©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Mitochondrial Metallopeptidase Reveals Neurolysin as a Homologue of Thimet Oligopeptidase (*)

(Received for publication, October 11, 1994; and in revised form, November 11, 1994)

Atsushi Serizawa (§) Pamela M. Dando (¶) Alan J. Barrett(¶)(**)

From the Department of Biochemistry, Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a metallopeptidase from rat liver. The peptidase is primarily located in the mitochondrial intermembrane space, where it interacts non-covalently with the inner membrane. The enzyme hydrolyzes oligopeptides, the largest substrate molecule found being dynorphin A; it has no action on proteins, and does not interact with alpha(2)-macroglobulin, and can therefore be classified as an oligopeptidase. We term the enzyme oligopeptidase M. Oligopeptidase M acts similarly to thimet oligopeptidase (EC 3.4.24.15) on bradykinin and several other peptides, but hydrolyzes neurotensin exclusively at the -Pro+Tyr- bond (the symbol + is used to indicate a scissile peptide bond) rather than the -Arg+Arg- bond. The enzyme is inhibited by chelating agents and some thiol-blocking compounds, but differs from thimet oligopeptidase in not being activated by thiol compounds. The peptidase is inhibited by Pro-Ile, unlike thimet oligopeptidase, and the two enzymes are separable in chromatography on hydroxyapatite. The N-terminal amino acid sequence of rat mitochondrial oligopeptidase M contains 19 out of 20 residues identical with a segment of rabbit microsomal endopeptidase and 17 matching the corresponding segment of pig-soluble angiotensin II-binding protein. Moreover, the rat protein is recognized by a monoclonal antibody against rabbit soluble angiotensin II-binding protein, all of which is consistent with these proteins being species variants of a single protein that is a homologue of thimet oligopeptidase. The biochemical properties of the mitochondrial oligopeptidase leave us in no doubt that it is neurolysin (EC 3.4.24.16), for which no sequence has previously been reported, and which has not been thought to be mitochondrial.


INTRODUCTION

Wünsch and Heidrich (3) designed the synthetic substrate, Pz(^1)-Pro-Leu-Gly-Pro-D-Arg, containing the collagen-like sequence -Pro-Leu-Gly-Pro-, as a substrate for clostridial collagenase. Avian and mammalian tissues contain a Pz-peptide-hydrolyzing enzyme that is not a collagenase, and was for a time simply termed Pz-peptidase, but is now known as thimet oligopeptidase (EC 3.4.24.15)(4) . Thimet oligopeptidase is a primarily cytosolic, thiol-activated metalloendopeptidase, which is confined to action on oligopeptides of up to about 17 amino acid residues(5) . It cleaves neurotensin at the -Arg+Arg- bond and is potently inhibited by a series of Cpp-tripeptidyl-pAb compounds(6, 7) . Since the determination of the amino acid sequence of thimet oligopeptidase (8, 9) a family of homologous metalloendopeptidases has been discovered (for review (10) ). The family contains exopeptidases, oligopeptidases, and endopeptidases and is thus of exceptional diversity. Two homologues, oligopeptidase A and peptidyl-dipeptidase A, have been identified in both Escherichia coli and Salmonella typhimurium(11) , and peptidase F of Lactococcus lactis is also related(12) . Eukaryotic members in addition to thimet oligopeptidase include mitochondrial intermediate peptidase (EC 3.4.24.59), which is a matrix protein in the mitochondria of rat and yeast(13, 14) , and saccharolysin (EC 3.4.24.37; formerly peptidase yscD), also from yeast(15) .

Two additional homologues of thimet oligopeptidase that are of particular importance here are pig-soluble angiotensin II-binding protein and rabbit microsomal endopeptidase. Soluble angiotensin II-binding protein was discovered in the course of work directed at the identification of receptors for angiotensin II. This protein is clearly a homologue of thimet oligopeptidase, but is a separate gene product(9, 16) . Kato et al.(16) demonstrated that pig-soluble angiotensin II-binding protein has the capacity to degrade radiolabeled angiotensin. A monoclonal antibody that was raised against rabbit-soluble angiotensin II-binding protein by Soffer et al.(17) has been valuable in the present study.

It was also noted by McKie et al.(9) and Kato et al.(16) that the deduced sequence of pig-soluble angiotensin II-binding protein is so similar to that of the rabbit enzyme termed microsomal endopeptidase (18, 19) as to indicate that the proteins are species variants of a single enzyme. The microsomal metalloendopeptidase has been suggested to be responsible for the post-translational processing of -carboxyglutamic acid-containing blood coagulation factors.

A further metallopeptidase that we need to introduce is one that has been termed neurotensin-degrading enzyme, and for which the recommended name is now neurolysin (EC 3.4.24.16). The enzyme is present in the cytosol and membrane fractions of brain, kidney, and other tissues, and it has been proposed to have the physiological function of destroying neurotensin and other bioactive peptides(20, 21) . In many respects, the activity of neurolysin resembles that of thimet oligopeptidase, but neurolysin is distinguished by its cleavage of neurotensin at the -Pro+Tyr- bond(22) , its sensitivity to inhibition by Pro-Ile(23) , and lack of thiol activation. Despite much work on the enzyme, no amino acid sequence data have been reported for neurolysin.

Heidrich et al.(24, 25) described Pz-peptidase activity in rat liver mitochondria, and Tisljar and Barrett (26) established that the mitochondria enzyme is distinct from thimet oligopeptidase, although there are similarities in substrate specificity. In particular, both enzymes hydrolyze a quenched fluorescence substrate related in structure to the Pz-peptide, QF02.

In the present paper, we describe the isolation and characterization of the mitochondrial peptidase, which we term oligopeptidase M. Our study of oligopeptidase M leads us to conclude that oligopeptidase M, soluble angiotensin II-binding protein, microsomal endopeptidase, and neurolysin are all one. In consequence of this, we find ourselves in possession of detailed knowledge of the primary structure and enzymology of a new member of the thimet oligopeptidase family of metallopeptidases.


EXPERIMENTAL PROCEDURES

Materials

Bz-Gly-Ala-Ala-Phe-pAb, and the quenched fluorescence (QF) substrates QF01, QF02, QF34, and QF37 were provided by Dr. C. G. Knight of the Strangeways Research Laboratory, following synthesis as described (27, 28, 29) or by standard methods. QF02 was also purchased from Calbiochem-Novabiochem Ltd. (United Kingdom) (product 03-32-5024). Z-Thioprolylthiazolidine was given by Dr. Tadashi Yoshimoto (School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan). W-1 was from Sigma. Bradykinin, the dynorphin-related peptides, neurotensin, and Pro-Ile were from Sigma. Thimet oligopeptidase was purified from rat testis (5) or E. coli expressing the rat testis enzyme(30) , as specified in the text. Pig prolyl oligopeptidase (EC 3.4.21.26) was given by Dr. S. R. Stone (Department of Haematology, MRC Centre, University of Cambridge, Cambridge CB2 2QH, U.K.).

Antibodies

Monoclonal antibody M3C to rabbit-soluble angiotensin II-binding protein was given by Dr. R. L. Soffer, Cornell University Medical College, New York. Biotinylated anti-mouse immunoglobulin G and avidin-peroxidase were from Vector Laboratories, Peterborough U.K.

Antiserum against recombinant rat thimet oligopeptidase was raised in three rabbits, each of which was injected subcutaneously with 50 µg of the recombinant enzyme at 0, 1, 2, and 7 months. The immunogen was emulsified with complete Freund's adjuvant for the first injection and incomplete adjuvant subsequently. Sera from bleeds taken 8 days after the third and fourth injections were combined. Immunoglobulin G was prepared by triple precipitation from 2.7 M ammonium sulfate, and the final precipitate was dissolved in phosphate-buffered saline and dialyzed against the same. Specific antibody was affinity purified by use of a column in which the recombinant enzyme was immobilized on activated CH-Sepharose 4B (Pharmacia). The antibody eluted with 0.1 M glycine-HCl, pH 2.3, was termed R646/P.

Antiserum against oligopeptidase M was raised in a sheep by two intramuscular injections of 50 µg emulsified in Freund's complete adjuvant, 1 month apart. Sera from bleeds taken at 7, 10, and 14 days after the second injection were combined. Immunoglobulin G was prepared by triple precipitation with ammonium sulfate as above and termed K118.

Assays for Oligopeptidase M and Thimet Oligopeptidase

Quenched fluorescence assays were made with QF02 as substrate, essentially as described previously for thimet oligopeptidase(28) , except that the temperature was 30 °C. When activity only of mitochondrial oligopeptidase was to be measured, the activator of thimet oligopeptidase, mercaptoethanol or dithiothreitol, was omitted.

Selective assays for either oligopeptidase M or thimet oligopeptidase were made on the basis that mammalian tissues contain three enzymes that hydrolyze QF02, prolyl oligopeptidase (a serine peptidase), thimet oligopeptidase, and oligopeptidase M (see ``Results''). The activity of prolyl oligopeptidase was eliminated by use of its inhibitor, Z-thioprolylthiazolidine (0.05 µM) (31) , and immunoinhibition allowed discrimination between the two metallopeptidases (see below).

Selective immunoinhibition of the respective enzymes was achieved by preincubation of the sample with either (a) an equal volume of a 240 µg/ml solution of antibody R646/P for 10 min at room temperature, for thimet oligopeptidase, or with 10 mg/ml of antibody K118, for oligopeptidase M. Tests with the pure enzymes established that these conditions produced 100% inhibition of thimet oligopeptidase with 0% inhibition of oligopeptidase M (R646/P) and 5% inhibition of thimet oligopeptidase with 100% inhibition of oligopeptidase M (K118), respectively.

Values of K for the hydrolysis of quenched fluorescence substrates by oligopeptidase M were determined in continuous fluorimetric assays controlled by the FLUSYS software(32, 5) . The method included restandardization of the fluorimeter at each substrate concentration to compensate for quenching by the substrates at high concentrations(33) , and comparative measurements were made with recombinant rat testis thimet oligopeptidase. Values for V were converted to k on the basis that the highest specific activities determined during a number of preparations of the homogeneous enzymes correspond to those of the fully active enzymes. These values (in the standard assay with QF02) were 4.0 unit/mg for oligopeptidase M and 6.0 unit/mg for thimet oligopeptidase. Values for k/K were determined directly when activity could be determined at substrate concentrations far below K, by use of the equation: k/K = v/E(0)S(0).

Values for K were determined with correction for the effect of substrate on the assumption of simple competition(7) .

Subcellular Fractionation of Rat Liver

All steps were at 0-4 °C. Rat liver (10-20 g) was rinsed with saline, chopped with scissors, and homogenized in 4 ml/g of 10 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose. The tissue was homogenized with two up and down strokes in a motor-driven Potter-Elvehjem homogenizer running at 1,000 revolutions/min, and the homogenate was centrifuged at 1,000 times g for 5 min. The post-debris supernatant was centrifuged at 7,500 times g for 10 min to yield a mitochondrial fraction, centrifuged again at 15,000 times g for 10 min to give a crude lysosome/peroxisome pellet, and finally recentrifuged at 100,000 times g for 1 h to give microsomes (pellet) and cytosol.

Marker enzymes were cytochrome oxidase for mitochondria(34) , beta-galactosidase and cathepsins B and L for lysosomes(35, 36) , catalase for peroxisomes(34) , and glucose-6-phosphatase for endoplasmic reticulum(37) . A mitochondrial matrix marker was 2-oxoglutarate dehydrogenase(38) . Protein was determined by use of the Bio-Rad kit, and all results were calculated as described by de Duve et al.(39) .

Purification of Oligopeptidase M from Rat Liver Mitochondria

Sprague-Dawley (CFY) rats were starved overnight and killed by cervical dislocation, and livers were removed immediately. All subsequent steps were at 0-4 °C. Mitoplasts were first prepared by a method similar to that of Kalousek et al.(40) . Rat liver (406 g) was homogenized in 4 ml/g of 10 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose, 5 mM 2-mercaptoethanol, and 0.1 mM phenylmethanesulfonyl fluoride (TS buffer), followed by centrifugation as described above to yield a post-debris supernatant. The first supernatant was recentrifuged for 15 min at 10,000 times g, and the pellet (crude mitochondria) was resuspended in 1 volume of TS buffer. Digitonin (as a 5% w/v stock solution) was added to 0.045% to lyse lysosomes and peroxisomes(41) . After 5 min with gentle stirring, a further three volumes of TS buffer was added, and the mitochondria were washed twice by centrifugation (10,000 times g, 15 min) and resuspension in TS buffer.

The washes were discarded, and the pellet (purified mitochondria) was diluted to a protein concentration of 100 mg/ml with TS buffer. Digitonin stock solution was added to make 0.55% digitonin, and after 15 min with occasional stirring three volumes of TS buffer was added, and the resulting mitoplasts were washed twice by centrifugation (25,000 times g, 15 min) and resuspension in TS buffer.

The mitoplast pellet was diluted to a protein concentration of 50 mg/ml in TS buffer, and W-1 stock solution (10%, w/v) was added to make 1.1%. After 30 min with occasional stirring, the suspension was diluted with an equal volume of TS buffer, and centrifuged at 100,000 times g for 1 h. The supernatant formed the source for the chromatographic isolation of oligopeptidase M.

DEAE-Cellulose Chromatography

A column (26 times 380 mm) of DEAE-cellulose (Whatman DE52) was equilibrated with 10 mM Tris-HCl, pH 8.0, containing 0.05% Brij-35, 5 mM 2-mercaptoethanol, 0.1 mM ZnCl(2), and 0.1 mM phenylmethanesulfonyl fluoride. The sample was run on the column with a 2-liter linear gradient from 0 to 0.3 M NaCl in the buffer. 15-ml fractions were collected at a flow rate of 1 ml/min.

Fractions containing activity against QF02 were combined, concentrated to about 40 ml by ultrafiltration over an Amicon PM-30 membrane, and dialyzed against 10 mM sodium phosphate buffer, pH 6.8, containing 5 mM 2-mercaptoethanol, 0.05% Brij-35, and 0.1 mM ZnCl(2) (PB buffer).

Hydroxyapatite Chromatography

A column (16 times 80 mm) of hydroxyapatite (Bio-Rad Bio-Gel HT) was equilibrated with PB buffer, and the sample was run with a 400-ml linear gradient from 10 to 300 mM sodium phosphate. Fractions (3.0 ml) were collected at 7.5 ml/h. The active fractions were combined and concentrated by ultrafiltration to 2-3 ml.

Sephacryl S-300 and HiTrap Blue Chromatography

The preparation was next run on a column (26 times 650 mm) of Sephacryl S-300 in 10 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 0.05% Brij-35, 5 mM 2-mercaptoethanol, and 0.1 mM ZnCl(2). Fractions (5 ml) were collected at 24 ml/h. The active fractions were combined and diluted with an equal volume of 10 mM Tris-HCl, pH 8.0, containing 0.05% Brij-35, 5 mM 2-mercaptoethanol, and 0.1 mM ZnCl(2). The solution was passed through a 1-ml column of Pharmacia HiTrap Blue in 10 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl, 0.05% Brij-35, 5 mM 2-mercaptoethanol, and 0.1 mM ZnCl(2).

Mono Q Fast Protein Liquid Chromatography

The procedure was completed by Pharmacia fast protein liquid chromatography on a Mono Q (HR 5/5) column equilibrated in 10 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl, 0.05% Brij-35, 5 mM 2-mercaptoethanol, and 0.1 mM ZnCl(2). Elution was with a linear gradient from 0.1 to 0.2 M NaCl run at 0.5 ml/min over 30 min. Fractions of 0.5 ml were collected. Active fractions were combined to form the final product.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting

Electrophoresis was in 10% polyacrylamide gels (42) . Separated protein bands were electroblotted on to Immobilon P membrane (Millipore) for immunochemical detection, N-terminal amino acid sequence analysis, and glycoprotein detection(43) . Automated N-terminal sequence analysis was done by Dr. R. A. Harrison, MRC Molecular Immunopathology Unit, Cambridge. The immunoblots were developed with the monoclonal antibody against angiotensin II-binding protein (M3C) as primary antibody and biotinylated anti-mouse immunoglobulin G as second antibody.


RESULTS

Purification of Oligopeptidase M

The results of a typical purification of oligopeptidase M are summarized in Table 1and Fig. 1. A preparation on the scale described typically yields 0.4 mg of the pure enzyme, with a specific activity of 3.0 units/mg in the assay with QF02. The enzyme can be stored in solution at 4 °C for some weeks with little loss of activity.




Figure 1: Purification of oligopeptidase M from rat liver mitochondria. A, SDS-polyacrylamide gel electrophoresis of samples obtained during the purification of oligopeptidase M. Samples were a, mitochondria; b, W-1 extract of mitoplasts; c, DEAE-cellulose fraction; d, hydroxyapatite fraction; e, Sephacryl S300 HR fraction; f, Hitrap Blue-Sepharose fraction; g, Mono Q fraction (final product). Lane h contained a reference sample of thimet oligopeptidase. B, immunoblot of lanes g and h from a gel duplicating A, above, developed with monoclonal antibody M3C against rabbit-soluble angiotensin II-binding protein.



When run in SDS-polyacrylamide gel electrophoresis, oligopeptidase M (Fig. 1A, lane g) appears as a single band of protein corresponding to a molecular mass of 76 kDa, about 2 kDa less than that of thimet oligopeptidase (lane h). A persistent contaminant (seen as the strongest band in lane e) was identified by N-terminal sequence analysis (data not shown) as serine-tRNA ligase and was almost completely removed by the HiTrap Blue column.

The immunoblot of a similar gel developed with monoclonal antibody M3C to soluble angiotensin II-binding protein showed a strong reaction with oligopeptidase M and none with thimet oligopeptidase (Fig. 1B).

A parallel electroblot was stained for glycoproteins by the periodate-Schiff method, with transferrin as positive control. The reaction of oligopeptidase M was completely negative.

The N-terminal sequence determined for oligopeptidase M by automated sequencing from a blot is shown in Fig. 2, aligned with those of related enzymes.


Figure 2: N-terminal sequence of oligopeptidase M. The N-terminal sequence of oligopeptidase M (a) is compared with those of rabbit microsomal endopeptidase (b), pig-soluble angiotensin II-binding protein (c), and rat thimet oligopeptidase (d). Basic residues indicative of a mitochondrial-targeting function for the N-terminal parts of sequences (b) and (c) are printed in bold type, and residues identical to those in oligopeptidase M are printed in white on black. Residue X was unidentified.



pH Dependence of Activity of Oligopeptidase M

The activity of oligopeptidase M on QF02 was determined across the pH range 6.8-8.0 in both 10 mM sodium phosphate and 10 mM Tris-HCl buffers. In both buffers, maximal activity was seen at pH 7.5-7.6.

Effects of Thiols and Thiol-blocking Reagents

The effect of dithiothreitol on the activity of oligopeptidase M was determined in the range 0-1 mM. At no concentration of dithiothreitol was there any stimulation of the enzymatic activity, but above 0.5 mM there was inhibition, increasing to 30% at 1 mM. The activity of oligopeptidase M was decreased to 31% in the presence of 1.0 mMN-ethylmaleimide, and to zero by 50 µMp-chloromercuribenzoate or 10 µM HgCl(2).

Specificity of Cleavage of Peptides

Bonds hydrolyzed in a range of peptide substrates were determined as described previously (5) . As can be seen in Fig. 3A, both thimet oligopeptidase and oligopeptidase M hydrolyzed the Pz-peptide, Bz-Gly-Ala-Ala-Phe-pAb, QF01, QF02, bradykinin, and dynorphin A in the same way. In contrast, some peptides were cleaved differently (Fig. 3B). The hydrolysis of neurotensin solely at the -Pro+Tyr- bond by oligopeptidase M was particularly striking, having been reported previously only for neurolysin(22) . The hydrolysis of dynorphin A by oligopeptidase M also was distinctive, since this peptide was not cleaved by thimet oligopeptidase, and indeed proved to be a potent inhibitor (data not shown). Moreover, the -Leu^5+Arg- bond cleaved in dynorphin A was resistant in dynorphin A. QF34 is a quenched fluorescence analogue of dynorphin A, which again shows the cleavage of the -Leu+Arg bond hydrolyzed in the natural peptide. Suppression of hydrolysis of this bond by substitution of Leu by Ile shifted the cleavage positions by both enzymes toward the C terminus, and created a remarkably good substrate for oligopeptidase M (see Table 3below).


Figure 3: Specificity of oligopeptidase M. The pattern of cleavage of peptide bonds by oligopeptidase M is shown in comparison to that of thimet oligopeptidase (Dando et al., 1993). A shows bonds cleaved similarly by both enzymes. B shows differing activities of the enzymes, for thimet oligopeptidase and for oligopeptidase M. The symbol &cjs1229; indicates a weak cleavage by thimet oligopeptidase.





Kinetic Parameters for Hydrolysis of Quenched Fluorescence Substrates

The kinetic parameters of hydrolysis of the four quenched fluorescence peptides by oligopeptidase M and thimet oligopeptidase are shown in Table 2. In considering the values obtained for oligopeptidase M with QF34, it must be remembered that two bonds were cleaved (Fig. 3), but despite this, QF37 had a much higher specificity constant.



Inhibitors

In addition to the thiol-blocking agents mentioned above, oligopeptidase M was totally inhibited by 1,10-phenanthroline (1 mM, instantaneous) and EDTA (30 min of incubation with 10 mM).

The dipeptide Pro-Ile was introduced into the study because oligopeptidase M was found to have properties in common with neurolysin, for which Pro-Ile has been described as a rather specific inhibitor(23) . We confirmed that the dipeptide has little effect on thimet oligopeptidase. Oligopeptidase M was markedly inhibited, although with a K(i) value of 0.54 mM, which was higher than that of 0.09 mM determined for neurolysin by the previous workers, using potentially less precise, discontinuous assays.

Oligopeptidase M also was inhibited by Cpp-tripeptidyl-pAb inhibitors of thimet oligopeptidase, although much more weakly than thimet oligopeptidase (Table 3).

Subcellular Distribution of Oligopeptidase M in Rat Liver

Subcellular fractions of rat liver were prepared and assayed for marker enzymes as described under ``Experimental Procedures.'' As is shown in Fig. 4, activity against QF02 was detected in mitochondria and cytosol, but not significantly in other fractions. Since QF02 is hydrolyzed by prolyl oligopeptidase and thimet oligopeptidase as well as oligopeptidase M(26) , further experiments were made to identify the mitochondrial and cytosolic enzymes hydrolyzing QF02. A typical immunoinhibition experiment with antibody R646/P (for thimet oligopeptidase) and antibody K118 (for oligopeptidase M) showed that the QF02 hydrolyzing activity of the mitochondrial fraction was 91% oligopeptidase M and 9% thimet oligopeptidase, with zero prolyl oligopeptidase. The cytosolic activity was 29% oligopeptidase M, 68% thimet oligopeptidase, and 3% prolyl oligopeptidase. Activity of all samples tested was at least 97% inhibited by the combination of the three inhibitors. Qualitatively similar results were obtained with the corresponding fractions of rat kidney (not shown), and again, QF02 hydrolyzing activity was completely abolished by the combination of the three inhibitors.


Figure 4: Subcellular distribution of activity against QF02. Subcellular fractionation of rat liver was as described under ``Experimental Procedures.'' Significant QF02 hydrolyzing activity was detected only in mitochondria and cytosol. Further experiments described in the text showed that the mitochondrial activity was almost entirely due to oligopeptidase M, whereas both thimet oligopeptidase and oligopeptidase M contributed to the cytosolic activity. Note that no significant activity was present in the microsomal fraction, despite the similarity in N-terminal amino acid sequence between oligopeptidase M and the endopeptidase previously described as microsomal.



Confirmation of the distribution of oligopeptidase M and thimet oligopeptidase in mitochondria and cytosol was sought from chromatography on hydroxyapatite, the only method discovered so far by which the two enzymes are readily separated. 15 ml of cytosol fraction (as above) was run on hydroxyapatite (15 times 115 mm) as described above for the purification of oligopeptidase M. Two completely separate peaks of activity against QF02 were detected in the effluent fractions, at 75 mM and 140 mM phosphate. The first peak contained 65% of the total activity, hydrolyzed neurotensin at the -Arg+Arg- bond, and was inhibited by antibody R646/P; it was thus identified as thimet oligopeptidase. The second peak comprised 35% of the activity, hydrolyzed neurotensin at the -Pro+Tyr- bond, and was inhibited by 10 mM Pro-Ile. Accordingly, the second peak was attributed to cytosolic oligopeptidase M. In contrast, when activity from mitoplasts was run on hydroxyapatite during the purification of oligopeptidase M, no peak attributable to thimet oligopeptidase was ever seen.

Localization of Oligopeptidase M within Mitochondria

Mitochondria were prepared by sedimentation from post-debris supernatant at 7500 times g and diluted to 50 mg protein/ml. Digitonin was then added to part of the suspension to make 0.12 mg digitonin/mg mitochondrial protein, to form mitoplasts by destruction of the mitochondrial outer membranes(44) . Oligopeptidase M remained almost entirely (94%) associated with the sedimentable mitoplasts. The accessibility of oligopeptidase M in both mitochondria and mitoplasts to inhibitors was then determined. There was potent inhibition (85-88%) by the low molecular mass inhibitor Pro-Ile in both, comparable to 90% inhibition of oligopeptidase M in solution. This indicated that Pro-Ile, like the substrate QF02, readily gains access to oligopeptidase M where it is located in mitochondria. In contrast, the high molecular mass inhibitor, antibody K118, inhibited the oligopeptidase in mitoplasts by 90%, but had no significant affect on that in mitochondria. From this we conclude that oligopeptidase M is within the mitochondrial outer membrane, which forms a barrier to the antibody, but is permeable to QF02 and Pro-Ile. It is known that the outer membrane is permeable to molecules of less than 5000 Da, whereas the inner membrane tends to be impermeable(45) .

In a second experiment, the nature of the association of oligopeptidase M with mitoplasts was investigated. Mitochondria and mitoplasts were suspended (at 5 mg protein/ml) in sodium acetate buffer, pH 5.0, at 0 °C for 30 min. Each suspension was then recentrifuged at 15,000 times g for 10 min, and the supernatants were dialyzed and assayed for total and specific activity of oligopeptidase M.

Activity was efficiently solubilized (77%) from mitoplasts by the acidic buffer, but there was little solubilization (6%) from mitochondria. A mitochondrial matrix marker, 2-oxoglutarate dehydrogenase, was retained in the sedimentable mitoplasts.


DISCUSSION

The mitochondrial enzyme that hydrolyzes the Pz-peptide was discovered by Heidrich et al.(24, 25) , and further studied by Tisljar and Barrett(26) . We here term this enzyme oligopeptidase M and have assayed it with QF02, a quenched fluorescence substrate, that contains the collagen-like sequence -Pro-Leu-Gly-Pro-, as does the Pz-peptide. The substrate is also hydrolyzed by thimet oligopeptidase (4, 5) and prolyl oligopeptidase(28) . QF02 has been used in the laboratory of Checler et al.(46) to assay neurolysin. We used selective inhibitors of thimet oligopeptidase, prolyl oligopeptidase, and oligopeptidase M to resolve the three enzymes and found that the activity against QF02 in rat liver and kidney could be totally blocked by the combined action of the three inhibitors.

The subcellular fractionation of rat liver showed that QF02 hydrolyzing activity was essentially confined to the mitochondrial and cytosolic compartments. The activity in mitochondria was due to oligopeptidase M, with a small contribution from thimet oligopeptidase. In contrast, more of the cytosolic activity was due to thimet oligopeptidase than to oligopeptidase M.

The N-terminal 20-residue sequence determined for rat oligopeptidase M was used in a search of GenBank and other data bases with the FASTA and BLAST programs(47, 48) . The only significant scores obtained were those for the rabbit liver microsomal endopeptidase and pig liver-soluble angiotensin II-binding protein (Fig. 2). The microsomal endopeptidase has been studied by Davie and co-workers (18, 19) as a possible post-translational processing enzyme for blood coagulation factors. The soluble angiotensin II-binding protein is one that was discovered incidentally in the search for receptors of angiotensin(17, 49) . The similarities throughout the complete sequences of rabbit microsomal endopeptidase and pig-soluble angiotensin II-binding protein are so great (91% identical residues) that we and others have suggested that they are species variants of a single endopeptidase(9, 16) . The exact properties of this enzyme have been unclear, however. Hydrolysis of angiotensin II by the pig liver-soluble angiotensin II-binding protein has been detected(16) , and the microsomal enzyme has been shown to cleave some synthetic peptides modeling the site of processing of -carboxyglutamic acid-containing blood coagulation factors(18, 19) .

It now seems that microsomal endopeptidase and soluble angiotensin II-binding protein represent species variants of the mitochondrial and cytosolic enzyme that we here characterized under the name oligopeptidase M. The ``microsomal'' localization of the putative coagulation factor processing enzyme may well have been artifactual, since the enzyme was isolated from frozen rabbit liver after prolonged treatment with a Waring blender(18) . Fragments of mitochondria could well have been present in the high speed pellet from such a preparation. The soluble angiotensin II-binding protein is presumably the cytosolic fraction of oligopeptidase M that we also have detected.

Assuming that the initiator Met residues are as shown on Fig. 2(discussed in (49) ), oligopeptidase M is synthesized with an N-terminal sequence of 28 residues that conforms to expectation for a mitochondrial-targeting sequence(50, 51) .

Neurolysin is abundant in rat kidney, and hydrolyzes QF02(52, 22) , and yet we have found that all of the QF02-hydrolyzing activity of rat kidney is inhibited by the combination of inhibitors of prolyl oligopeptidase, thimet oligopeptidase, and oligopeptidase M. This suggests that one of these enzymes is neurolysin, and indeed, the properties reported in the literature for neurolysin are so similar to those described here for oligopeptidase M that there can be very little doubt that they are the same. Thus, both enzymes are proteins of M(r) about 75,000, with chromatographic properties closely similar to those of thimet oligopeptidase except for greater retention on hydroxyapatite(53, 54, 22) . Additionally, neurolysin has metallopeptidase characteristics with no activation by thiols (22) and has specificity generally similar to that of thimet oligopeptidase, including bonds hydrolyzed in QF02 and bradykinin(47) , but with distinctive cleavage of neurotensin at Pro+Tyr(56, 22) , and neurotensin to yield neurotensin(55) . Oligopeptidase M and neurolysin are the only enzymes apart from thimet oligopeptidase that have been found to be inhibited by the Cpp-tripeptidyl-pAb compounds described by Orlowski et al.(6) , but in both cases the K(i) values are 10-100-fold higher than for thimet oligopeptidase(22) . Inhibition by Pro-Ile (23) also clearly links oligopeptidase M and neurolysin. Like oligopeptidase M in rat liver, neurolysin has a partially cytosolic distribution in brain and neural cells of several species(53, 54, 55) . Neurolysin has not previously been described as mitochondrial, but the success of Barelli et al.(22) in purifying the enzyme from a 6,000 times g pellet from rat kidney homogenate that would have been largely mitochondria is entirely consistent with our finding of oligopeptidase M in this organelle. We conclude from all these lines of evidence that there is no significant doubt that oligopeptidase M is identical with neurolysin, and we propose to use the term neurolysin for the enzyme in future.

Neurolysin represents an interesting new addition to the thimet oligopeptidase family of peptidases, termed family M3 by Rawlings and Barrett (57, 58, reviewed in (10) ). A phylogenetic tree for the family (58) suggests that neurolysin and thimet oligopeptidase diverged from a common ancestor about 500 million years ago, during the evolution of animals. Many of the similarities between neurolysin and thimet oligopeptidase, such as molecular size, oligopeptidase specificity, hydrolysis of the Pz-peptide and QF02, sensitivity to a number of inhibitors that affect no other enzymes(56) , and binding of angiotensin II with similar characteristics(9) , are attributable to the structural similarities of homologous proteins. With the present report, the recognized membership of family M3 becomes thimet oligopeptidase (EC 3.4.24.15), neurolysin (EC 3.4.24.16), saccharolysin (EC 3.4.24.37), mitochondrial intermediate peptidase (EC 3.4.24.59), oligopeptidase A, bacterial peptidyl-dipeptidase, and the peptidase F of lactococci.

Neurolysin has for some time appeared to be an enzyme with the potential for important biological activities. As a result of our study, we now know the amino acid sequence and evolutionary relationships of the enzyme. The demonstration of its primarily mitochondrial location suggests new types of experiments relating to its function. For example, it will be of interest to know whether the excellent inhibitors of neurolysin that are now available (56) will disrupt mitochondrial physiology.


FOOTNOTES

*
This work was supported by the Snow Brand Milk Products Co., Ltd, Sapporo, Japan and the Medical Research Council (United Kingdom). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Permanent address: Sapporo Research Laboratory, Snow Brand Milk Products Co., Ltd., Sapporo 065, Japan.

Present address: Peptidase Laboratory, Department of Immunology, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, United Kingdom.

**
To whom correspondence should be addressed. Tel.: 1223-832312; Fax: 1223-836122.

(^1)
The abbreviations used are: Pz, 4-phenylazobenzyloxycarbonyl-; Cpp, N-[1-(RS)-carboxyl-3-phenylpropyl]-; Lys(Dnp), N^6-(2,4-dinitrophenyl)-L-lysine; Mca, (7-methoxy-coumarin-4-yl)acetyl; Mcc, 7-methoxycoumarin-3-carboxylyl-; pAb, p-aminobenzoate; QF01, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys; QF02, Mcc-Pro-Leu-Gly-Pro-D-Lys(Dnp)NH(2); QF34, Mca-Gly-Gly-Phe-Leu-Arg-Arg-Ala-Lys(Dnp), and QF37, McaGly-Gly-Phe-Ile-Arg-Arg-Ala-Lys(Dnp)NH(2); W-1, polyoxyethylene ether W-1. EC numbers and recommended names are those given by Nomenclature Committee of the International Union of Biochemistry and Molecular Biology(1, 2) .


ACKNOWLEDGEMENTS

We are grateful to Dr. C. G. Knight for synthesis of peptides, to N. D. Rawlings for help with amino acid sequence analysis, to Dr. R. L. Soffer for the gift of monoclonal antibody M3C, to Dr. S. R. Stone for prolyl oligopeptidase, and to Dr. T. Yoshimoto for Z-thioprolylthiazolidine.


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