Endoproteinase activity in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Insect Bwchem, Vol 10, pp. 363 to 366 ©Pergamon Press Ltd 1980 Printedm Great Britain

0020-1700/80/0801--0363 $02 00/0

ENDOPROTEINASE ACTIVITY IN THE POSTERIOR MIDGUT OF R H O D N I U S P R O L I X U S STAL (HEMIPTERA: REDUVIIDAE) JON G HOUSEMAN and A E. R. DOWNE Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

(Received 2 October 1979)

Abstract--Tests with homogenates of digestive m~dgut of Rhodmus prohxus Still showed that hydrolysis of N-benzoyl-DL-argmme naphthylamme (BANA), N-benzoyl-oL-argmine-p-mtroanillde (BAPNA) and casein was activated by EDTA and thlol reagents (cysteme, glutathlone, mercaptoethanol, and dithlothreltol) but the hydrolysJs of N-benzoyl-L-tyrosme ethyl ester (BTEE) was.inhibited lodoacetamlde and tosyl-L-lysme chloromethyl ketone inhibited BAPNA and casein hydrolysis but &d not affect BTEE hydrolysis Iodoacetamlde also inhibited BANA hydrolysis Horse serum inhibited hydrolysis of all tested substrates but soybean trypsin inhibitor inhibited only casein and BTEE actwitles Metal ions Ca 2+, Mg 2 + and Mn 2+ had no affect on hydrolysis of BTEE, BANA or casein when crude mldgut homogenate was used These results suggest that at least two protelnases (one, cathepsln B-like, and another which is a thlol- and EDTAsenslt~ve endoprotelnase) are present in the posterior mldgut of R prohxus Key Wordlndex Rhodmusprohxuv, haematophagous insects, digestion, endoprotelnase, cathepsm B-hke protemase

INTRODUCTION

(HOUSEMAN, 1978) This term has recently been changed to cathepsln B (BARRETT, 1977) and this INITIAL Investigations of proteinase activity from the change applies to the name suggested for the R digestive mldgut of Rhodmus prohxus St~tl prohxus protelnase demonstrated an acid protelnase that hydrolyzed The purpose of this lnvestlgatton was to determine ff azocaseln at an optimal p H of 5.5 (PERSAUD and more than a single endoprotelnase is present in the DAVEY, 1971) Recent stu&es showed that proteolytlc m i d g u t o f R prohxus Various activator and inhibitor activity increases In the presence of thlol reagents chemicals were used to further characterize the (HousEMAN, 1978, GARCIA et al., 1978) Dtgestlve protelnase activities mldgut homogenates hydrolyzed the low molecular weight trypsin substrates N-benzoyl-oL-arglnlne-pnitroanihde (BAPNA) and N-benzoyl-oL-arglnme MATERIALS AND METHODS fl-naphthylamlne ( B A N A ) (HOUSEMAN, 1978) and the chymotrypsin substrate N-benzoyl-L-tyroslne ethyl lnwcts ester (BTEE) (GOODING, 1968, 1969, GARCIA et al., A colony of Rhodmus prohxus Still, originally supphed by 1978) Atomic Energy Canada, Chalk River, Ontario, was GARCIA et al (1978) found "only one enzyme to maintained in the laboratory using the method of KWAN and DOWNE (1977) explain the endopeptldase activity of R prohxus" and suggested the name " R prohxus thlol protease." The Preparatzon of mtdgut homogenate authors examined the effects of various activators and The digestive midgut, the region between the crop and the lnhlbltors on midgut protelnase hydrolysis of whole rectum, was removed from adult male and/or female insects protein substrates, casein and azocaseln, and the low five to ten days after feeding to repletion on a rabbit and molecular weight substrate BTEE Activity stored frozen ( - 15°C), in batches of twenty, untd required determinations were not carried out m a parallel The mldgut material was thawed and, unless stated manner for low molecular weight and protein otherwise, homogenized in 0 1 M potassium phosphate substrates and mldgut preparations were treated with buffer, pH 5 5 The supernatant, approximately 2 5 ml, after tetrathlonate to increase stablhty of the BTEEcentrlfugatlon at 15,000 g for 10 mm at 4°C, was used for protemase actwlty determinations For BTEE assays hydrolyzlng protelnase supernatants from 3 or 4 preparations were pooled, dialyzed Rhodnius prohxus proteinase has been shown to be against several changes of buffer, and concentrated by thlol and E D T A (ethylenedlamlnetetraacetlc acid) ultrafiltratlon using an Amlcon PM 10 membrane filter After achvated and to hydrolyze casein, B A P N A and concentration this material was recentnfuged and the B A N A (HOUSEMAN, 1978) During this study supernatant used m actlwty determinations untreated mldgut homogenate was used and no Enzyme as~ay~ investigations were done using lnhlbltors to identify the protelnase The name 'cathepsln Bl-hke Protemase determinations using BANA (Sigma Chemical Co.) were carried out as previously described (HOUSEMAN, protelnase' was proposed for this protelnase 363

364

JON G HOUSEMANAND A E R DOWNE

1978) Unless stated otherwise the reaction buffer, 1 5 ml pH 5 5, consisted of 0 1 M potassmm phosphate, 3 mM EDTA and 1 5 mM dlthlothreltol (DTT) EDTA and DTT were present in excess to ensure complete activation. In some cases high concentrations of protein caused the reaction mixture to become cloudy after addmon of the colour reagent. When this occurred colour was extracted using 2 5 ml of n-butanol and absorbance of the organic phase determined (BARRETT, 1972). The method of BARRETT (1972) was used to determine hydrolysis of BAPNA (Sigma Chemical Co ) The reaction mixture, unless stated otherwise, consisted of 1 5 ml of 0 1 M potassmm phosphate, 5 mM EDTA and 3 mM DTT, pH 5 5 Prehmmary results showed that the amounts of EDTA and DTT were m excess of the minimal concentration for complete actuation The reaction was stopped by the ad&tlon of 0 5 ml of 30~ (v/v) acetic Rod Hydrolysis of BTEE (Sigma Chemical Co) was determined using a mo&ficat~on of the method of HUMMEL (1959) Mldgut homogenate was added to 0 5 ml of 0 1 M potassmm phosphate buffer, pH 5 5, m a 1 ml cuvette After mcubatlon for 5 mm at 30°C to ensure temperature equlhbratlon, 0 5 ml of BTEE, 1 mM m 50~o (v/v) methanol, was added The absorbance, determined using a Gdford 2400-S spectrophotometer equipped with dual thermospacers, at the beginning and the end of the reaction was used for actwlty determinations Casem (techmcal grade, Sigma Chemical Co) was dissolved m 0 1 M NaOH and after centrlfugatlon at 10,000g for 15 mm the supernatant was dmlyzed, at 4°C, against several changes of 0 15 M NaCI untd the pH of the protein solution was neutral The dmlyzed protein was recentnfuged and adjusted to 20 mg of protem/ml Casein hydrolysis was determined by adding midgut homogenate to 0 3 ml of phosphate buffer, pH 5 5, containing or lacking both 10 mM EDTA and 5 mM DTT and incubated for 5 mm at 30°C prior to the addition of 0 3 ml of casein substrate The reaction was stopped by the ad&tlon of 0 6 ml of 20~o (w/v) tnchloroacetlc acid (TCA) After centrffugatlon at 15,000 g for 10 mm the absorbance, at 280 nm, of TCA-soluble material was used for activity determinations Controls were run to account for spontaneous breakdown of substrate and endogenous substrate m m~dgut preparations Unless stated otherwise all reacttons were carried out at 30~C and controls were run to account for any increase m absorbance by actuator or inhibitor chemicals All determinations were done m duphcate, except for BTEE determinations which used 6 to 10 rephcates One enzyme unit (E U ) is defined as the amount of materml required to hydrolyze 1 #moles of substrate of 30 °, or 37~C/mm/ml Extmctton coefficients for BAPNA (A~e~l o = 8 , 8 0 0 ERLANGER et al, 1961), BANA (Ae520=27,000 BARRETT, 1978) and BTEE (Ae2s6=964 HUMMEL, 1959) were used to determine/~moles of substrate hydrolyzed One casein enzyme umt (C E U ) is defined as the increase of one absorbance umt, at 280 nm/mm/ml of reaction mixture at 30~C

Protein determinations

Protein in midgut homogenates was determined using the method of BRAMrtALL et al (1969) All other protein determinations used the method of LOWRY et al (1951) Bovine serum albumin (fraction V, Sigma Chemical Co ) standard was used for both methods

RESULTS The BTEE-hydrolyzing proteinase is reported to be unstable to freezing and handling (GARCIA et a l , 1978). During p r e h m i n a r y investigations only freshly prepared mldgut h o m o g e n a t e was used. We found, however, no apparent loss o f actwlty in h o m o g e n a t e s prepared from whole frozen midguts. The effect o f 5 m M equivalents (i.e. 5 m M m o n o t h i o l and 2.5 m M &thtol) o f cysteine, reduced glutathlone, 2-mercaptoethanol and D T T m the reaction mixture on the hydrolysis o f B A P N A , BTEE and B A N A was determined in the absence o f E D T A . Hydrolysis o f BTEE was determined at 30°C and 37°C Thiol reagents strongly activated hydrolysis o f B A P N A and B A N A but inhibited s o m e w h a t hydrolysis o f BTEE at both 30 ° and 37°C (Table 1). The effects o f 1 m M and 3 m M E D T A , m the presence o f D T T on hydrolysis o f B A P N A , B A N A and casein and m the absence o f D T T on casein and BTEE were determined E D T A activated hydrolysis o f B A N A , B A P N A , thiol-actwated and unactwated casein hydrolysis but inhibited hydrolysis o f BTEE (Table 2) 2-Iodoacetamide (IAA, Baker Chemical Co ), 0 5 and 1.5 m M , 0.5 m M tosyl-L-lysine chloromethyl ketone (TLCK, Sigma Chemical Co.) 50 and 150 #g o f horse serum (Nutritional Blochemicals) protein and soybean trypsin inhibitor (Type II-S, Sigma Chemical Co ) protein was a d d e d to E D T A - p h o s p h a t e buffer for B A P N A , B A N A and thiol-acttvated casein assays and to p h o s p h a t e buffer for unactlvated casein and BTEE This was incubated for 5 m m with mldgut homogenate. DTT, in 25 #1, was added to give the appropriate thlol concentration in the thlol assays and this was incubated for an additional 5 m m prior to addition o f substrate Hydrolysis o f B A P N A , B A N A , thlol-actlvated and u n a c t w a t e d casein was inhibited by I A A but hydrolysis o f BTEE did not show any marked decrease (Table 2) T L C K Inhibited the hydrolysis of BAPNA, thiol-activated and unactwated hydrolysis o f casein but hydrolysis o f BTEE was not affected (Table 2) It was not possible to

Table 1 Effect of thlol reagents on the hydrolysis of three substrates by dlgestwe mldgut homogenate from R prohxus BTEE 30°C (No EDTA) Buffer alone 5 mM Cysteme 5 mM Glutathlone 5 mM Mercaptoethanol 2 5 mM DTT

100a 68 73 88 82

Relative activities BTEE 3T'C BANA 30°C (No EDTA) (3 mM EDTA) 100b 94 100 79 88

100c 720 770 470 1160

BAPNA 30~C (5 mM EDTA) 100d 3571 2400 429 3042

100~o activities, _+ range/2 for duphcate assay and SUM for BTEE assays, per mg ofmldgut protein a 0 0790 _+ 0 0017 E U ( N = 9 ) , b 0 1 3 3 0 + 0 0 0 3 2 E U ( N = 9 ) , c 0 0 0 2 1 + 0 0 0 1 0 E U ( N = 2 ) , d 0 0 0 0 1 _+ E U (N=2)

Endoprotemases of R prohxus

365

Table 2 Effects of EDTA, IAA and TLCK on the hydrolysis of four substrates by &gestlve mldgut homogenate from

R. prohxus

Buffer 1 mM EDTA 3 mM EDTA 0 5 mM IAA* 1 5 mM IAA* 0 5 mM TLCK*

BANA (1 5 mM DTT)

BAPNA (3 mM DTT)

100 298 a 315 a 2b

100c 185 200 6

0b

Relative actwmes Casein (2 5 mM DTT)

Casein (No DTT)

BTEE (No DTT)

100 1200 1340 58c 26c 15e

100f 120 145 94 61 35

100 86g 75 s 91 g 91 g 98 h

0

0

* Reaction mixtures contained the following EDTA concentrations BANA, 3 mM, BAPNA, 5 mM, and casein with DTT, 5 mM 100% actlvmes, + range/2 for duplicate assays and SEM for BTEE assays, per mg ofmldgut proteins a 0 0114 + 0.0009 E U (N = 2), b 0 0329 + 0 0009 E U (N = 2), c 0 0061 + 0 0002 (N = 2), d 0 0588 + 0 0039 C E U (N = 2), e 0 0783 + 0.0042 (N = 2), f0 0321 + 0 0028 (N = 2), g 0 0790 + 0 0019 (N =9), h 0 065 t + 0 0021 (N = 6) Hydrolysis of BANA with TLCK was not determined because of chloromethyl ketone interference with assay procedure

d e t e r m i n e if T L C K inhibited B A N A hydrolysis, because T L C K interferes with the assay procedure (BARRETT, 1972). Serum inhibited hydrolysis o f all tested substrates while soybean trypsin i n h i b i t o r shghtly decreased only BTEE, thlol-actlvated a n d u n a c t i v a t e d casein hydrolysis (Table 3). A d d i t i o n of the soybean i n h i b i t o r to the B A N A reaction mixture precipitated the substrate. The effect of CaC12, MgCI 2, 1 m M a n d 3 m M in 0 1 M s o d i u m acetate, p H 5.5, o n hydrolysis o f thiolactivated B A N A a n d casein a n d u n a c t i v a t e d B T E E a n d casein by m i d g u t h o m o g e n a t e p r e p a r e d in acetate buffer, was determined. D T T was a d d e d to B A N A a n d t h l o l - a c t w a t e d casein assays after h o m o g e n a t e a n d i n c u b a t e d with the metal ion for 5 m m at 30°C p r i o r to a d d i t i o n of the substrate M e t a l ions tested did n o t m a r k e d l y inhibit or activate hydrolysis o f any substrate except for 25~o inhibition of thlol-activated casein hydrolysis in the presence o f 3 m M M n C I 2.

DISCUSSION Results suggested t h a t at least two e n d o p r o t e m a s e s were present in the m i d g u t o f R. prolixus. The first proteinase was cathepsin B-hke a n d hydrolyzed low

molecular weight substrates B A P N A a n d B A N A . It was activated by thiol a n d E D T A a n d inhibited by serum, I A A a n d T L C K . Soybean trypsin i n h i b i t o r did n o t affect B A P N A hydrolysis. These results were consistent w~th the characteristics of cathepsin B-like proteinase The second proteinase hydrolyzed BTEE, was inhibited by thiol reagents, E D T A , horse serum a n d soybean trypsin i n h i b i t o r b u t n o t affected by either I A A or T L C K . T h e E D T A thiol-sensltive proteinase h a d similar characteristics to some metaloprotemases. U n t i l the proteinase is purified a n d substrate speoficities determined n o n a m e has been suggested for the second R. prolixus e n d o p r o t e m a s e . M a n g a n e s e ion a p p e a r e d to inhibit only thiolactivated casein hydrolysis while u n a c t l v a t e d casein, B T E E a n d B A N A hydrolysis was n o t affected Calcium a n d m a g n e s i u m ion did n o t affect hydrolysis of any substrate The lack of a n affect by metal ions may result from using crude m i d g u t h o m o g e n a t e which m a y c o n t a i n e n d o g e n o u s metal ions, r a t h e r t h a n purified enzyme. Thlol-activated a n d u n a c t i v a t e d casein hydrolysis showed similar results, except for m a n g a n e s e inhibition, t h r o u g h o u t the study due to the presence of b o t h proteinases in the crude m l d g u t h o m o g e n a t e . T h e present results t h a t were o b t a i n e d for u n a c t i v a t e d casein hydrolysis c o m p a r e with those reported by

Table 3 Effect of natural protemase mhlbltors horse serum and soybean trypsin Inhibitor on the hydrolysis of four substrates by digestive midgut homogenate from R prohxus BANA (3 mM EDTA l 5 mM DTT) Buffer 50/~g Serum protein 150/zg Serum protein 50 #g Soybean inhibitor protein 150 #g Soybean inhibitor protein

100a 97 37

BAPNA (5 mM EDTA 3 mM DTT) 100b 81 73 100 104

Relative activities Casein (5 mM EDTA 2 5 mM DTT) 100c 64 57 81 63

Casein (No EDTA No DTT) 100a 108 88 104 87

BTEE (No EDTA No DTT) 100e 82 36 75 65

100% activities, + range/2 for duplicate assays and SEM for BTEE assays, per mg mldgut protein a 0 0329 + 0 0009 E U (N=2),d00411 + 00013CEU (N=2),e00848 + 0 0021 E U (N =9S The effect of soybean trypsin inhibitor on BANA hydrolysis was not determined because substrate would not remain m solution m the presence of the inhibitor

(N=2),bO0061 + 0 0 0 0 2 E U ( N = 2 ) , c 0 0 7 8 3 + 0 0 0 4 2 C E U

366

JON G HOUSEMANAND A E R DOWNE

GARCIA et al. (1978) These authors did not report addition of thlol in any whole protein assays N o reason was given for the omission of thiol while studying what they have termed a thiol protelnase or in the study of control of proteinase production in R. prohxus (GARCIA et a l , 1977) In only one instance is thlol-acttvated B T E E hydrolysis reported by GARCIA et al. (1978) and in this case it is not clear whether gut material was treated with tetrathlonate, which could create apparent thiol activation When the affect of thlol on B T E E hydrolysis was determined at 37°C, the reaction temperature reported by GARCIA et al. (1978), no thlol activation was detected. During their single step purification procedure using mercurial affinity chromatography R. prohxus cathepsln B-like proteinase was probably also purified. Cathepsm B adsorbs to this affinity material (BARRETT, 1977) Serum inhibits both R prohxus endoprotelnase activities, and the alkahne protemases in other haematophagous insects GOOOING (1977) suggested that there would be an advantage to having serum inhibitors as the secretagogue controlling protelnase production in bloodsucking insects. This hypothesis would apply to R prohxus even though its protelnases differ from other haematophagous insects The discrepancies between the findings ofGARCIA et al (1978) and ourselves, with BTEE, illustrate the Importance of ensuring that the hydrolysis of protein and low molecular weight substrates is carried out in a parallel manner using untreated mldgut homogenate during preliminary investigations. It is possible that these discrepancies may have resulted from the use of cltrated blood as a meal source rather than a live host. GARCIA et al (1978) report that mldgut homogenate was clear and colourless while we obtained a coloured preparation It is also not known if different strains of R prohxus contain different protemases It is likely that more protemases will be detected in R prohxus. GOODING and ROLSETH (1976) found four endoproteinase and three exoproteanase fractions during partial purification of digestive enzymes from Glossina morsttans morsttans To date no exoprotelnase has been described from R. prohxus midgut homogenates Further work will be carried out in this laboratory to identify digestive protemases in R. prohxus and factors controlling their production. Acknowledgements--We thank DAROE GREGGSand Mrs ANNE HUTCHISONfor maintenance of the insect colony and JOAN ROBERTSfor typmg the manuscript The research was

supported by a grant from the National Research Council of Canada (No 6499)

REFERENCES

BARRETTA J (1972) A new assay ofcathepsln BI and other thtol protemases Anal B1ochem 47, 280-293 BARRETT A J (1977) Cathepsm B and other thlol protemases In Protemases from Mammahan Cells and Tissues (Ed by BARRETTA J ) p 181 North Holland, Amsterdam, New York BRAMHALLS, NOACK N , WU M and LOEWENBERGJ R (1969) A simple colorlmetrlc method for determination of protein Anal Bloehem 31, 146-148 ERLANGERB F , KOKOWSKYN. and COHENW (1961) The preparation and properties of two chromogemc substrates for trypsin Arch Btoehem. Btophys 95, 271-278 GARCIAE. S, MACARINIJ D and GARCIAM L M (1977) Control of protease secretion in the intestine of fifth lnstar larvae of Rhodmus prohxus J Insect Physlol 23, 247-252 GARCIA E S, GUIMARAESJ A and PRADO J L (1978) Purification and properties of a sulfhydryl-dependent protease from Rhodmus prohxus m,dgut Arch Btochem Btophys 188, 315-322 GOOOING R H (1968) Dlgestwe protemases from bloodsucking insects XII1 Int Congr Entomol Proc pp 381-382 GOODINGR H (1969) Studies on the protemases from blood sucking insects Proc ent Soc Ont 100, 139-145 GOODING R H (1977) Dlgestwe processes of haematophagous insects XII Secretion of trypsin and carboxypeptldase B by Glossma morsttans morsttan~ Westwood (Diptera Glossmldae) Can J Zool 55, 215-222 GOODING R H and ROLSETH B M (1976) Digestive processes of haematophagous insects XI Partial purification and some propert,es of six proteolyt~c enzymes from the tsetse fly Glossma morsttans morsttans Westwood (Diptera Glosslnidae) Can J Zool 54, 1950-1959 HOUSEMAN J (1978) A thiol-activated dlgestwe protemase from adults of Rhodnms prohxus Stal (Hemiptera Reduvndae) Can J Zool 56, 1140-1143 HUMMEL C W (1969) A mod,fied spectrophotometnc determination of chymotrypsin, trypsin and thrombm Can J Btochem Physzol 37, 1393-1399 KWAN L. R and DOWNEA E R (1977) The effects of 5fluorouracil on reproduction in Rhodmus prohxus J Med Ent 14, 270-275 LOWRYO H , ROSEBROUGHN. J, FARRA L. and RANDALL R J (1951) Protein measurement with the fohn phenol reagent J Btol Chem 193, 265-275 PERSAUD C E and DAVEY K G (1971) The control of protease synthesis in the intestine of adults of Rhodnms prohxus J Insect Physml 17, 1429-1440