vol. 20, No. 3, pp. 313-318, 1990

0020-1790/90$3.00+ 0.00 Copyright© 1990PergamonPress pie

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IDENTIFICATION OF CATHEPSIN B, D AND H IN THE LARVAL MIDGUT OF COLORADO POTATO BEETLE, LEPTINOTARSA DECEMLINEATA SAY (COLEOPTERA: CHRYSOMELIDAE) NORMANM. R. THIE and JON G. HOUSEMAN* Ottawa-Carelton Institute of Biology, Biology Department, University of Ottawa, Ontario, Canada K1N 6N5 (Received 17 August 1989; revised and accepted 28 November 1989)

Abstract--The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-AlaArg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor. Key Word Index: cathepsin B, cathepsin D, cathepsin H, potato beetle, proteinase, digestion, Leptinotarsa decemlineata

INTRODUCTION Proteinases are classified according to their catalytic mechanism and the four classes include serine proteinases with a serine or histidine in the active centre, cysteine proteinases with a cysteine in the active centre, aspartic proteinases with an acidic amino acid residue and metalloproteinases with an essential metal involved in the catalytic mechanism (Barrett and McDonald, 1980; McDonald and Barrett, 1980; Storey and Wagner, 1986). Digestive serine proteinases, including chymotrypsin and trypsin, and the aspartate proteinase pepsin were first studied in insects since these were the principal digestive enzymes in vertebrates and reviews (House, 1974; Law et al., 1977; McFarlane, 1985) reported trypsin as the principal digestive proteinase in insects. Insects use a variety of different proteinases to hydrolyse ingested proteins including catheptic cysteine and aspartate proteinases in Hemiptera (Houseman and Downe, 1980, 1981, 1982a, 1983), pepsin-like enzymes in some Diptera (Greenberg and Paretsky, 1955; Sinha, 1975; Pendola and Greenberg, 1975) and trypsin-like enzymes with maximal activity at pH values greater than 9.0 are frequently reported in Lepidoptera (Applebaum, 1985). Coleoptera appear to use either tryptic serine proteinases or cysteine enzymes for digestion of ingested protein. Initial investigations of casein hydrolysis by Tenebrio molitor showed maximal hydrolysis between pH 6.2 and 6.4 and between 6.5 and 6.9 for

*Author to whom all correspondence should be addressed.

Tribolium confusum and Tribolium castaneum (Birk et al., 1962). In whole T. molitor gut preparations

proteolytic activity increased in the presence of thiol compounds and was stabilized by the chelating agent EDTA (ethylenediamine tetraacetic acid) and proteolytic activity could be divided into two components based on resistance or susceptibility to soybean trypsin inhibitor. The susceptible fraction contained tryptic activity and the resistant contained carboxypeptidase B and aminotripeptidase. The latter also contained an unknown esterase activity which could be attributed to either carboxypeptidase B or cathepsin B, a thiol activated and EDTA stabilized (Applebaum et al., 1964) cysteine proteinase. Proteolytic activity in T. molitor was also resolved into an ~t- and//-proteinase. The ct-proteinase differed from either vertebrate chymotrypsin and trypsin and although the fl-proteinase shared a number of characteristics with vertebrate trypsin, its cleavage specificity towards insulin and peptides differed (Zwilling, 1968; Zwilling et al., 1972). The Tenebrio trypsin-like enzyme was purified and resembled bovine trypsin in its size, kinetic parameters, substrate specificity and inhibition by either naturally occurring or synthetic inhibitors and the only differences were conformational (Levinsky et al., 1977). Using partially purified preparations, synthetic and natural substrates, pH optima for maximal hydrolysis of each substrate and diagnostic inhibitors and activators, three trypsin-like, four chymotrypsin-like enzymes (Baker, 1981a), aminopeptidase (Baker and Woo, 1981) and carboxypeptidases (Baker, 1981b) were characterized in the Black carpet beetle Attagenus megatoma. Trypsin and chymotrypsin were

313

314

NORMANM. R. Tree and JON G. HOUSF.MAN

identified in the beetle, Pterostichus melanarius, using partial purification and synthetic substrates in combination with pH optima and inhibition by PMSF (phenyl methyl sulfonylfluoride) and TPCK (tosylphenylalanine chloromethyl ketone) (Gooding and Huang, 1969). Aminopeptidase and trypsin have been detected in the elaterid beetle Pyrophorus divergens (Colepicolo-Neto et al., 1987) and hydrolysis of trypsin and chymotrypsin substrates in four different Carabus sp. beetles was detected although the pH at which maximal activity occurs was not determined (Vaje et al., 1984). Grass grub, Costelytra zealandi, proteases were partially purified and trypsin, chymotrypsin, elastase, aminopeptidase, carboxypeptidase A and B were identified based on synthetic substrate hydrolysis and effects of a range of inhibitors diagnostic for different protease classes (Christeller et aL, 1989). Similar enzymes are present in the alimentary tract of Sitophilus oryzea, S. zeamais and S. granarius and Cyclas formicarius elegantus (Baker, 1982; Baker et al., 1984). Proteolytic activity in Bruchid beetles appears to differ from that found in other insects in the order and when vertebrate trypsin and chymotrypsin assay procedures were used only weak proteolysis could be detected in either Callasobruchus chinensis or Acanthoscelides obtectus (Applebaum, 1964). When activity was assayed by visualizing proteolytic peptide products using SDS--polyacrylamide gel electrophoresis, optimal activity in C. maculatus occurred at pH 5.4 and effects of a variety of potential activator and inhibitor compounds indicated that a thiol (cysteine) proteinase resembling cathepsin B was active in the gut (Gatehouse et al., 1985). Kitch and Murdock (1986) and Campos et al. (1989) used partial purification and both synthetic and natural substrates in combination with a variety of potential activators and inhibitors to further identify the C. maculatus proteinase as a cysteine proteinase that may resemble cathepsin B. A major gut proteinase from A, obtectus was partially purified and its identity as a cysteine proteinase was also confirmed (Wieman and Nielsen, 1988). In a survey of 11 different Coleopteran species Murdock et al. (1987) used haemoglobin hydrolysis at pH 5.5 and inhibition by the cysteine protease inhibitor E64, to demonstrate that cysteine proteinases are commonly used by Coleoptera. When intact proteins are used to measure or characterize proteolytic activity in crude gut preparations it is impossible to identify individual proteases since activity detected is attributable to all proteases present. The unique chemical structure of synthetic substrates allows a tentative identification of individual enzymes contained in midgut preparations and although cysteine class proteinases are involved in beetle digestion their identities remain unknown. The objective of this study is to identify digestive proteinases in the alimentary tract of the potato beetle, Leptinotarsa decemlineata, using synthetic substrates, pH optima for substrate hydrolysis and effects of potential activators and inhibitors. MATERIAl,S AND METHODS

Insects Adult Colorado potato beetles, Leptinotarsa decemlineata, were kept in a covered glass tank (62 × 30 × 30 era),

with a soil depth of 10 cm, in a walk-in incubator at 25°C, 50% relative humidity with a 16:8 (light:dark) photoperiod. Insects were maintained on fresh potato plant cuttings. Eggs deposited on potato leaves were collected and placed in separate containers with filter paper and developing larvae were transferred to larger containers. The midguts from late third and early fourth instar larvae were collected and stored at -15°C. When required, they were thawed, homogenized in 0.15 M NaC1 and supernatants, after centrifugation at 13,500g, 4°C for l0 rain, were used for both protein and protease activity determinations. Enzyme assays All substrates, potential inhibitors and activators were purchased from Sigma Chemical Co. Assays were carried out in duplicate at 30°C, using a thermoregulated Beckman DU 65 spectrophotometer. Buffers, 0.1 M in the final reaction mixture, contained, unless stated otherwise, 1 mM DTT (dithiothreitol) and 3 mM EDTA (ethylenediamine tetraacetic acid) for Bz-Arg-NHNa (N-benzoyl-DL-arginine-naphthylamide), Bz-Arg-Np (benzoyl-oL-arginine-pnitroanilide) and Arg-NHNa (L-arglnine-fl-naphthylamide), hydrolysis. The pH for maximal activity was determined using tris [tris(hydroxymethyl)aminomethane], bis-tris [bis(2- hydroxyethyl)imino-tris (hydroxymethyl)methane], potassium phosphate, citrate-potassium phosphate and succinate. For hydrolysis of ~-N-benzyloxycarbonyl-L-AlaL-Arg-L-Arg-4-methoxy-/i'-napthylamide (Z-Ala-Arg-ArgMeONHNa) buffers were: maleate, suceinate, potassium phosphate and bis-tris propane (1,3-bis[tris(hydroxymethyl)methyl-amino]propane), 50 mM in the final reaction mixture containing 1.0 mM DTT and 1.5 mM EDTA. Total proteolysis was measured by haemoglobin (Hb) hydrolysis following the procedure of Houseman and Downe (1982b). One haemoglobin enzyme unit is defined as the increase of one absorbance unit at 280 nm, per min per ml at 30°C. Cathepsin B activity was measured with Bz-Arg-NHNa and the specific substrate Z-Ala-Arg-ArgMeONHNa (Smith and Van Frank, 1975) using the method of Barrett (1972, 1976) as modified by Houseman (1978), or with Bz-Arg-Np 0rlouseman et al., 1985). Midgut homogenate was added to 1.5 ml of buffer and allowed to incubate at 30°C for 5 min and the reaction was started by adding 40 #1 of substrate, 40 mg Bz-Arg-Np or Bz-ArgNHNa/ml or 0.5mg/ml Z-Ala-Arg-Arg-MeONHNa in dimethyl sulphoxide. With Bz-Arg-NHNa and Z-Ala-ArgArg-MeONHNa the reaction was stopped by addition of 2ml mersayl-Brij reagent (Barrett, 1976) containing 0.25 mg/ml fast garnet GBC salt (Sigma Chemical Co.) and absorbance at 520 nm determined. With Bz-Arg-Np the reaction was terminated by addition of 0.5 ml of 30% (v/v) acetic acid and absorbance at 410 nm determined. Cathepsin H activity was determined in a manner similar to cathepsin B, Arg-NHNa, 40 mg/ml in dimethyl sulphoxide, was added to 1.5 ml buffer in which midgut homogenate had preincubated, and the reaction was stopped by the addition of 2 ml mersayl-Brij reagent. To account for any increased absorbance the homogenate may contribute and any spontaneous breakdown of substrate, controls were run by adding homogenate after the stop reagent. One enzyme unit is defined as hydrolysis of 1 nmol of substrate per rain determined using molar extinction coet~eients of 27,000 for naphthylamide Fast garnet GBC colour development (Barrett, 1977a), and 8800 for Bz-Arg-Np (Erlanger et al., 1961). To further identify proteolytic activities in crude gut preparations, potential activators and inhibitors were ineluded in the reaction mixture and allowed to incubate at 30°C for 5 min with gut homogenate prior to addition of substrate. Thiol compounds, 5 mM equivalents, DTT, cysteine, mercaptoethanol and glutathione, were tested in the absence of the chelating agent EDTA, the effects of EDTA, 1 and 3 raM, were tested in the presence of 1.0 mM DTT.

Potato beetle proteinases Five and 10#g pepstatin, added in 25/tl of methanol, 0.5 and 1.0 #g E-64 [trans-epoxysueeinyl-L-leucylamido(4guanidino)butane], 0.5 and 1.0 mM (and 10 mM for Z-Ala-Arg-Arg-MeONI-INa) iodoacetamide, 0.5 mM tosylL-lysine chloromethyl ketone (TLCK), 0.75 #g leupeptin were dissolved in appropriate buffer, and 10 and 50# 8 soybean trypsin inhibitor (STI), dissolved in 0.15 M NaC1 were included in the reaction mixture and their effects on substrate hydrolysis determined.

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Protein was determined using a bovine serum albumin standard (fraction V, Sigma Chemical Co.) by the method of Bramhall et al. (1969).

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0 RESULTS Maximal haemoglobin hydrolysis by potato beetle gut homogenates occurred at pH 4.5 in citratepotassium phosphate buffer (Fig. 1). In the presence of 1 mM DTT and 3.0 mM EDTA, synthetic substrates Bz-Arg-NHNa (Fig. 2), in bis-tris buffer, Bz-Arg-Np (Fig. 3) in either bis-tris or citratepotassium phosphate buffer, were maximally hydrolysed at pH 6.0 and Arg-NHNa optimal hydrolysis occurred at pH6.5 and 7.0 with bis-tris and potassium phosphate buffers, respectively (Fig. 4). Maximal Z-Ala-Arg-Arg-MeONHNa hydrolysis, in the presence of I mM DTT and 1.5mM EDTA occurred at pH 6.5 in maleate, and 7.5 with potassium phosphate and bis-tds propane buffers (Fig. 5). Results with potential activators and inhibitors are given in Table 1. Thiol compounds increased substrate hydrolysis of Arg-NHNa, Bz-Arg-NHNa, BzArg-Np, with DTT being the best activator, and had no effect on Z-AIa-Arg-Arg-MeONHNa hydrolysis. Mercaptoethanol inhibited Hb hydrolysis, cysteine showed slight activation and DTT and glutathione had no effect of hydrolysis of the natural substrate. The chelating agent EDTA had no effect on either the natural substrate or synthetic substrates when tested in the presence of DTT. Iodoacetamide inhibited hydrolysis of Arg-NHNa, Bz-Arg-NHNa, Bz-ArgNp and Z-AIa-Arg-Arg-MeONHNa and had no effect on Hb hydrolysis. E-64 inhibited hydrolysis with all substrates tested and leupeptin inhibited all

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3

4

5

6

7

8

9

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pH Fig. 2. Effect of pH on hydrolysis of Bz-Arg-NHNa by larval midgut of L. decemlineata. Buffers were succinate (0), bis-tris (&) and tris (A).

but Arg-NHNa hydrolysis. The aspartic proteinase inhibitor pepstatin inhibited only Hb hydrolysis and STI had no detectable effect on midgut homogenate hydrolysis of any tested substrate. The effect of STI on Bz-Arg-NHNa could not be determined due to precipitation in the assay mixture when the colour reagent was added. DISCUSSION

Results from these studies indicate that at least three proteinases are present in the midgut of larval Colorado potato beetles and include cathepsin B, H and D, although their location either in the gut lumen or gut wall has not been determined. The cysteine proteinase, catbepsin B, hydrolyses substrates Bz-Arg-Np, Bz-Arg-NHNa and Z-AIaArg-Arg-MeONHNa and a thiol activator is required although activity may or may not require a chelator (Barrett and McDonald, 1980). Z-AIa-Arg-ArgMeONHNa is more specific for B, while Bz-Arg-Np and Bz-Arg-NHNa are hydrolysed by both cathepsin B and H and as a consequence cannot be used to 12

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Fig. l. Effect of pH on hydrolysis of haemoglobin by larval midgut of L. decemlineata. Buffers were citrate-potassium phosphate (D) and tris (A).

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pH Fig. 3. Effect of pH on hydrolysis of Bz-Arg-Np by larval midgut of L. decemlineata. Buffers were citrate-potassium phosphate (Q), bis-tris (&) and tris (A).

316

NORMANM. R. THInand JON G. HOUSEMAN

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pH Fig. 4. Effect of pH on the hydrolysis of Arg-NHNa by larval midgut of L. decemlineata. Buffers were succinate (O), potassium phosphate (11), bis-tris (&) and tris (A). distinguish the two enzymes in whole midgut preparations. Cathepsin H is an aminoendopeptidase, which is also activated by thiol compounds, and can be distinguished by hydrolysis of specific substrate Arg-NHNa and by the absence of leupeptin inhibition which does inhibit cathepsin B (Barrett and McDonald, 1980). Potato beetle midgut hydrolysis of all four synthetic substrates was optimal between pH 6.0-7.5, was activated by thiol compounds, except for Z-Ala-Arg-Arg-MeONHNa, inhibited by TLCK and thiol blocking agents IAA and E-64 and not affected by the protease inhibitor STI indicating that cysteine proteinases and not trypsin or chymotrypsin like enzymes are present in the larval midgut. ArgN H N a hydrolysis and the absence of leupeptin inhibition and Z-Ala-Arg-Arg-MeONHNa hydrolysis which was inhibited by leupeptin indicates that both catbepsin B and H activities are present in these preparations. The aspartic proteinases cathepsin D and pepsin both hydrolyse protein under acid conditions and are inhibited by the aspartate specific proteinase inhibitor pepstatin (Barrett, 1977b). Pepsin has maximal activ-

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Fig. 5. Effect of pH on hydrolysis of Z-Ala-Arg-ArgMoOHNHNa by larval midgut of L. decemlineata. Buffers were succinate (Q), maleate (~7), potassium phosphate ( I ) and bis-tris propane (A).

ity at pH 2.0 while cathepsin D is active under less acid conditions ranging from 2.8 to 5.0 depending on the substrate used. The pH optima for Hb hydrolysis, inhibition by pepstatin and absence of STI inhibition are all consistent with the designation of cathepsin D. Inhibition of aspartate proteinases, such as cathepsin D, by thiol compounds reducing one or more strained disulphide bonds in the enzyme has been reported previously (Barrett, 1977b) and in a similar manner TLCK inhibition may result from alkylation of an essential group. During characterizations using crude midgut preparations and general proteinase substrate Hb other proteinases contained in the assay mixture may remain active. Decreased Hb hydrolysis detected in the presence of E-64 and leupeptin may result from the residual activity of thiol activated enzymes in the assay mixture. Previous reports of digestive cysteine proteinases in insects, in particular cathepsin B, have been based either on hydrolysis of Hb or Bz-Arg-NHNa, thiol activation and inhibition by E-64 which are not adequate characteristics to accurately identify which cysteine proteinase is present since both substrates are hydrolysed by more than one enzyme. By using specific substrates for cathepsin B and H, and the differential effect of leupeptin (Barrett and Kirschke, 1981) both cysteine proteases cathepsin B and H were detected. Cathepsin H was originally designated as cathepsin Bla or cathepsin B3 and was first described as catbepsin H by Kirschke et al. (1976). As a consequence previous reports of cathepsin B activity, based on Bz-Arg-NHNa hydrolysis, in Hemiptera must remain tentative, and indicate only the presence of cysteine proteinase. The Coleoptera appear to include at least two different groups, those dependent on cysteine and aspartic proteinases and those using serine proteinases. Houseman and Downe (1983) proposed that use of catheptic proteinases in Hemiptera is a consequence of a plant sap feeding ancestor which, as a result of adaptations to this low protein food source, lost the ability to produce alkaline proteases. With a return to a high protein food source, associated with predatory habit, or seed feeding, catheptic lysosomal proteinases became the dominant enzymes for digestion. Terra et al. (1988) state that "the finding of digestive alkaline proteases in several Heteroptera families (Nepidae: Khan, 1964; Belostomatidae: Rees and Offord, 1969; Miridae: Laurema et al., 1985) disagrees with the above-mentioned hypothesis" but fails to identify these cases as specialized salivary proteinases involved in extraintestinal proteolysis and that proteolytic activity in heteropteran salivary secretions has been reported by other authors (Babtist, 1941; Kretovitch et al., 1943; Nuorteva, 1954; Bronskill et al., 1958; Edwards, 1961; Hori, 1970). These enzymes have both a different origin and function from catheptic gut enzymes and are an adaptation to a feeding mechanism requiring penetration and subsequent liquification of the ingested food to permit its ingestion through haustellate mouthparts and do not preclude the origin and use of gut catheptic proteinases as originally postulated. As an alternative it was suggested (Terra et al., 1988) that in both Hemiptera and Coleoptera catheptic proteinases are an adaptation to feeding on food

Potato beetle proteinases

317

Table I. The effect of potential activators and inhibitors on substrate hydrolysis by larval midgut homogenate from the Colorado potato beetle, Leptinotarsadecem//ncata. Buffers, unless stated other wise were: Arg-NHNa, 0.1 M bis-tris, pH 6.5 at 30°C, Bz-Arg-NHNa and Bz-Arg-Np, 0.1 M bis-tris, pH 6.0 at 30°C, all three containing I mM DTT and 3 mM EDTA, Z-AIa-Arg-Arg-MeONHNa, 50 mM male.ate,pH 6.5 containing 1.0 mM DTT and !.5 mM EDTA and 0.1 M citrate-potassium phosphate, pH 4.5, for Hb hydrolysis % Relative activity Arg-NHNa

Buffer only 2.5 mM DTT* 5 mM Mercaptoethanol* 5 mM Glutathione* 5 mM Cysteine* ! mM EDTAt 3 mM EDTAt 0.5 mM TLCK

Bz-Arg-NHNa

Bz-Arg-Np

100 170a 166" 146' 158'

100 521e 341e 234c 448c

91 b

102f

103i

92 b

104f

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51 d 52 d

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.

.

.

100

Hb

Z-Ala-Arg-ArgMeONHNa

483 h 288 h

100 881 59t

271 h

100t

103h

33J

118t 1041 941 43m

100 106P 99P 90p 94p 102p 101p 31q

41 k 40 k

101 m 97 m

9S q 86 q

.

60q

0.75 # g Leupeptin 5 # g Pepstatin 10 # g Pepstatin

94 c 106c 106c

21 f 104f 100f

40 k 103k 97 k

62 m 27 n 36n

37 q 101 q 100q

0.5 #g E-64 !.0/~g E-64 10 #g STI 50 #g STI

43c 41c 103c 104c

9s 11s ---

49j 48j 93j 93j

48° 49° 109° 107°

61q 61q 101q 102q

The following values (mean + range/2 in E U per m g protein) represent 100% activity: a, 5.71 _+0.104; b, 15.3 + 0.01; c, 17.9 _+ 0.4; d, 7.42 _+ 0.04; e, 2.37 + 0.11; f, 7.40 _ 0. i; g, 5.08 + 0.01; h, 2.28 + 0.12; i, 4.02 _+0.07; j, 5. i 7 _+ 0.07; k, 1.94 _+ 0.05; I, 39.38 _+ 1.61; m, 62.12 _+ 1.91; n, 23.79 + 2.18; o, 23.65 _+ 1.45; p, 21.81 _+ 0.08; q, 5.21 _+ 0.08. *Tested in the absence of E D T A . tBuffer contained I m M D T T .

sources rich in trypsin inhibitors a n d use the example t h a t Bruchid beetles, which c o n t a i n cathepsin B like enzymes, feed o n a food source rich in trypsin inhibitors. U s e o f these enzymes overcomes the potential detrimental effects o f the ingested inhibitors. O n the o t h e r h a n d cysteine proteinase inhibitors are present in cowpea seeds a n d they are effective at inhibiting C. maculatus proteinase ( G a t e h o u s e et aL, 1985). O t h e r insects such as h a e m a t o p h a g o u s insects, such as the m o s q u i t o Aedes aegypti (Huang, 1971), a n d Sitophilus weevils (Baker, 1982) still use tryptic enzymes even t h o u g h b l o o d serum a n d p l a n t seeds c o n t a i n high levels o f trypsin inhibitor. It is difficult to speculate o n the origins a n d role o f catheptic proteinases in Coleoptera until sufficient species in representative groups have been adequately studied a n d assessed in order to determine w h e t h e r their presence is related to ingestion o f low protein diet or proteinase inhibitors by ancestoral beetles.

Acknowledgements--This work was supported by an operating grant from The Natural Sciences and Engineering Research Council of Canada (A3618) awarded to J. G. Houseman. N.M.R. Thie was supported in part by an Employment and Immigration Canada Challenge 89 grant. REFERENCES

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Comparative Physiology, Biochemistry and Pharmacology of Insects (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 4, pp. 279-31 I. Pergamon Press, London. Applebaum S. W., Birk Y., Harpaz I. and Bondi A. (1964) Comparative studies on proteolytic enzymes of Tenebrio molitor L. Comp. Biochem. Physiol. 11, 85-103.

Baker J. E. (1981a) Resolution and partial characterization of the digestive proteinases from larvae of the black carpet beetle. In Current Topics in lnsect Endocrinology and Nutrition (Edited by Bhaskaran G., Friedman S. and Rodriguez J. G.), pp. 283-315. Plenum Press, New York. Baker J. E. (1981b) Isolation and properties of digestive carboxypeptidases from midgut of larvae of the Black carpet beetle Attagenus megatoma. Insect Biochem. 11, 583-591. Baker J. E. (1982) Digestive proteinases of Sitophilus weevils (Coleoptera: Curculionidae) and their response to inhibitors from wheat and corn flour. Can. J. Zool. 60, 3206-3214. Baker J. E. and Woo S. M. (1981) Properties and specificities of a digestive aminopeptidase from larvae of Attagenus megatoma (Coleoptera: Dermestidae). Comp. Biochem. Physiol. 69B, 189-193. Baker J. E., Woo S. M. and Mullen M. A. (1984) Distribution of proteases and carbohydrases in the midgut of larvae of the sweet potato weevil Cyclas formicarius elegantus and response of proteinases to inhibitors from sweet potato. Ent. exp. appl. 36, 97-105. Baptist B. A. (1941) The morphology and physiology of the salivary glands of Hemiptera-Heteroptera. Q. Jl microsc. Sci. 83, 91-139. Barrett A. J. (1972) A new assay of cathepsin BI and other thiol proteinases. Analyt. Biochem. 47, 280-293. Barrett A. J. (1976) An improved colour reagent for use in Barrett's assay of cathepsin B. Analyt. Biochem. 76, 374-376. Barrett A. J. (1977a) Cathepsin B and other proteinases. In Proteinasesfrom Mammalian Cells and Tissues (Edited by Barrett A. J.), p. 181. North Holland, Amsterdam. Barrett A. J. (1977b) Cathepsin D and other carboxyl proteinases. In Proteinases from Mammalian Cells and Tissues (Edited by Barrett A. J.), p. 209. North Holland, Amsterdam. Barrett A. J. and Kirschke H. (1981) Cathepsin B, cathepsin H and cathepsin L. Meth. Enzym. 80, 535-561. Barrett A. J. and McDonald J. K. (1980) Mammalian

Proteuses. A Glossary and Bibliography Vol. 1. Endoproteinases. Academic Press, New York.

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