endopeptidase 24.15 from rat testes

Biochem. J. (1989) 261, 951-958 (Printed in Great Britain)

Endopeptidase 24.15 from rat testesIsolation of the enzyme and its specificity toward synthetic and natural peptides, includingenkephalin-containing peptides

Marian ORLOWSKI,* Sandra REZNIK,* Julia AYALA* and Adrian R. PIEROTTIt* Department of Pharmacology and t Research Center in Neurobiology, Mount Sinai School of Medicine of the City Universityof New York, New York, NY 10029, U.S.A.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70000, was purified tohomogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobicamino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicatedby the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate bindingand catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymicactivity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2" ions. The rateof reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. Theseresults are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethylpeptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potentcompetitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone,bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced fromstudies with synthetic peptides. Dynorphin A (1-8)-peptide, /8-neoendorphin, metorphamide, and Met-enkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme iscatalytically and immunologically closely related to the previously identified brain enzyme.

INTRODUCTION

Previous work in this laboratory led to the identifica-tion and partial purification from rat brain of a metallo-endopeptidase predominantly associated with the solublefraction of homogenates [1]. A membrane-bound form ofthe enzyme constituting approx. 20 % ofthe total activity,and having properties similar to those of the solubleform, was found associated with brain particulate frac-tions, including synaptosomes [2]. The finding that endo-peptidase-24.15 is highly active in brain, pituitary andtestis, and that it does not apparently cleave proteins andlarge peptides containing more than 20 amino acidresidues, suggests that the enzyme is involved in themetabolism of bioactive peptides.

Endopeptidase 24.15 together with endopeptidase24.11 [3-6] seem to constitute the main two brainmetalloendopeptidases. Unlike endopeptidase-24. 11,also misleadingly called 'enkephalinase', because of itscleavage of the Gly-Phe bond in both Leu- and Met-enkephalin [7], endopeptidase 24.15 does not attack thetwo enkephalins. Indeed, experiments with purified pre-parations of the enzyme from rat brain and also experi-ments with synaptosomal membranes have shown thatthe enzyme converts some larger opioid peptides such as

dynorphin A"-8, /3-neoendorphin and Met-enkephalin-

Arg-Gly-Leu into Leu- and Met-enkephalin respectivelyin reactions inhibited by cFE-AAF-pAB, a substrate-related specific inhibitor of the enzyme [2,8].

Isolation of a homogeneous preparation of endo-peptidase 24.15 from rat brain by conventional enzyme

purification methods presented difficulties [1]. Even thebest enzyme preparations contained an inactive proteincontaminant of almost the same isoelectric point andmolecular mass, that could not be removed by ionexchange chromatography, preparative gel electro-phoresis or chromatofocusing. Since rat testes are rich inendopeptidase 24.15, we directed our efforts towardsisolation of the enzyme from this source. Most of theenzyme activity in rat testes is associated with the solubleprotein fraction of homogenates, whereas the membrane-bound form of the enzyme constitutes only about 8 % ofthe total activity. Here we report the isolation of a

homogeneous preparation of the enzyme, and the resultsof studies of its interaction with synthetic substrates,natural peptides, and inhibitors. Evidence is presentedthat the testis and brain enzymes are immunologically andcatalytically closely related if not identical. The homo-geneous testis enzyme, like its brain counterpart, convertsdynorphin A"-8, fl-neoendorphin, metorphamide andMet-enkephalin-Arg8-Gly7-Leu8 into the correspondingenkephalins in a reaction inhibited by cFP-AAF-pAB.

Abbreviations used: Bz, NA-benzoyl; cFE-AAF-pAB, N-[(IRS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-pAB; cFP-AAF-pAB, N-[(IRS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-pAB; DFP, di-isopropyl fluorophosphate; DTT, dithiothreitol; LHRH, luteinizing hormone-releasing hormone; pAB,p-aminobenzoate; PCMB, p-mercuribenzoate; PMSF, phenylmethanesulphonyl fluoride; PAGE, polyacrylamide-gel electrophoresis. Thenomenclature proposed by Schechter & Berger [26] is used to describe the position (P) of the residues in the substrate and the correspondingsubsites (S) in the active site of the enzyme.

t To whom correspondence and reprint requests should be addressed.

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M. Orlowski and others

This is in contradiction with a report by Toffoletto et al.[9] that these reactions are catalysed by endo-oligo-peptidase A but not by endopeptidase 24.15.

MATERIALS AND METHODS

MaterialsEndopeptidase 24.15 substrates and inhibitors were

synthesized as described previously [1,10,11]. Frozen rattestes from Sprague-Dawley rats were obtained from PelFreeze Inc. (Rogers, AR, U.S.A.). All other reagentswere obtained from Fisher Scientific Co. or from SigmaChemical Co.

Determination of enzyme activityEnzyme activity was determined in a coupled enzyme

assay in the presence of excess of aminopeptidase N (EC3.4.11.2) using Bz-Gly-Ala-Ala-Phe-pAB (0.8 mM) as thesubstrate [1]. The enzyme cleaves the Gly-Ala bondyielding two products, Bz-Gly and Ala-Ala-Phe-pAB.The second of these products is hydrolysed by theaminopeptidase with the release of pAB which is thenquantified after diazotization.

In crude enzyme preparations the Ala-Phe bond ofthe substrate can be cleaved by endopeptidase 24.11.Accordingly, in such preparations the activity was

determined in the presence of 25 /LM-N-[(1 RS)-carboxy-2-phenylethyl]-Phe-pAB, a specific inhibitor of endo-peptidase-24.1 1 (Ki 7.1 x 10-8 M) [12]. To account for thepossible substrate degradation by other enzymes presentin crude tissue homogenates the degradation of thesubstrate was also measured in parallel reaction mixturescontaining, in addition to the inhibitor of endopeptidase-24.1 1, also 25 /SM of cFP-AAF-pAB, a specific inhibitorof endopeptidase-24. 15 [1 1]. The difference in the rate ofsubstrate degradation between incubation mixtures con-

taining both inhibitors and those containing only theinhibitor of endopeptidase-24. 11 was taken as a measure

of endopeptidase-24.15 activity. Activity is expressed inunits, 1 unit being defined as the amount ofenzyme whichcatalyses the release of 1 ,umol of product/h. Specificactivity is expressed in units/mg of protein as determined,by the method of Lowry et al. [13].

Determination of kinetic constantsThe steady-state parameters, Km and kcat. (= Vle,

where e = total enzyme concentration) were determinedfrom initial velocity measurements at various substrateconcentrations. Michaelis-Menten kinetics wereobserved with all substrates. Km values were calculatedfrom double-reciprocal plots by a linear-regressionprogram. Correlation coefficients were generally 0.99 or

better. In the calculation of data an Mr of 70000 was

assumed, with one catalytic site per enzyme molecule.K1 measurements were carried out by the method of

Dixon [14] at three different substrate concentrations andat six different inhibitor concentrations (plots of 1/v

versus [1]) using a computer program. Coefficients ofdetermination (r2) of better than 0.99 were generallyobtained.

Polyacrylamide-gel electrophoresisDisc PAGE was carried out under non-dissociating

conditions in a 0.05 M-Tris/HCl buffer (pH 8.3). Enzymeprotein (5-10 ,ug) was layered on the top of the gel anda current of 4 mA/tube was applied for a time period

necessary for the tracking dye to reach the bottom of thegel. Preparative slab PAGE was carried out under similarconditions in gels of 3 mm thickness using a slab gelapparatus (model 100; Aquebougue Machine Shop, Box205, Aquebougue, NY 11931, U.S.A.). Electrophoresisunder dissociating conditions and Mr determinationswere carried out in the same apparatus using a dis-continuous buffer system [15]. Gels (8 %o) contained 0.1 00SDS, and electrophoresis was run after the enzymehad been exposed to a 1 00 solution of SDS and 20%2-mercaptoethanol and heated to 70 °C for 10 min. Mrstandards contained 2.5 jig of each protein (trypsinogen,ovalbumin and bovine serum albumin).

Isolation of the enzymeAll purification steps were carried out at 4 'C. De-

ionized water was used for the preparation of buffers.Frozen rat testes (100 testes; 172 g) were defrosted,homogenized in 4 vol. of 0.01 M-Tris/HCl buffer (pH 7.6)containing 0.32 M-sucrose and 0.5 mM-2-mercaptoethanol(buffer A) and centrifuged at 30000 g for 120 min. Thesupernatant was collected and applied to the top of aDEAE-cellulose column (DE-52; 200 ml bed volume)equilibrated with buffer A (without sucrose). The columnwas washed with a total of 1000 ml of buffer A. Elutionwas then started with a linear gradient establishedbetween 400 ml of buffer A and 400 ml of the same buffercontaining 0.3 M-NaCl. Fractions (8 ml) were collectedand tested for protein by absorbance at 280 nm and forenzyme activity using Bz-Gly-Ala-Ala-Phe-pAB as thesubstrate. The enzyme emerged after 360 ml of the elutingbuffer had passed through the column. Fractions withinthe central part of the activity peak were pooled andassayed for protein [13] and for activity as describedabove. The pooled fractions were concentrated to about40 ml and then applied to the top of a Sephadex G-100column (5.0 cm x 100 cm) equilibrated with a 0.05 M-Tris/HCI buffer, pH 8.0. Elution was carried out with thesame buffer and fractions of about 20 ml were collected.The enzyme emerged, preceded by a large inactive proteinpeak, after about 780 ml of the eluting buffer had passedthrough the column. Fractions in the central portion ofthe active peak were pooled and applied to the top ofa DEAE-Sephacel column (15 ml) equilibrated with a0.02 M-Tris/HCl buffer (pH 8.0) containing 0.5 mM-2-mercaptoethanol. The column was washed with the samebuffer (220 ml) and then eluted with a linear gradientestablished between 150 ml of the same buffer and150 ml of the buffer containing 0.3 M-NaCl. Fractions ofabout 3 ml were collected. The enzyme emerged from thecolumn after about 100 ml of the eluting buffer hadpassed through the column. A large amount of inactiveprotein was eluted before and after the main activitypeak. Fractions containing enzyme with the highestspecific activity were combined and desalted by passingthrough a Sephadex G-25 column (150 ml) equilibratedwith 0.01 M-Tris/acetate buffer (pH 7.5).

Samples of the enzyme after step 4 of the purificationprocedure were subjected to PAGE as described above.Staining with Coomassie Brilliant Blue revealed thepresence of two major protein bands (Fig. 1). Parallelunstained gels were cut into 2 mm slices, and the dis-tribution of activity was examined by homogenizing theslices in 0.25 ml of Tris/HCI buffer (0.05 M, pH 7.0), anddetermining the activity in gel extracts as describedabove. Activity was associated with the slower-moving

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Rat testis endopeptidase 24.15

protein band. For the isolation of homogeneous enzyme,aliquots of the enzyme after step 4 of the purificationprocedure containing about 1 mg of protein (Table 1)were concentrated to about 0.5 ml and subjected topreparative slab PAGE. The location of protein bandswas visualized by staining gel strips from the edges andthe centre of the slab gel with Coomassie Brilliant Blue.Good separation of the two protein bands was con-sistently obtained. The slab gel was sliced into 2 mmstrips. The gel strips containing activity were placed indialysis bags filled with 1.5 ml ofTris/HCl buffer (0.05 M,pH 8.3). The enzyme was electroeluted from the gel byplacing the dialysis bags in a flat slab gel electrophoresisapparatus containing the same buffer and applying acurrent of 140 V for 5 h. The buffer containing theenzyme was recovered from the dialysis bag (1.35 ml)and the solution was tested for activity and protein bythe method of Peterson [16]. Enzyme purity was deter-mined by PAGE under non-dissociating and denaturingand dissociating conditions and also by testing theimmunoreactivity toward an antibody raised in rabbits(see below). Enzyme samples obtained by this procedure(usually about 35-40 gg of protein) were apparentlyhomogeneous by several criteria (see below).

Raising of antibodiesAliquots of the homogeneous enzyme, obtained after

preparative slab PAGE, containing about 20 ,ug of pro-tein were used for immunization. The enzyme was mixedwith complete Freund's adjuvant and injected intra-dermally at multiple sites into the back of a New Zealandwhite rabbit. The injections were repeated at least two orthree times after 14-20 day intervals. Blood was collectedinto heparinized tubes from the ear vein 10 days after thelast injection, at which time a rather high titre ofanticatalytic antibodies was present. Aliquots (0.2 ml) ofthe plasma recovered by centrifugation were stored at-20 °C.

Electrophoretic transfer of proteins from SDS/poly-acrylamide gels was carried out essentially as described[17,18] using a Trans-Blot electrophoretic transfer cell(Bio-Rad Laboratories). A 5% bovine serum albuminsolution (20 mM-Tris/HCl/500 mM-NaCl, pH 7.5) wasused as the blocking solution. The first antibody wasused in a 1:500 dilution in a buffer containing 1%bovine serum albumin and 0.05 % Tween 20 in 20 mM-

Tris/HCl/0.5 M-NaCi (pH 7.5). An affinity-purified goatanti-rabbit horseradish peroxidase conjugate (Bio-Rad)wa' used as the second antibody and visualization wasaccomplished according to the manufacturer's recom-mendations.

(a) (b) (c)

Fig. 1. PAGE of the enzyme from testis

Gel (b) (10 ug of protein) represents the pattern obtainedafter step 4 of the purification procedure. Gel (a) and (c)(each containing about 5,ug of protein) represent re-spectively the homogeneous enzyme and the impurity eachisolated after preparative PAGE.

Table 1. Summary of purification of the enzyme frogi rat testes

SpecificVolume Protein Activity Total activity Purification

Purification step (ml) (mg/ml) (units/ml) (units) (units/mg) (fold) Recovery (%)

1. Supernatant2. DE-52 chromatography

(pH 7.6)3. Sephadex G- 1004. DEAE-Sephacel

chromatography(pH 8.0)

5. Preparative PAGE

610 10.777 6.6

130 0.9635 0.38

1.35 0.025

49 29890194 14940

83 10790129 4515

30.4

4.5829.4

86.5339

41 1220

6.4

18.974

266*

10050

3615

0.14* Aliquots of the enzyme after step 4 containing about 350 units were subjected to preparative slab PAGE. Only about 10% of

homogeneous enzyme was recovered by electrolution as described in the Materials and methods section.

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RESULTSA summary of the purification procedure is given in

Table 1. The specific activity of the enzyme fractionsobtained after step 5 of the purification procedure(Table I) represented a more than 260-fold purification.Disc PAGE after step 4 showed the presence of twowell-separated protein components (Fig. 1). Activitywas associated with the slower-moving protein band.Attempts to separate the two proteins by chromato-graphy on various ion-exchange columns, chromato-focusing or chromatography on a hydroxyapatite columnwere not successful. The enzyme was therefore isolated insmall batches by preparative slab PAGE. When theelectrophoresis time necessary for the tracking dye toreach the bottom of the gel was doubled, the two bandswere widely separated, allowing the excision of the bandcontaining the enzyme, without contamination with theinactive protein. Attempts to obtain a similar separationin purified preparations from rat brain were not suc-cessful. The isolated enzyme solution gave a single bandin PAGE under non-denaturing conditions (Fig. 1).PAGE under denaturing and dissociating conditions

(Fig. 2) also revealed the presence of a single proteinband. The molecular mass of the enzyme calculated fromits relative mobility with respect to marker proteins wasapprox. 70000. This value is rather close to that obtained

(a) (b)

_A

(c) (d)

..

for the brain enzyme (67 000) on the basis of measure-ments of the elution volume from calibrated columns ofSephadex G-100 [1]. Immunoblots also showed thepresence of a single immunoreactive component in theisolated enzyme. These data taken together indicate thepresence of a single polypeptide chain. Crude testissupernatants contained an additional, somewhat fastermoving, immunoreactive band. The origin of this bandand its significance is currently being investigated.Double immunodiffusion experiments (Fig. 3) [19]

gave single precipitation lines with the testis enzyme,suggesting the presence of a single antigenic component.The antiserum was also tested by the same method for itsreactivity toward the enzyme in a crude rat brain extract.As shown in Fig. 3, patterns of identity were obtained forboth the purified testis enzyme and the enzyme present inrat brain supernatant.The effect of general protease inhibitors on the activity

of the enzyme is summarized in Table 2. High concen-trations of leupeptin weakly inhibited the enzyme. Noinhibition was obtained with pepstatin, antipain andchymostatin. Indeed, some increase in enzyme activitywas observed in the presence of pepstatin and chymo-statin. The detailed mechanism of this activation has notbeen further studied. DFP and PMSF, irreversible inhibi-tors of serine proteases, had no effect on activity. Amongseveral thiol blocking agents, PCMB and N-ethyl-maleimide partially inhibited the enzyme, while iodo-acetamide and iodoacetic acid at rather high (mM)concentrations had no effect. Consistent inhibition wasobtained with the metal chelators such as EDTA andEGTA. Unlike crude enzyme preparations which weremoderately activated by addition of DTT (0.4-1.0 mM),the isolated homogeneous enzyme was inhibited by DTT.The profile of inhibition indicated that the enzyme

belongs to the class of metalloendopeptidases. Consistentwith this conclusion was the finding that dialysis of theenzyme against EDTA (Table 3) led to loss of activity.Activity could be restored by addition of some divalentmetal ions. Complete reactivation was obtained afteraddition of zinc ions. The highest activity in reactivationexperiments was obtained at Zn2+ concentrations as low

2

I

Fig. 2. PAGE and immunoblots of the enzyme after step 5 of thepurification

Electrophoresis was carried out under denaturing anddissociating conditions as described in the Materials andmethods section using 2.5 ,ug of protein. Lanes (a) and (b)are immunoblots of the enzyme. Lane (c) contains theisolated enzyme stained with Coomassie Brilliant Blue andlane (d) contains the standard marker proteins: 1, bovineserum albumin; 2, ovalbumin; 3, trypsinogen.

(a). ; (b)

!. .. .. ..

Fig. 3. Ouchterlony double-immunodiffusion patterns of enzyme

The rabbit antiserum was placed in the centre well andallowed to diffuse toward the purified enzyme (wells 1, 2and 4). Wells 3 and 5 contained a pH 5.0 supernatant ofthe soluble fraction of a rat brain homogenate afteradjustment of pH to 7.6. The amounts of active enzymeare 0:14 units in (a) -and 0.07 units in (b)

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as 1 ,cM. Indeed, at this concentration enzyme activitywas somewhat higher than that of the undialysed enzyme,suggesting that some of the enzyme was stripped of itsmetal content during purification. Higher Zn2+ concen-trations led to a progressive decrease of activity. Theextent of reactivation was greater after addition of Co2"than ofZn2+, although the concentration ofCo2" requiredfor maximal activation was 100 times greater (100,M)than that of zinc. These data indicate that the Co2"

Table 2. Effect of inhibitors on enzyme activity

Activity was determined with Bz-Gly-Ala-Ala-Phe-pAB(0.8 mM) as substrate. Activity in the presence of pepstatinwas determined at 0.4 mm substrate concentration. Inhibi-tors were preincubated with the enzyme for 12 min and thereaction was then started by addition of substrate. Dataare mean values obtained from two to four determinations.

Finalconcentration Relative

Inhibitor (mM) activity (%)

NoneLeupeptin

Pepstatin

AntipainChymostatinDFPPMSFPCMBlodoacetamideN-EthylmaleimideIodoacetic acidEDTAEGTADTT

0.040.120.0080.240.0130.0131.00.21.01.01.01.01.01.00.51.0

100906814217199108102100351075510040417148

enzyme is more active than the Zn2+ enzyme toward Bz-Gly-Ala-Ala-Phe-pAB as the substrate. At an optimalconcentration of each of the two metals, the Co2" enzymecleaved the substrate 2.5 times faster than the Zn2"enzyme. The enzyme could also be reactivated by additionof Mn2+, albeit at much higher concentrations than withcobalt or zinc (1.0-10 mM), while incomplete reactivationwas obtained with calcium ions (0.05-2.0 mM). Thesedata suggest that the enzyme is apparently a zinc-containing endopeptidase.The specificity of the enzyme was studied with a series

of synthetic substrates. The data summarized in Table 4indicate that the apparent Km and the turnover rateconstant (kcat), and accordingly the specificity constant(kcat./Km) are affected both by the structure of the aminoacid residues that form the scissile.bond as well as by thestructure of residues beyond the immediate vicinity ofthis bond. The effect of replacing a Gly residue in the P3'position by residues of increasing hydrophobicity isevident from the kinetic parameters obtained with sub-strates 1-3 (Table 4). Thus, replacing a Gly residue by aLeu or Phe residue causes a progressive decrease in theapparent Km. This effect is also associated with an increasein the turnover rate constants and consequently thespecificity constants. Replacement of the Gly residue bya Phe residue causes a 4-fold decrease in Km, a 9-foldincrease in kcat and almost a 40-fold increase in thespecificity constant. These data suggest the presence of ahydrophobic binding pocket at the S3' subsite of theactive site.The effect of structural changes in the amino acid

residue at the P1' position is indicated by the changes inkinetic parameters obtained with substrates 3, 4 and 5.Replacement of an Ala residue in this position by eithera Phe or a Gly residue decreased both the rate of thereaction as well as the specificity constants.The specificity of the enzyme toward the residue

contributing the carbonyl moiety to the scissile bond isindicated by the kinetic parameters obtained with sub-strates 6 and 7. Substrates having a Phe residue in the P1position have markedly lower apparent Km values and

Table 3. Effect of metal ions on enzyme activity after dialysis against EDTA

The enzyme after step 5 of the purification procedure was dialysed against three changes (1000 ml each) of a 1 mm solution ofEDTA (pH 7.0) in deionized water, and then against three changes of a 1 mM solution of Tris/HCl (pH 7.0). Activity was thendetermined with Bz-Gly-Ala-Ala-Phe-pAB as the substrate. Activity was measured without preincubation with the metal ions.

Concn. Activity RelativeEnzyme Additions (#M) (units/ml) activity (%)

Not dialysedDialysed

NoneNoneZnC12

CoCl2

- 71- 22.80.5 45.61 91.65 48.3

50 42.8500 16.1

1 80.95 130.0

50 212.2100 232.1500 192.71000 151.22000 98.5

1003264129686023114183299327271213139

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956 M. Orlowski and others

Table 4. Kinetic parameters of endopeptidase-24.15-catalysed hydrolysis of synthetic substrates

Data are mean values + S.E.M. obtained from four determinations. Data for substrate 2 are the mean from two determinations.The site of cleavage is indicated by an arrow.

[S] Km kcat. kcat./KmP4-P3-P2-PI-P1/-P2/-P3f-P4I (mM) (mM) (s-') (s-'. M-1)

12345678910

Bz-Gly-Ala-Ala-Gly-pABBz-Gly-Ala-Ala-Leu-pABBz-Gly-Ala-Ala-Phe-pABBz-Gly,-Phe-Ala-Phe-pABBz-Gly-Gly-Ala-Phe-pAB

Boc-Phe-Ala-Ala-Phe-pABBz-Gly-Phe-Ala-Ala-Phe-pAB

Bz-Gly-D-Phe-Ala-Ala-Phe-pABBz-Gly-Phe-D-Ala-Ala-Phe-pAB

Bz-Gly-Ala-Phe-pAB

0.96-4.80.64-3.20.36-1.20.06-0.40.72-3.60.13-0.8

0.027-0.08InactiveInactiveInactive

3.45 + 0.271.540.83 +0.0370.39+0.131.36+0.210.10+0.01

0.058+0.007

5.317.048.319.24.54

94.1179

1.5x 10'1.1 x 10l5.8x 1044.9 x 1043.3 x 1039.4 x 1053.1 x 106

Table 5. Rate of degradation of natural peptides by endopeptidase 24.15

Conventional one-letter abbreviations are used for amino acids. Degradation rates were determined in incubation mixturescontaining peptide (neurotensin 0.086 mm, all others 0.16 mM), enzyme and Tris/HCI buffer (0.1 M, pH 7.0). Aliquots (100 ,u)of the incubation mixtures (440 ll) were subjected after various time intervals to h.p.l.c. on a C,, uBondapack column and therate of peptide degradation was calculated from the rate of disappearance of the substrate peak. Sites of cleavage are indicatedby arrows. Km values used for calculation of kcat and kcat/Km values were those reported previously for the brain enzyme[8, 21], and should be therefore considered as an approximation, since the corresponding values for the testis enzyme have notbeen determined.

Peptide Degradation rate Km kcat kcat./Kmand structure (,umol/h per mg) (mM) (s-') (s-l M-1)

Dynorphin A-8Y-G-G-F-L-R-R-I

,-Neoendorphin ;Y-G-G-F-L-R-K-Y-P

Bradykinin tR-P-P-G-F-S-P-F-R

LHRHpGlu-H-W-S-Y-G-L-R-P-G-NH2

NeurotensinpGlu-L-Y-E-N-K-P-R-R-P-Y-I-L

2233

1647

1126

0.06 86.4 1.4 x 106

0.038 37.9 9.98 x 105

0.067 33.8 5.0 x 10'

388 0.095 12 1.26 x 10'

285 0.037 7.9 2.1 x 10'

greatly increased kcat and kcat /Km values compared withthose having a Gly residue in this position (substrate 3).This indicates preferential cleavage of substrates havinga bulky hydrophobic group in the P, position.The stereospecificity of the enzyme toward peptide

bonds formed between L-amino acids is indicated by thefinding that replacement of an L-amino acid residue ineither the P, or P,' position by a D-isomer renders thesubstrates (substrates 8 and 9, Table 4) resistant todegradation.

It is of interest that Bz-Gly-Ala-Phe-pAB (compound10, Table 4), an analogue of Bz-Gly-Ala-Ala-Phe-pAB inwhich one of the Ala residues has been deleted, wascompletely resistant to hydrolysis. This finding indicatesthat hydrolysis requires the presence in the substrate ofa minimum of five peptide bonds, and consequently sug-gests the presence of an extended substrate-binding site.

Like the rat brain enzyme the isolated testis enzymewas inhibited by a series of previously synthesized active-site-directed N-carboxymethyl peptide inhibitors [9, 10].Both enzymes were inhibited in a competitive manner

with similar K, values. Thus, for example N-[( 1 RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-pAB inhibited thepartially purified brain enzyme and the isolated testisenzyme with Ki values of 0.016 and 0.026 /LM respectively.

It was important to determine whether the isolatedtestis enzyme, like its brain counterpart, is capable ofgenerating enkephalins from several enkephalin-contain-ing peptides (Table 5). Incubation of the enzyme withdynorphin A'-8, ,-neoendorphin, Met-enkephalin-Arg6-Gly7-Leu' and metorphamide led to a rapid conversion ofthese peptides to the corresponding enkephalins in areaction completely inhibited by cFP-AAF-pAB, anactive-site-directed inhibitor of the enzyme (K1 0.027/kM). The formation of Leu-enkephalin from dynorphinA1-8 and Met-enkephalin from metorphamide (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH,) and inhibition of thesereactions by cFP-AAF-pAB is shown in Fig. 4. Amongthe bioactive peptides studied dynorphin A'-8 was one ofthe most rapidly cleaved. It can be calculated that thisopioid peptide was converted to Leu-enkephalin with aVmax of about 74 ,tmol/min per mg of enzyme. The

1989


1.01

0.5,

(a) (b)

AcOH li

c D

,)r

0a1

0 10 0 10Time (min)

Fig. 4. Formation of Leu-enkephalinMet-enkephalin from metorpl24.15

Reaction mixtures contained dynitop panel) or metorphamide (0.2 ntestis endopeptidase 24.15 (9 ng) ar7.0). After incubation at 37 °C (5 rand 15 min for metorphamide) reaby addition of 5 ,u1 of glacial acetich.p.l.c. using a linear gradient be0.1 0% phosphoric acid. The initial (

nitrile was 10% and the concentr35 % during 25 min for metorpham30 min for dynorphin A 8̀ (a) Coincubation with the enzyme; (c) incthe presence of 25 ,uM-cFP-AAF-p

reaction with f,-neoendorphin v

however, both these peptides hadthe order of 106 (s-' M-'). Theefficiently bradykinin, LHRH arconsistent with its specificity dedwith model synthetic substrates. Sby the testis enzyme with theproducts in a pattern similar to tIwith the brain enzyme [1]. Althouand the amino acid composition 4been determined it can be assundegrade the peptide in a similar

DISCUSSIONPrevious efforts to isolate a hot

of endopeptidase 24.15 from ratthe presence of an inactive contarvirtually the same molecular masThis led us to direct our effortsthe enzyme from rat testes, a tissu24.15 activity. The purification v

reported previously [1] by elimii

cipitation step and hydrophobic chromatography and(c) introducing two ion-exchange chromatography steps at

two different pH values. An additional purification stepCL .: <,consisting of preparative PAGE followed by electro-

3L= "elution of the enzyme was needed to obtain a homo-o X geneous preparation of the enzyme. While the yield in the

U,X, last step was rather low, it could be used repeatedly onfractions from a single purification run to obtain small

en l + batches of a homogeneous enzyme each containing aboutL IL 40 jug of protein. The isolated enzyme gave a single band0 10 20 30 in PAGE under non-dissociating and denaturing and

dissociating conditions, and homogeneity was also sug-gested by Ouchterlony immunodiffusion experiments and

(c) Western blots using a polyclonal antiserum raised inrabbits. The electrophoretic pattern of the isolated en-

eMtorp m zyme indicated the presence of a single polypeptide chainOL with a molecular mass of about 70 kDa, similar to thatLL estimated for the brain enzyme (67 kDa). The catalytic

and immunological properties of the brain and testisenzyme are closely similar if not identical, although

C) zturnover rate constants for the testis enzyme were muchhigher as expected from the high purity of the isolated

i . protein. Kinetic parameters determined with synthetic0 10 20 30 substrates having a minimum of five peptide bonds gave

values for specificity constants ranging from 1.5 x 10' tofrom dynorphin A'-8 and 3.1 x 106O'1 * -1. The results indicated the presence of anhamide by endopeptidase extended binding site with major determinants of speci-

ficity being a hydrophobic residue in the P, position anda hydrophobic binding pocket in the S3' subsite. The

orphin A't8 (0.16 mM; importance of these sites is indicated by the finding thatadM; bottom panel), rat replacement of Phe residues in the P.and P3' positions byind Tris/HC (0.1 M, pH Gly residues caused a decrease in the specificity constantsnin for dynorphin A' 8 by more than three orders of magnitude (substrates 7Lctions were terminated versus 1 in Table 4). While an Ala residue in the P,'

acid and subjected to position seems to be favoured over a Gly or a Phe residue,,tween acetonitrile and the S' subsite seems to readily accomodate a basicconcentration of aceto- 1yation was increased to amino acid residue as shown by the rate of cleavage ofide, and to 30% during dynorphin A' and ,-neoendorphin (Table 5).ntrol at zero time; (b) The specificity of the enzyme determined with modelubation with enzyme in synthetic substrates is clearly reflected in studies withAB. bioactive peptides. Prominent among these is the rapid

conversion of dynorphin A'`8 and ,b-neoendorphin intoLeu-enkephalin and metorphamide into Met-enkephalin.

vas somewhat slower; The cleavage of the Phe'-Ser6 bond in bradykinin, thespecificity constants in Arg8-Arg' bond in neurotensin and the Tyr'-Gly6 bondenzyme also cleaved in LHRH also reflects the same enzyme specificity.

nd neurotensin at sites Indeed, recent studies have shown that endopeptidaseuced from experiments 24.15 is the primary enzyme responsible for cleavage ofubstance P was cleaved the Arg8-Arg9 bond in neurotensin [20], and the primaryrelease of six peptide enzyme responsible for cleavage of the Tyr'-Gly' bondhat previously obtained in LHRH in membrane preparations from hypothalamusigh the rate of cleavage and pituitary [21]. Experiments have also shown thatof the products has not specific inhibitors of the enzyme [10,11] caused aned that both enzymes dramatic increase in the half life of intravenously ad-manner. ministered LHRH (A. Lasdun, S. Reznik, C. Molineaux

& M. Orlowski, unpublished work).Though we have not yet been able to determine the

metal contents of the enzyme, several lines of evidencemogeneous preparation indicate that it is a metalloendopeptidase. This is basedbrain failed because of on the finding that metal chelators (o-phenanthroline,ninating protein having EDTA, EGTA) consistently inhibit the enzyme, thatss and isoelectric point. dialysis against chelators leads to loss of activity whichtoward the isolation of can be restored by Zn2+ and Co2" ions, that the Co2"te rich in endopeptidase enzyme is more active than the Zn2+ enzyme, and finallyvas modified from that that substrate-related N-carboxymethyl peptides act asnating the pH 5.0 pre- specific and potent enzyme inhibitors [10,11]. All these

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findings are characteristic for Zn2+ peptidases. An ad-ditional argument for classification of the enzyme as ametalloendopeptidase is inhibition of the isolated enzymeby low concentrations of DTT (0.4 mM). Crude enzymepreparations [1] from brain and testis are weakly acti-vated by low concentrations of DTT (0.4-1 mM), butinhibited at higher concentrations (over 1.5 mM), whereasthe isolated enzyme is inhibited even at low DTTconcentrations. While the causes of this behaviour are notclear, we have observed that enzyme preparations afterstep 4 of the purification procedure have a tendency toform heterodimers with the contaminating impurity (ourunpublished work). Accordingly the activating effect ofDTT in crude preparations may result from preventingformation of such heterodimers.

Finally it is important to discuss the relationship ofendopeptidase 24.15 to endo-oligopeptidase A, an en-zyme described in rabbits by Camargo et al. [22-24].While the two enzymes cleave bradykinin and neuro-tensin at the same sites, endo-oligopeptidase A wasclaimed to be a cysteine endopeptidase [22,24], to beinactive toward LHRH [25], not to hydrolyse the Gly-Alabond in N-benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate(Bz-GAAF-pAB), a synthetic substrate used for thedetermination of endopeptidase 24.15 activity [1], nor tobe inhibited by cFE-AAF-pAB [9]. Furthermore, it wasrecently claimed [91 that conversion of metorphamideand dynorphin A`8 into Met- and Leu-enkephalin re-spectively is catalysed by endo-oligopeptidase A but notby endopeptidase 24.15, and that this reaction is notinhibited by cFE-AAF-pAB. In addition, the sameauthors report that an antiserum against endo-oligo-peptidase A does not inhibit the cleavage of Bz-GAAF-pAB by endopeptidase 24.15. This led to the conclusionthat endopeptidase 24.15 and endo-oligopeptidase A aretwo distinct enzymes.Our present study shows, in confirmation of our

previous reports with less pure enzyme preparations andcrude membrane fractions [2,8], that a homogeneouspreparation of endopeptidase 24.15 converts severalenkephalin-containing peptides to the correspondingenkephalins, and that these reactions are inhibited byspecific inhibitors of endopeptidase 24.15. Indeed, con-version of dynorphin A'-8 to Leu-enkephalin by isolatedendopeptidase 24.15 proceeds at a rate more than 10-20times greater than that reported by Toffoletto et al. [9]for endo-oligopeptidase A. Furthermore, independentwork by Checler et al. [20] showed that cleavage of theArg8-Arg9 bond of neurotensin, attributed by Camargoand his group to endo-oligopeptidase A [9], is actuallycatalysed by endopeptidase 24.15 in a reaction inhibitedby cFE-AAF-pAB.

Thus, the two enzymes catalyse the hydrolysis of thesame bonds in neurotensin, bradykinin, dynorphin A"-8,fl-neoendorphin and metorphamide, but differ with re-spect to their mechanistic classification (cysteine versusmetalloendopeptidase), activity toward LHRH and syn-thetic endopeptidase 24.15 substrates and inhibitors. Inspite of these differences the possibility that the twoenzymes are actually identical needs to be considered.The procedure used for the purification of brain endo-oligopeptidase A [24] also results in an enrichment ofendopeptidase 24.15 activity, although the enzyme is stillcontaminated with the same inactive component men-

tioned above. Since endopeptidase 24.15 is a metallo-enzyme, the classification of endo-oligopeptidase A,claimed to be a thiol peptidase [9,22,24], would need tobe re-examined, by studying activation of the enzyme bymetal ions after dialysis under conditions of strict ex-clusion of traces of such ions. The claim that endo-oligopeptidase A is inactive toward LHRH [25], that itdoes not cleave synthetic endopeptidase 24.15 substrates,and that it is not affected by specific endopeptidase 24.15inhibitors [9] would also require a thorough re-exam-ination. Species differences in enzyme properties (es-pecially rabbit versus rat) would also need to beexamined.

This work was supported by a grant from the NIH (DK25377). S. R. is a trainee on Medical Scientist Training GrantGM-07280 from NIH.

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1989

Received 4 January 1989f15 March 1989; accepted 28 March 1989

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endopeptidase 24.15 from rat testes

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