Properties of arylamidases found in cytosol, microvilli and in luminal contents of Rhynchosciara americana midgut caeca

Insect Biochem, Vol 12, No 4. pp. 413-417. 1982 Printed m Great Britain

0020-1790/82/040413-05503.00/0 © 1982 Per,qamon Press Ltd

P R O P E R T I E S O F ARYLAMIDASES F O U N D IN CYTOSOL, M I C R O V I L L I A N D IN L U M I N A L C O N T E N T S O F R H Y N C H O S C I A R A A M E R I C A N A M I D G U T CAECA CLI~LIA FERREIRA and WALTER R. TERRA* Departamento de Bioquimica, Instltuto de Quimlca. Unlversldade de S~o Paulo, C P 20780, $5.o Paulo, Brasd (Recewed 24 November 1981)

A~tract--Dlfferentlal centnfugation of homogenates of midgut caecal cells obtained by mild and by vigorous homogenizing have been carried out and hydrolase activities using ammoacyl-fl-naphthylamldes and phenylalanylglycme (Phe-Gly) as substrates have been determined in the isolated fractions The data suggest that arylamldases and Phe-Gly hydrolase occur in the mlcrovllh and in the cytosol of caecal cells Tns buffer actwates mlcrovlllar arylamidases and shghtly inhibits hydrolases from the caecal cytosol and lumen, whereas p-hydroxymercunbenzoate inhibits the mlcrowllar enzymes and has little effect on the soluble enzymes. This, as well as the relaUve actlwty upon several different substrates, shows that arylamidases from the caecal cytosol and lumen are similar and that both differ from caecal mlcrovlllar arylamidases Electrophoretic data showed that 4 of the 5 Intracellular soluble mldgut caecal arylam~dases occur in the luminal fluid The data support the assumption that the majority of the soluble arylamidases from the caecal cells are enzymes en route to their being secreted and only a minor amount would have a typical mtracellular function K e y Word Index. Rhynchosezara amerwana, cytosohc arylamidases, luminal arylamidases, microwllar arylamidases, mldgut arylamldases, protein digestion, terminal digestion

INTRODUCTION

DIGESTION of proteins in insects is rather poorly understood. This is a consequence of a lack of comprehensive data on the distribution of peptlde hydrolases among different regions of the mldgut and also results from the lack of knowledge of the physical and kinetic properties of these hydrolases As far as we know, Rhynchosclara a m e r w a n a larvae are the best known insects in regard to the distribution of digestive hydrolases among different mldgut regions (TERRA et a l , 1979; FERREIRA and TERRA, 1980, 1982). In these larvae, a trypsin-like proteinase occurs in cells and in the luminal spaces outside and inside the peritrophlc membrane, i.e. in the ecto- and endo-peritrophic spaces. Ammopeptldases (assayed with L-leucine p-nltroanthde) are found in the ectoperitrophlc fluid and in cells, mainly of the midgut caeca, whereas carboxypeptldases are restricted to cells chiefly from midgut caeca There, ammopeptldases and carboxypeptldases occur in the cytosol and are bound to the membrane covering the cell microvilli. From an enzymological standpoint, protelnases are by far the most studied insect peptide hydrolases, whereas few data exist on insect carboxypeptldases (LAw et a l , 1977, review) Although some insect ammopeptldases have been purified and partially characterized (WARD 1975a,b,c, GOODING and ROLSETH, 1976, BAKER and Woo, 1981), they have always been prepared from the soluble portion of mldgut homogenates. This approach, in spite of producing relevant molecular data on ammopeptldases, is less useful in clarifying the physiological significance of the * To whom any correspondence should be addressed 413

various amlnopeptldases, since there should be soluble enzymes which are lysosomal, cytosolic or secreted Into the ectoperltrophic fluid. Furthermore, except for studies on R a m e r i c a n a (FERREIRA and TERRA, 1980, 1982), no study so far has taken into account the existence of amlnopepttdases bound to the mlcrovillar membranes This paper compares the amlnopeptldases from cytosol and plasma membrane of mldgut caecal cells, and from the mldgut caecal luminae R. a m e r w a n a mldgut caeca were chosen for this study because they contain virtually all the ectoperitrophic fluid and about 78°o of the total gut aminopeptidase restricted to the cells (cf. TERRA et al., 1979). MATERIALS AND METHODS Materials

L-Ammoacld oxldase (Type I1), L-argmme-fl-naphthylamlde (ArgflNA): ethylene&amme-tetraceUc ac:d (EDTAI" p-hydroxymercuribenzoate (PHMB): L-leucme-fl-naphthylamlde (LeuflNA). L-leucme-p-nitroamhde (LPNA): OLmethlonme-p-naphthylamlde (MetflNA), p-nitrophenylphosphate (NPP), peroxldase (Type IlL IA0-phenanthroline, L-phenylalanylglycme (Phe-Glyl. L-prohne-fl-naphthylamlde (ProflNA) were purchased from Sigma Chemical Company (U S A ) All the other reagents were analytical grade reagents from E Merck (Darmstadt, Germany) and J T Baker (U S A ) The solutions were prepared m glass double &stdled water Ammals Rh ~ncttos~ tara amert~ aria (Diptera, Sctandae) was reared in the laboratory according to LARAet al (19651 and only feeding female larvae at the end of the second period of the fourth mstar (TERRAet al. 1973~ were used

414

CLELaA FERREIRA AND WALTER R TERRA

Table 1. Arylamldase actlvmes (mUnlts/anlmal) of subcellular fractions of cells and of the luminal contents of the R americana mldgut caeca at d~fferent condmons* Assay con&tlons LPNA, phosphate LPNA, Tns LeuflNA, Trls

H

P1

162+44 213_+2 478_+25

527+98 836_+56 187_+8

P4

S

Caecal luminal contents

553+038 102_+09 199-+11

481 +__81 381 _ + 5 . 2 260___2.8

582_+57 247_+42 147+20

Madgut caecal cells fractions Pz P3 23.1 + 4 9 348_+30 796_+76

259+55 464_+41 123+2

* Midgut caeca cells homogcnates obtamed in mild conditions were submRted to dlfferentml ccntrffugatlon Fractaons collected. P~, pellet 600g, 10mln, P2, pellet 33009, 10mm, P3, pellet 25,0009, 10mm; P4, pellet 100,000 8, 1 hr, S, final supernatant Cell fractions and luminal contents were assayed at 37~C in either 1 mM LPNA or l mM LeuflNA m the presence of 0.1 M phosphate buffer pH 8 0 or 50 mM Trls buffer pH 8 0 Activities are means + S D based on duplicate analyses carried out m two preparations obtamed from 200 larvae each Other details in Materials and Methods

Preparatzon of samples Midgut caeca were dissected out and their luminal contents were collected as previously reported (TERRA et al, 1979). Differential centrlfugatlon of midgut caccal homogcnates was accomphshed using the followmg procedure MIdgut caeca were homogemzed under mdd (t~ght-fittlng Dounce homogemzer, 5-10 strokes) or vigorous (Ommmaxer (SorvaU) at 15,000 rev/mln for 20 scc) conditions at 4°C in a sufficient volume of pH 7.0 ISOtonic KCI solution to contam 3 mg protem/ml The homogenatcs, after bemg filtered through a piece of nylon mesh of 45 ~um pore size, were adjusted to contam 2 mg protcln/ml and centrifuged at 4°C The followmg fractions were collected: PI, pellet 600 g, l0 mln; P2, pellet 3300 g, 10 mm; Pa, pellet 25,000 g, 10ram; P4, pellet lO0,O00g, l hr; S, final supcrnatant All the pellets were &spersed m homogenlzmg medmm with the aid of a Dounce homogemzcr before use. M]crovllh from midgut caecal cells were prepared by the calcium procedure of SCHMITZ et al (1973) according to FERREIRA and TERRA (1980). Purified mlcrowth, subcellular fractions and lummal contents of caeca could be stored for at least one year at - 2 0 ° C wRhout noticeable change in the activity of the enzymes assayed H ydrolase assays and protem determlnatwn Hydrolasc assays were accomphshed under the condRlons spec]fied in Tables and Legends of Figures. Naphthylamme hbcrated from ammoacyl-fl-naphthylamldes, mtroandme from LPNA and phcnylalanme from Phe-Gly were determmed by the methods of HoPSU et al (1966), ERLANGER et al (1961) and NICHOLSON and KIM (1975), respectively In each determmat~on, incubations have been carried out for at least 4 different periods of time and the initial rates calculated A umt of enzyme as defined as the amount that catalyses the cleavage of I/~mole of substrate/ mm Protein was determmed as before (TERRA et al, 1979) Polyacrylamlde gel electrophores~s Samples were apphed to 7.5% acrylamlde gels prepared accordmg to DAWS 0964) in glass tubes of 5 mm internal dmmeter and 100 mm long The electrophoretic separation was ach]eved with a current of 2 5 mA/column at 4°C. After the runs the gels were fractlonated m an acrylamlde gel fractlonator (Autogeldwlder Savant Instruments, U.S A ) with 0.2 M phosphate buffer pH 8.0 Fractions of nmc drops (correspondmg to 2 mm of gel) were collected with the a]d of a fraction collector (2112Redlrac, LKBProdukter AB, Sweden) To each fraction 0 58 ml of 0 2 M phosphate buffer, pH 8.0 were added and after standmg overnight at 4°C the fractions filtered usmg Whatman No 41 filter paper Two ahquots of 0 2 m l were taken from each filtrate and after addmg 0.8 ml of buffered substrate (2 1 mM LPNA in 0.1 M phosphate, pH 8.0 m one ahquot and I 25 mM LcuflNA in Tns-HC! pH 8.0 in the other),

they were incubated at 37°C (60mln with LPNA and 120 mln with LeuflNA) The recovery of the arylamldase actlwty apphed to gels amounts to 95 _ 12°o (means plus S D, 8 determmattons)

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Fig 1 Dlstnbut]on of arylamldase acuvltles among subcellular fractions of R americana m]dgut caeca using &fferent substrates. Homogemzlng medmm 0.1 M KCI, pH 7 0 First column (A). mild homogemzmg Second column (B) vigorous homogemzmg. Assays (37°C) were accomplished m 50 mM Tns buffer pH 8 0 with substrates at the followrang concentrations' LeuflNA, l raM, ArgflNA, 0 56 mM, ProflNA, 0.125 raM, MetflNA, 0 125 mM; Phe-Gly, 5 mM Alkaline phosphatase is a plasma membrane marker of mldgut caeca cells (cf FERREIRAand TERRA, 1980) and was assayed with 4mM NPP in 50raM Glycme-NaOH pH 10 4 by the method of FERREIRAand TERRA(1980) The data are displayed accordmg to DE DUVE et al (1955) and are means + S.D based on determmatlons carried out on three independent preparations obtained from 200 larvae each

Midgut arylamldases

415

Table 2. Relatwe arylamidase actiwtles of the parUculate and soluble fractions of cells and tummal contents of R. americana midgut caeca upon several substrates* Source of enzyme Particulate fraction Soluble fraction Caecal contents

ProflNA

Met//NA

ArgflNA

Phe-Gly

Leu//NA

1.55 _ 0 05 1 58 _ 046 --

12.1 _ 1.0 202 + 8.6 19.1 _ 9.4

17.5 +__3 2 44 _ 15 67 + 16

21.7 + 7.8 59.1 + 8 8 62 _ 15

100 (6272 + 54) 100(584 _ 51) 100(831 _ 149)

* Data from particulate (Fraction P1) and soluble fractions were taken from experiments s~mdar to those described m Fig. IA, Caecal contents were assayed as soluble fractions m Fig. 1 Specific act]Vltles upon the substrates were calculated taking as 100 the specific actlwty (displayed m parentheses) upon Leu~NA Figures are averages + S D. from determlnat]ons carried out m at least three preparations obtained from 200 larvae each, except for caecal contents which preparations came from 50 larvae each

RESULTS Arylamidase acttwty in subcellular fractions and m luminal contents of rmdgut caeca

The particulate fractions of midgut caecal ceils are similar being most actwe upon Leu//NA in Tns buffer, intermediate in actwlty upon LPNA in Tris buffer and least active upon LPNA in phosphate buffer, whereas the contrary was true for the soluble fract]on (Table 1) Luminal contents of the midgut caeca displayed an arylam]dase actwlty similar to that of the soluble fraction. Hydrolysis of different ammoacyl-fl-naphthylamides and Phe-Gly (Fig. 1) occurred chiefly m P~ (changing to P3 after vigorous homogenization) and in the final supernatant. The data suggest arylam~dases and Phe-Gly hydrolases were both plasma membrane bound and in the cytosol. This is consistent with the fact that the plasma membrane, after mild homogemzauon produces large fragments sedimenting mainly at low grawty values. When hom-

08

ogenlzatlon is more vigorous, the resulting smaller plasma membrane fragments sediment only at higher gravity values, as ~s shown by the plasma membrane marker alkaline phosphatase (Fig. 1, see also EVANS, 1978). There was no evtdence for the occurrence of any slgmficant arylamidase (or Phe-Giy hydrolase) in lysosomes. If such were the case, arylamidase specific activity in fraction P3 (mild homogemzing conditions) should have been higher than fraction Pt and after vigorous homogemzmg some particle-bound arylam]dase should have been transferred to the soluble fraction (cf FERREIRAand TERRA, 1980) Table 2 supports the conclusions drawn from the data m Table 1: the membrane-bound enzymes are different from the cytosohc ones and these are s~mflar to those present m the luminal fired of m]dgut caeca. Since particle-bound hydrolases seem to be plasmamembrane bound enzymes (see above), midgut caecal m~crowlh were purified and used as a source of plasma membrane enzymes in the study of the effect of several compounds on them. Mlcrovilli prep-

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Fig 2 Arylamldase actwltles of the cytosol and luminal contents of the mldgut caeca after electrophoretlc separation at 4 C m polyacrylamlde gel columns A, cytosol with Leu~Na, B. cytosol with LPNA, C. caecal contents with Leu~NA, D. caecal contents with LPNA The profiles came from a preparation obtained from 200 larvae (caecal cells) or 50 larvae (caecal contents) Profiles obtamed with another preparation are very hke this one

416

CLt~LIA FERREIRA AND WALTER R. TERRA

arations are supposed to be much less contaminated by soluble enzymes than any of the particulate fractions obtained by differentml centrffugation (cf. FERREmA and TERRA, 1980). Arylamldase activity from the m~crovilli, cytosol and luminal fluids was inhibited (Table 3) by heavy metals (Zn ~+, Co 2+ and Mn z +)and by phenanthrohne, but was little affected by Ca 2+, Mg 2+ and EDTA. Microvillar arylam~dase activity was extensively mh~b~ted by PMHB, whereas cytosoi and luminal arylamidase actw~ty was little affected

Electrophorems of cytosol and lummal arylamtdases There were three major (peaks 2, 4 and 5) and two minor (peaks 1 and 3) arylam~dases from caecal cytosol (Fig. 2). Cytosol arylamidase 1 was much more active upon LeuflNA than upon LPNA and sometimes it became resolved from peak 2 as m Fig. lc for luminal arylamidases. Cytosol arylamidases 4 and 5 were poorly resolved, although they were differentiated due to their different activities upon LPNA. The migration rate and the ratio of the hydrolytic activities upon LeuflNA and LPNA support the assumption the luminal arylamidases are the same as the cytosolic ones, except for cytosol arylamidase 5, which was not found in the luminal fluid.

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DISCUSSION The role of m~dgut caeca arylamidases

Our data are not sufficient to support the assertion that the R. americana midgut caeca arylamidases are true aminopeptidases. Nevertheless, ~t is possible that they are, ff we take into account the fact that all the m~dgut arylamidases studied so far are true aminopeptidases (WARD, 1975a,b,c; BAKER and Woo, 1981). Arylamidase actwity in the ventricular cells is low (12.3% of total midgut activity), the majority of the gut arylamidase being distributed between gastric caeca cells (24.5% of total midgut activity in the m~crovilli and 20% of total m the cytosoi) and ectopentrophic fluid (43.2% of total) (TERRA et al., 1979; FERRHr,~ and T~aRA, 1980). The ectoperitroph~c fluid comprises the caecal contents and the film of fluid adsorbed to the outside surface of the peritroph~c membrane (TERRA et al., 1979). The fact that 74 2% of the gut arylam~dase acuv]ty ~s found m the caeca (cells and luminae, cf. T~RRA et al., 1979) suggests midgut caeca is the mare site for the intermediary and final digestion of proteins. Th~s ~s probably a consequence of the mare ectopentroph~c fluid flux (from ventriculus to caeca, see TERRA and FERREmA, 1981; F~RREmA et al., 1981) This flux directs, to the m:dgut caeca, the oligopeptides produced by the action of the proteinase on proteins, as soon as the ohgopeptides become sufficiently small to pass through the pentrophic membrane. The cytosolic arylamidases from the gastric caeca (except arylamidase 5) were found in the ectoperitro-. phic fluid and their amount in caecal cells was only 14.6% of total midgut arylamidase activity (calculated from the ratio of the area below peaks to the total area of the profile shown in Fig. 1B and on the known relative activity found in caecal cytosol), whde that m the lumen represented 43.2% of total midgut activity.

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Midgut arylam~dases These observatmns suggest that cytosohc arylamidases from caeca (except arylam~dase 5) are enzymes en route to their being secreted mto the midgut lumen a n d hence would not have an intracellular function. However, cytosolic arylamtdase 5 from caeca, which corresponds to 27~, of the total cytosolic arylam~dases (5 4~'o of m~dgut activity) a n d does not occur in the lummal fluid, should have an mtraceilular functmn. Microwllar arylamtdases from caeca are very actwe (24.5~o of m~dgut actwity) a n d are d~fferent from the lummal arylamsdases (see Results) It ~s posstble that their spectfictties are such as to complete the pept~de hydrolysis started by the luminal arylamldases. Acknowledgements--This work was supported by grants from the Funda~:,~o de Amparo /t Pesquisa do Estado de S~o Paulo (FAPESP). We are much indebted to M~ss LU~ZA Y. NAKAaAVASm for technical assistance. C F ~s a graduate fellow of FAPESP and W.R.T. ~s a staff member of the Bmchem~stry Department and a research fellow from the Conselho Nacmnal de Desenvolvimento Clentirico e Tecnol6g~co (CNPq).

REFERENCES BAKER J E. and Woo S. M (1981) Properties and spectficlties of a digestive ammopeptldase from larvae of Attagenus megatoma (Coleoptera.Dermestldae). Comp. Btochem. Phystol 69B, 189-193. DAVIES B J. (1964) DIsc electrophoresls--II. Methods and apphcatmn to human serum proteins Ann N.Y Acad Scl. 121,407-427. DE DUVE C, PRESSMAN B. C, GIANETTO R, WATTIAUX R and APPELMANSF (1955) Tissue fractmnatmn studies. 6 lntracellular d~stnbutmn patterns of enzymes m rat hver tissue. Biochem J 60, 6044517. ERLANGER B F., KOKOWSKY N and COHEN W (1961) The preparauon and properties of two new chromogemc substrates of trypsin. Archs Bzochem. Bzophys 95, 271-278 EVANS W. H. (1978) Preparatmn and charactenzatmn of mammahan plasma membranes. In Laboratory techtuques in biochemistry and molecular biology (Ed. by WORK T S. and WORK E.), Vol. 7, Part 1, Pocket edition. North-Holland, Amsterdam FERREIRA C, RIBEIRO A F and TERRA W. R. (1981) Free structure of the larval mldgut of the fly Rhynchosc,ara and its physmloglcal imphcatmns J Insect Physwl. 27, 559-570 FERREIRAC and TERRA W R. (1980) Intracellular dlstnbu-

417

tlon of hydrolases in mldgut caeca cells from an insect with emphasis on plasma membrane-bound enzymes Comp. Btochem Physiol. 66B, 467--473 FERREmA C. and TERRA W R (1982) Function of the midgut caeca and ventnculus, mlcrovllh bound enzymes from cells of different mldgut regions of starving and feeding Rhynchosctara amerzcana larvae Insect Btochem 12, 257-262. GOODING R H. and ROLSETH B. M. (1976) DIgestwe processes of haematophagous insects XI Partial purification and some properties of six proteolytlc enzymes from the tsetse fly Glossma morsttans morsztans Westwood (Diptera.Glossmldae). Can J Zool. 54, 1950-1959) Hopsu U K, MAKINE~ K K and GLENNER G G (1966) Purlficatmn of a mammahan pepndase selective for N-terminal arglnme and lysme residues ammopeptldase B Archs Bwchem Bwphys 114, 557-566 LARA F J S, TAMAKI H and PAVAN C (1965) Laboratory culture of R angelae Am Nat 99, 189-191 LAW J H, DUNN P E and KRAMER K J (1977) Insect proteases and peptldases. Adv Enzymol. 45, 389-425. NICHOLSON J. A and KIM Y S (1975) A one-step L-amino acid oxldase assay for intestinal peptlde hydrolase actlwty Analyt Bwchem 63, 110-117. SCHMITZ J, PREISER H, MAESTRACCI D, GHOSH B K, CERDA J and CRANE R K (1973) Purification of the human intestinal brush border membrane Bwchlm Btophys. Acta 323, 98-112 TERRA W R, DE BIANCHI A G . GAMBARINI A G and LARA F J S (1973) Haemolymph amino aods and related compounds during cocoon productmn by the larvae of the fly, Rhyncho,s¢tara americana J Insect Physlol 19, 2097-2106 TERRA W. R and FERREmA C (1981) The physmloglcal role of the pentrophlc membrane and trehalase digestive enzymes m the mldgut and excreta of starved larvae of Rhynchosclara J. Insect Physwl 27, 325-331 TERRA W. R, FERREIRA C and DE BIANCHI A G (1979) D~stnbutmn of d~gestwe enzymes among the endo- and ectopentrophlc spaces and mldgut cells of Rhynchosctara and its physmloglcal slgmficance J Insect Phvswl 25, 487-494 WARD C. W (1975a) Resolutmn of proteases tn the kerat,nolytlc larvae of the webbing clothes moth Aust J hwl Scl 28, 1-23 WARD C. W. (1975b) Ammopeptldases m webbing clothes moth larvae Properties and speclficmes of the enzymes of intermediate electrophoretlc mobdtty Bwchlm. Bwphys. Acta 410, 361-369. WARD C. W. (1975c) Amlnopeptidases in webbing clothes moth larvae Properties and spec~fiCltles of the enzymes of highest electrophoretlc mobdlty Aust J bwl Sct 28, 447-455.