A prolyl endopeptidase from murine macrophages, its assay and specific inactivation

ARCHIVESOF BIOCHEMISTRY AND BIOPHYSICS Vol. 225, No. 1, August, pp. 331-337, 198.3

A Prolyl Endopeptidase from Murine Macrophages, Its Assay and Specific Inactivation GEORGE D. J. GREEN AND ELLIOTT SHAW’ Biology

Department,

Brookhaven

Natiaal

Laboratory,

Upton, New York 11973

Received April 15, 1983

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro /%methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues. from different sources is susceptible both to thiol reagents and to diisopropyl fluorophosphate. An evaluation of the evidence obtained with a purified preparation from rat brain led to the conclusion that the enzyme is probably a serine protease (9). In the course of developing more selective inactivators of thiol proteases, we have been exploring the properties of peptidyl diazomethyl ketones since these have been found to be unreactive to serine and other mechanistic classes of proteases but very effective against thiol proteases (10). Furthermore, the reactivity of various reagents of this class toward thiol proteases depends on the extent to which the substrate preference of the protease is satisfied by the peptidyl portion of the reagent in a manner characteristic of affinity-labeling reagents (10). The diazomethyl ketone type of inhibitor therefore constitutes a powerful diagnostic tool and may also provide the means of blocking a given proteolytic

An endopeptidase cleaving on the carboxy1 side of proline was apparently first detected in uterus (1) and later in kidney (2). A separate line of investigation dealing with the metabolism of hormones in brain or pituitary extracts (3-6) also revealed a postproline cleaving enzyme. For a while these were judged to be separate enzymes based on differences in apparent molecular weight and sensitivity to proteinase inhibitors such as iodoacetamide and diisopropyl fluorophosphate. However, recent results suggest that a single enzyme with an apparent M, of 70,000 may account for these activities based on a reexamination of the molecular weight of the kidney enzyme (7) and the cross-reactivity of enzyme preparations from a variety of rat tissues with the monospecific antiserum prepared for the rat brain enzyme (8). The enzyme ‘Author dressed.

to whom correspondence should be ad331

0003-9861/83 $3.00 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

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AND

reaction in viva to deduce its metabolic role (11). We report the finding of a prolyl endopeptidase with properties similar to those previously reported in mouse peritoneal macrophages. The response of this enzyme to a number of peptidyl diazomethyl ketones has been examined and it is shown that benzyloxycarbonyl-Ala-AlaProCHNz is particularly effective in inactivating the protease. MATERIALS

AND

METHODS

Muter&&for cell culture. Multiwell plastic culture plates, medium 199, and serum were from Flow Laboratories, Inc., Rockville, Maryland. Biochemical reagents Heparin and penicillin/ streptomycin were obtained from Flow Laboratories. Brewers thioglycollate medium and la&albumin hydrolysate were from Difco, Detroit, Michigan; latex beads (1.09 pm diameter, 10% v/v), Micrococcus Zysodeikticus, NADH, N-ethyl maleimide, and pnitrophenyl-fl-n-glucuronide were from Sigma Chemical Company, Saint Louis, Missouri; DFP’ was from Aldrich Chemical Company, Milwaukee, Wisconsin; and pGlu-His-Pro flNA was a gift from Dr. J. E. Dixon, Department of Biochemistry, Purdue University, Lafayette, Indiana. Peptide diazomethyl ketones were prepared by the published procedures (10). Z-Ala-Ala-Pro $-methazy-bnaphthylumide. Prolyl 4-methoxy-2-naphthylamide (100 mg, Enzyme Systems Products) was treated with a 26% excess of ZAla-Ala hydroxysuccinimidate in dioxane solution with stirring at room temperature for 4 days. After removal of the solvent an ethyl acetate solution of the residue was extracted successively with dilute acid, bicarbonate, and finally dried over anhydrous magnesium sulfate. The residue, 115 mg, was recrystallized from ethyl acetate and hexane to yield 85 mg, mp 138-140°C. Anal. Calcd for CalHalN305 (525.6): C, 70.84; H, 5.94; N, 8.00. Found: C, 70.80; H, 6.10; N, 7.99. Macrop~e collection and culture. Macrophages from normal female mice (Swiss albino, 2 to 5 months old) were collected and cultured as described (11). Cultures were considered to be established at 24 h, when serum-containing medium was replaced with serum-free medium. Where applicable, inhibitors were added to cultures at this time. Aliquots of stock inhibitor solutions, in ethanol, were added to experi-

’ Abbreviation phate.

used: DFP, diisopropyl

fluorophos-

SHAW

mental cultures and ethanol was added to control cultures to provide a final ethanol concentration of 0.5% v/v. Cells were normally cultured 48 h in the presence of inhibitors before lysis. Adhered cells were judged to be greater than 95% macrophages by the criteria of latex particle ingestion. Cultures were routinely maintained at 3YC under 5% carbon dioxide and air for up to 3 days. Lysates were prepared in 0.5 ml 0.05 M sodium acetate, pH 7.2, after removal of media and washing with 1.0 ml phosphate-buffered saline; cells were scraped off the plates with a rubber policeman. Lysates for experimental use were prepared from cultures at any time up to 3 days after becoming established. Toxic effects of added reagents were examined after 48 h of culture by cell morphology and the assay of lactate dehydrogenase levels in the media and cell lysates. The ability of the cells to ingest latex beads, another test of viability, was carried out by addition of 10 ~1 of latex bead suspension to cultures and examination of cells after a 60-min period. In experiments where the rapid inactivation of prolyl endopeptidase was studied established cell cultures were exposed to the inhibitor, as described for the cultures above, for times ranging from 5 to 120 min. The medium was removed and the cells washed four times with 1 ml phosphate-buffered saline, incubating 5 min at 37°C before the removal of each wash. Cells were then lysed as described above. Assay of pro&l edopeptidase activity. Preliminary work used the substrate pGlu-His-Pro BNA. The appearance of the fluorescent product was monitored continuously with a recorder-equipped American Instruments fluorescence spectrofluorometer (Model SPF-125) or an American Instruments fluorocolorimeter as described by Rupnow et aL (5). Subsequent work used the substrate Z-Ala-Ala-Pro BmNA. The substrate was dissolved as a 5 X 1Om8M stock solution in absolute ethanol. Routinely, 10 ~1 of stock solution was incubated with an aliquot of enzyme and sufficient buffer (0.1 M phosphate, pH 7.4, containing 1 mM mercaptoethanol) to afford a final volume of 2 ml. The appearance of the fluorescent product, 4-methoxynaphthylamine, was monitored using excitation and emission wavelengths of 350 and 420 nm, respectively. Enzymatic studies. Inactivations of the prolyl endopeptidase in macrophage lysates were initiated by adding an aliquot of filtered lysate to dilutions of an inhibitor in 0.1 M phosphate buffer, pH 7.4, containing 1.0 mM mercaptoethanol, in a total volume of 1 ml. Stock solutions of the inhibitor were made up in absolute ethanol and final dilutions were not greater than 1% v/v (ethanol/buffer). Timed aliquots were removed and assayed as described above. The apparent pseudo-first-order rate constants for

A PROLYL

ENDOPEPTIDASE

the inactivation reactions were determined from the slopes of semilogarithmic plots of enzyme activity versus time. For reagents acting as competitive inhibitors of the prolyl endopeptidase, substrate concentration was varied over the range 5 X lo-’ to 1.5 X 10m6 M for different inhibitor concentrations and a Ki determined from the slopes of a double reciprocal plot. Molecular weight determination, Lysates of thiolglycollate-elicited peritoneal macrophages were filtered (0.45 pm, Millipore Corp.) and a l-ml aliquot was applied to a Sephacryl S-200 column (120 X 1 cm) equilibrated with 0.01 M phosphate, pH 7.5, containing 1 mM mercaptoethanol and 1 mM EDTA. The column was eluted with the same buffer at a flow rate of approx 7 ml/h and l-ml fractions were collected. The column was calibrated using ribonuclease, soy bean trypsin inhibitor, ovalbumin, bovine serum albumin, and aldolase as molecular weight standards. SubceUular localization of prolyl endopeptti~ A procedure modified from that of Snyder and Baggiolini (12) was used. Cells were harvested from thioglycollate-treated mice as described above. The peritoneal lavage was centrifuged at 12Og for 5 min at 4°C. The cell pellet was resuspended in 10 ml of 0.225% (w/v) KC1 to lyse erythrocytes, and after 2 min, 20 ml of 1.8% (w/v) KC1 was added to restore isotonicity. The cell suspension was recentrifuged at 120g for 5 min and the pellet resuspended in 8 ml 0.25 M sucrose buffered with 5 mM Tris-HCl, pH 7.4. The cells were disrupted with 15 strokes of a Dounce homogenizer. Intact cells and nuclei were removed by centrifuging at 1OOOgfor 3 min, at 4°C. The supernatant was fractionated by centrifugation at 25,000g for 30 min and 100,OOOgfor 60 min, both at 4°C. The two pellets were each suspended in 4 ml of the sucrose buffer. The final supernatant and the two suspended pellet fractions were assayed for protein, lactate dehydrogenase, lysozyme, @-glucuronidase, and prolyl endopeptidase. Protein was determined by the Bio-Rad protein assay. Lysozyme was assayed by monitoring the decrease in absorbance at 540 nm of a solution containing 500 ~1 of sample and 1.5 ml of a suspension of M. lym deikticus, 0.15 mg/ml, in 0.1 M sodium phosphate, pH 6.3. Lactate dehydrogenase was assayed by following the decrease in absorbance, at 340 nm, of a solution containing50 ~1 of sample fraction, 1.88 ml 0.2 M TrisHCI, pH 7.3, 60 ~1 6.6 mM NADH, and 60 pl 30 mM sodium pyruvate, both in the same Tris-HCl buffer. p-Glucuronidase was measured by incubating, for 60 min at room temperature, 200 ~1 of sample with 0.8 ml 0.1 M sodium acetate, pH 4.5, and 100 ~1 0.01 M pnitrophenyl-@-D-glucuronide in 0.1 M sodium phosphate buffer, pH 7.0. The reaction was stopped by the addition of 2.0 ml 0.2 M glycine containing 0.2 M NaCl. The absorbance increase, at 405 nm, compared

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MACROPHAGES

to a blank was read. All assays were performed using a Beckman spectrophotometer, Model UV 5230. RESULTS

AND

DISCUSSION

Prolyl endopeptidase activity was readily detected in peritoneal macrophage lysates from normal mice and those previously injected with thioglycollate media. The specific activities of prolyl endopeptidase, based on arbitrary activity units and protein concentration in the lysates, were approximately the same for both types of cell, measured on establishment of cultures. As peritoneal macrophages elicited by thiolglycollate broth are considered “activated” or stimulated whereas those from normal mice are not, this suggests the enzyme is not activatable by these conditions. Preliminary work used as substrate pGlu-His-Pro PNA, a substrate based on the thyrotropin-releasing hormone activity of the prolyl endopeptidase isolated from rat brain (5). Specificity information deduced from the effectiveness of a limited number of peptide diazomethyl ketones examined as potential affinity labels as described below, led to the synthesis of ZAla-Ala-Pro PmNA as a possible improved substrate. This substrate proved to be several times more sensitive than pGlu-HisPro ,0NA. Study of the hydrolysis of a range of substrate concentrations gave a K, of 2.6 X 10m6M for Z-Ala-Ala-Pro PmNA (data not shown), approximately lo-fold lower than that for pGlu-His-Pro PNA (5). This enabled the use of low substrate levels but still gave a high fluorescence yield. The molecular weight was estimated by gel filtration of a sample of macrophage lysate on a calibrated Sephacryl S-200 fine column. The enzyme activity eluted as a single peak, corresponding to an M, of 74,000. This is similar to the M,‘s of 70,000 reported for rat brain prolyl endopeptidase (5), 76,000 for the enzyme from bovine anterior pituitary (4), and 66,000 for the enzyme from rabbit brain (3). Subcellular

Localization

of the Enzyme

A simple fractionation scheme, performed with thioglycollate-elicited peri-

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AND

toneal macrophages, showed that the prolyl endopeptidase, under conditions that would sediment lysosomes and other cellular granules, remained in the soluble fraction. It therefore appears that the prolyl endopeptidase is a soluble cytoplasmic enzyme and is not lysosomal in origin, as demonstrated by the distinct separation of prolyl endopeptidase and lactate dehydrogenase from the lysosomal marker enzymes lysozyme and P-glucuronidase in this fractionation scheme (Table I). This result is consistent with observations on the distribution of the brain endopeptidase (5, 13). Enzyme Inuctivatkm

Studies in Vitro

A major thrust of our work was to treat the proline endopeptidase as a thiol protease and examine peptidyl diazomethyl ketones as a source of specific inhibitors of the enzyme, thus extending our studies on this chemical type of alkylating reagent as a source of inactivators uniquely effective with thiol proteases (10) in contrast to other classes. From the inhibitors studied, Z-Ala-Ala-ProCHNz emerged as the most effective inactivator of the macrophage enzyme. At an inhibitor concentration of 4 X lo-? M, 50% of the activity was lost in about 5 min followed by a slower phase. This biphasic response may have been due to an inadequate excess of inhibitor over enzyme concentration to provide pseudo-first-order conditions. Inactivation was followed to 97%. More detailed TABLE

I

SUBCELLULAR DISTRIBUTION OF PROLYL ENDOPEPTIDASE Percentage activity each fraction

Enzyme

Soluble fraction

25,000~ Pellet

Prolyl endopeptidase Lactate dehydrogenase Lysozyme @-Glucuronidase Protein

96.5 97.5 23.5 35 65.5

3 1.5 69 63 23.5

in

l@W%7 Pellet 0.5 1 7.5 2 11

SHAW

kinetic studies await a supply of purified enzyme. This reagent has been shown not to inhibit cathepsin B or other thiol proteases (10) except at concentrations many orders of magnitude greater. At the concentrations used in this study, the prolyl endopeptidase could be selectively inactivated while cathepsin B would remain active. Several other reagents of the same class, which did not contain a proline resZ-Pheidue, e.g., Z-Ala-AlaCHNz, AlaCHN2, and Z-LysCHN,, acted as competitive inhibitors of macrophage prolyl endopeptidase, showing tight binding to the enzyme (&‘s for Z-Ala-AlaCHN2, Z-Phe-AlaCHN2, and Z-LysCHN2 were 2.2 X 10p7, 1.2 X 10e6, and 2.0 X low7 M, respectively). Slow inactivation of the enzyme was also observed with Z-PheAlaCHN2 and Z-Ala-AlaCHN2, in addition to the competitive effect. This loss of activity seemed of a more complex kinetic nature, not being directly related to inhibitor concentration. Similarly, the reagent Z-Gly-Gly-ProCHN2 gave complex inhibition, apparently including both inactivation, presumably due to alkylation of a thiol group, and competitive inhibition. As reported for other prolyl endopeptidase preparations (3-5) the macrophage enzyme was also rapidly inactivated by Nethylmaleimide (5 min, 5 X 10e3M) and by DFP (5 min, 1.0 mM). DFP is normally considered a specific reagent for serine proteases and is strongly indicative of an active site serine residue. These results suggest that the macrophage enzyme is closely related to the other prolyl endopeptidases with both a cysteine and serine residue in or near the active site of the enzyme. It is interesting that the enzyme binds the reagents Z-Lys-CHN2, Z-Ala-AlaCHN2, and Z-Phe-AlaCHN2 very tightly yet these lead to only slow inactivation, whereas Z-Ala-Ala-ProCHN2 rapidly inactivates the enzyme. Similarly Z-Phe-Ala PmNA is not a substrate for the enzyme (unpublished observations) while Z-AlaAla-Pro PmNA is a sensitive substrate as described above. This probably reflects a specific requirement for a proline residue in order for cleavage or alkylation to ef-

A PROLYL

ENDOPEPTIDASE

ficiently take place, even though the enzyme will bind strongly to other amino acid sequences. This predominant specificity for cleaving after a proline residue is abundantly demonstrated in the cleavage sites of many peptides reported to date (4, 6, 14-19) with a minor activity after alanine residues (8), a property understandable in terms of steric compatability and encountered in other peptide binding sites (20). That the sequence Z-Ala-Ala-Pro- is effective both in an inhibitor and in a substrate suggests that both reagents bind in a similar manner and that an activated cysteine residue is part of the active site. This appears to us to be true particularly in view of the demonstration that peptidyl diazomethyl ketones have inactivated only thiol proteases thus far (10). The positive result with DFP tends to be given more weight because of long tradition but more than once it has been shown to be misleading, for example, because of impurities (21). It may be that the partial inhibition by iodoacetamide or iodoacetic acid was thought to be ambiguous evidence for thiol function (9). This type of reagent is deficient in the case of a hindered thiol group, for example, in cathepsin L with which millimolar iodoacetamide gives only incomplete inactivation (22) whereas a substrate-derived diazomethyl ketone, such as Z-Phe-PheCHNz, rapidly inactivates at a 5 PM concentration (23) and can be followed to completion. A final resolution of the question of the catalytic mechanism of this protease may require sequence information or spectroscopic evidence for a thiol-acyl enzyme intermediate as was provided by Lowe and Williams in the case of papain (24). Inactivation of Cellular Endopeptidase by Incubation with Diazomethyl Ketones Inactivation of the macrophage prolyl endopeptidase in intact cells in culture was followed by the assay of enzyme activity present in cell lysates after addition of, and culture with, various inhibitors. Z-AlaAla-ProCHNz, the most effective inhibitor found in in vitro inactivation studies, ap-

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MACROPHAGES

peared to rapidly inactivate the cellular enzyme at concentrations of 5 X lo-’ and 5 X lop6 M with half-times of approx 17 and 4 min, respectively (Fig. 1). These results are similar to the rates of inactivation seen in the inactivation study using cell lysate enzyme. The inhibitor therefore seems able to rapidly enter the cell. Incubation with lower concentrations of Z-Ala-Ala-ProCHNe (e.g., 1 X lo-’ M) or other diazomethyl ketones for a period of 48 h shows a concentration-dependent effect on cellular enzyme levels, assayed in lysates at the end of the culture period (Fig. 2). The effectiveness of the reagents in culture reflected their effectiveness in the inactivation studies, Z-Ala-AlaProCHN2 being more effective than Z-GlyGly-ProCHNz and Z-Phe-AlaCHNz. ZGly-Gly-PheCHNe had no inhibitory effect when present in culture for 48 h at a concentration of 5 X 10m5M. In cultures, ZPhe-AlaCHNz appeared to inactivate the enzyme whereas in kinetic studies based on initial velocity measurements with macrophage lysate it behaved primarily as a competitive inhibitor. Loss of enzyme in

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20

’

30

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40

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50

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TIME(mm1

FIG. 1. Prolyl endopeptidase inactivation in intact cells on brief exposure to Z-Ala-Ala-ProCHI$. Cells were incubated with 5 X lo-’ M Z-Ala-Ala-ProCHNZ for the periods indicated, washed thoroughly, and lysed before enzyme activity was measured.

336

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~1 w

0 IO”

AND

Z-Ala-Ala-ProCHN2 Z-Gly-Gly-ProCHN2 Z-Ala-AlaCHN2 Z-Phe-AlaCHN2

10-e INHIBITOR CONC ,k?-’

10-G

FIG. 2. Prolyl endopeptidase activity in lysates from cells cultured in the presence of various peptidyl diazomethyl ketones (0, Z-Ala-Ala-ProCHNa; A, n , Z-Ala-AlaCHNa; 0, Z-PheZ-Gly-Gly-ProCHNz; AlaCHN,). Cells cultured 48 h in the presence of inhibitor or ethanol (0.5% v/v) before lysis and assay of enzyme as described in the text.

cultures treated with inhibitors compared to control cultures would seem to represent a slow rate of inactivation occurring over the duration of the culture and not due to competitive inhibition. The extensive washing of the adhered cells, followed by lysing in phosphate-buffered saline, plus further dilution during assay would dilute any cellular inhibitor remaining sufficiently to prevent it from having an effect in the assay. At these levels the reagents were not toxic to the cells as determined by morphology, lactate dehydrogenase release, and the ability of the cells to phagocytose latex particles. However, it is interesting to note that higher concentrations of two reagents, Z-Ala-Ala-ProCHNz (5 X lop6 M) and Z-Gly-Gly-ProCHNz (5 X 10V5M), were toxic to the cells during 48-h cultures, but other diazomethyl ketones at similar concentrations showed no toxic effects. The cause of this apparent specific toxicity is not known. Lower concentrations of the reagents, with which complete inactivation of prolyl endopeptidase is observed, were not toxic over a 48-h period. Recovery of Cellular Enzyme Levels afler Inhibition bg Z-Ala-Ala-ProCHN,

Cells were cultured for periods up to 48 h after exposure to 5 X 10e6M Z-Ala-Ala-

SHAW

ProCHNz for a 90-min culture period. Cells washed and lysed immediately after this exposure to inhibitor showed very low levels of prolyl endopeptidase activity. Levels of activity in the lysates of cells cultured for periods following inhibitor exposure recovered to 39% of control values after 20 h and 65% of control values after 43 h. This recovery was completely stopped by the addition of cycloheximide (2 pg/ml), an inhibitor of protein synthesis. Cells treated with cycloheximide alone showed some loss of prolyl endopeptidase activity over the 48-h culture period possibly due to enzyme turnover. Cells treated with ZAla-Ala-ProCHN, followed by cycloheximide were devoid of prolyl endopeptidase activity during the culture period. Since the enzyme is irreversibly inactivated by the inhibitor, appearance of activity after inhibitor treatment suggests that new enzyme is being synthesized by the cultured cells. The specific activity of the prolyl endopeptidase was measured as the amount of 4-methoxy 2-naphthylamine liberated per milligram protein per minute, by an aliquot of filtered lysate assayed with ZAla-Ala-Pro OmNA. Typical values for lysate prepared from cells established in culture were of the order of 0.2 ~Mols @mNA mg-’ min-‘. Although the ability to metabolise hormones played a large role in the discovery of postproline endopeptidase activity, as pointed out by others (8), its widespread distribution argues against a specific function in hormone regulation. The enzyme may have an important role in protein turnover which in some cases makes large demands on the degradative capacity of cells. For example collagen, a proline-rich protein, is a major secretion product of fibroblasts, but 20% or more of the newly synthesized material is digested intracellularly prior to secretion (25). Since the proline endopeptidase apparently does not act on proteins (6,14) but only on peptides, it could be involved at later stages of protein turnover after initial cleavage by other endopeptidases. The availability of the specific inhibitor, Z-Ala-Ala-ProCHN2,

A PROLYL

ENDOPEPTIDASE

should greatly aid in clarifying the role of this enzyme. With respect to the function of other cellular thiol proteases, Z-Phe-Ala-CHNs has been useful for the in vitro and in vivo inactivation of cathepsins B and L (10, 11, 22,26). However, in view of the competitive inhibition of the postproline endopeptidase by Z-Phe-AlaCHNz, it may be advantageous to use a different inactivator for cathepsins B and L. Continuing investigation of the properties of these proteases recently provided improved irreversible inhibitors for cathepsin B, for example Z-Phe-Ser(Opzl)CHNz (27). The larger substituent in the primary specificity site increases the binding to cathepsin B but has the opposite effect with respect to the macrophage postproline endopeptidase, for which the relatively high concentration of 10e4 M inhibitor is required to produce a half-time of inactivation of 18 min whereas a lOOO-fold less will readily inactivate cathepsin B. Thus, this inhibitor, being more selective, is superior to Z-Phe-AlaCHNz for the cellular inhibition of cathepsin B. As the study of thiol proteases progresses, it can be expected that evolution of the knowledge of their specificity can be matched in inhibitor design to permit their selective inactivation in vivo. A recent publication (28) reports independent observation of the postproline endopeptidase in macrophages and extends earlier findings of the presence of the enzyme in bronchopulmonary lavage fluid (18).

FROM

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E., PEARCE,

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(1979) Biochemistry

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