Inactivation of endothelin I by deamidase (lysosomal protective protein).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 5, Issue of February 15, pp. 2872-2875.1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication Inactivation of Endothelin I by Deamidase (Lysosomal Protective Protein)* (Received for publication, November 12, 1991)

Herbert L.Jackman, PaulW.Morris$, Peter A. Deddish, Randal A. Skidgel, and Ervin G . Erdoss From the Laboratory of Peptide Research and Departments of Pharmacology, Anesthesiology,and $Biochemistry, University of Illinois College of Medicine, Chicago, Illinois 60612

Deamidase cleaves esterand peptide bonds in various substrates and deamidates protected COOH-terminal amino acids. It preferentially hydrolyzes peptides which contain hydrophobic amino acids in the P1' and/ or PI position. Because the COOH-terminal end of endothelin I contains the hydrophobic sequence -Ile19-Ile20-Trp21-OH, we investigated whether human deamidase, purified from platelets, could inactivate this peptide. We found that deamidase readily cleaved off Trp21 with an acid pH optimum, a K , = 22 YM,a kat of 1454 min", and a &JKm of 68 p ~ " min- We also found the enzyme to be present in target cells of endothelin, in vascular smooth muscle cells. Extracts of cultured vascular smooth muscle cells cleave both the synthetic fluorescent substrate 5-dimethylaminonaphthalene-1-sulfonyl(Dns)-Phe-Leu-Argand endothelin I by releasing the COOH-terminal amino acid. The reaction was inhibited by diisopropyl fluorophosphate, benzyloxycarbonyl-Gly-Leu-Phe-CHZCl, and p chloromercuribenzenesulfonate, which inhibit the purified deamidase, but not by inhibitors of some other peptidases. The rate of hydrolysis of endothelin I in the soluble, 100,000 X g final supernatant of the homogenized smooth muscle cells was2.1 pmol/h/mg and 3.1 pmol/h/mg for Dns-Phe-Leu-Arg. Thus, smooth muscles, platelets, and many other tissues which contain the deamidase can inactivate endothelin by cleaving the COOH-terminaltryptophan.

.

muscle that can elevate the blood pressure (1, 2). ET1 andits congeners also contractintestinaland pulmonary smooth muscles, affect cardiac and renal function, and areimplicated in theregulation of transmembrane signaling and gene expres21 amino acids, sion (3). All matureendothelinscontain including 4 cysteine residues linked bytwo intramolecular disulfide bonds and the unusually hydrophobic COOH-terminal end of Ile'9-Ile'0-Trp'1 (3). Recently, we isolated and purified an enzyme from human platelets which cleaves COOH-terminal free or protected amino acids (4). It also deamidates peptides such as tachykinins by converting the COOH-terminal Met-NH, to Met-OH. Thus, it was called deamidase, but the enzyme is identical with the so-called lysosomal protective protein (5,6) and has many properties in common with cathepsin A (4). The deamidase activity of the homogeneous protein has a neutral pH optimum, while the carboxypeptidase-type action is more effective at an acidic pH. The enzyme preferentially cleaves substrates where the P1 residue is a hydrophobic amino acid. Since our best synthetic substrates employed to assay the enzyme have hydrophobic amino acids in either the P1 and Ppor PI' and PIpositions' (4),we tested ET1 as a substrate. The high potency and widespread effects of ET1 require tight control of its bioavailability, and that usually includes enzymatic degradation. Since the removal of the Trp from the hydrophobic COOH terminus of ET1 terminates the vasoconstrictor activity (7, 8 ) ) we hypothesized that this peptidase may play a role in the inactivation of ET1. Indeed, we found that thedeamidase inactivated ET1 by removal of Trp". EXPERIMENTALPROCEDURES

Materiaki-5-Dimethylaminonaphthlene-1-sulfonyl-~-phenylalanyl-L-leucyl-L-arginine (Dns-Phe-Leu-Arg) was synthesized by coupling Dns-Phe toLeu-Arg using standard techniques (9). Deamidase was purified from human platelets as described (4). Trans-epoxysuccinyl-~-leucylamido-(4-guanidino)-butane (E64) and otherlaboratory reagents were obtained from Sigma, and benzyloxycarbonyl (Z)-GlyLeu-Phe-CH&l was from Enzyme Systems Products (Livermore, CA). Enzyme Assays-The activity of the deamidase was determined with ET1 substrate by separating and quantitating the products in HPLC. Each reaction tube contained either purified deamidase or the final supernatant of homogenized cell preparations as enzyme source, ET1 (8.6 p M or 10 pM), 100 mM buffer (sodium acetate, pH 5.5, MES, pH 6.25, or HEPES pH 7.0), and water in a final reaction Endothelin I (ET1)' belongs to a group of peptides which volume of 100 pl. Reactions were carried out at 23 or 37 'C for 2-20 exert a potent and long lasting stimulationof vascular smooth min, stopped with 100 p1 of ethyl alcohol, and stored at -20°C until analyzed by HPLC. In kinetic studies, the concentration of ET1 . inhibition studies, reaction mixtures * This work wassupported in part by National Institutes of Health ranged from 2.5 to 30 p ~ For Grants HL 36082, HL 36473, H36081, and DK 41431. The costs of with or without inhibitors were preincubated at 23"C for 15 min publication of this article were defrayed in part by the payment of followed by addition of ET1 toinitiate the reaction. The carboxypeptidase activity of the deamidase was determined page charges. This article must therefore be hereby marked "advertisement" in accordance with 18U.S.C. Section 1734 solelyto indicate with Dns-Phe-Leu-Arg by the technique reported previously for DnsAla-Arg (10). Briefly, the enzyme was incubated with 0.2 mM Dnsthis fact. 5 To whom correspondence should be addressed Dept. of Phar- Phe-Leu-Arg (final volume, 250 pl) at pH 5.5 or 7.0 at 37 "C for 5-60 macology (M/C 868), University of Illinois at Chicago, 835 S. Wolcott min. The reaction was stopped by the addition of 150 pl of 1 M citric acid, pH 3.1, and the product was extracted into 1 ml of chloroform. Ave., Chicago, IL 60612. Tel.: 312-996-9146;Fax: 312-996-1225. The abbreviations used are: ET1,endothelin I; HPLC, high The fluorescence was measured at 340 nm excitation and 495 nm emission wavelength (10). Deamidase activity was taken as the difperformance liquid chromatography; Dns, 5-dimethylaminonaphthalene-1-sulfonyl; E64, trans-epoxysuccinyl-L-leucylamido- ference between total activity and the activity inhibited with 1 mM (4-guanidino)-butane;2, benzyloxycarbonyl; DFP, diisopropyl diisopropyl fluorophosphate (DFP). DFP usually inhibited 93-100% fluorophosphate; PCMS, p-chloromercuribenzenesulfonate;MES, 2- of the reaction. (N-morpho1ino)ethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-lP. A. Deddish, H. L. Jackman, R. A. Skidgel, and E. G. Erdos, piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyunpublished data. ethylenenitri1o)ltetraacetic acid.

2872

Inactivation of Endothelin

2873

HPLC and Amino Acid Analysis-The HPLC analysis was done as described previously (4). In all HPLC analyses, injection of authentic ET1 was used to identify the ET1peak. The peptide fractions collected after separation by HPLC were analyzed for amino acid content.Solvent wasremoved from the fractions, and the peptides were hydrolyzed in 6 N HC1 followed by phenylthiocarbamyl derivatization. Amino acid analysis was performed in an Applied Biosystems Inc. model 420/130chromatograph following the company's protocols. Peptide samples were pretreated with 1-dodecanethiol in hexane to aid tryptophan recovery, which ranged from 45 to 55% at 250 p m o l / ~ p l e . Cell Culture-Rat vascular smooth muscle cells were harvested from rat lung and established as a primary culture. The cells were then maintained untilcell growth allowed for splitting and seeding of new culture flasks. Cells from six confluent T75 culture flasks were harvested, homogenized by~nication, and after subcellular fractionation (lo), thesoluble (&), final supernatant obtained after centrifugation at 100,000 X g for 60 min was retained for experimental use. Bioassay-The inactivation of ET1 by purified deamidase at pH 5.5 was also determined by bioassay on the guinea pig ileum. The reaction was carried out as described above, except it was stopped prior to the assay by cooling in a 4 "C ice bath instead of using ethyl alcohol. Aliquots of the reaction mixtures were injected into a tissue bath containing the isolated guinea pig ileum, and the isotonic contractions were recorded (11).

ET1 was incubated with the deamidase, and the new peak and the peak coeluting with the unhydrolyzed ET1 substrate peak were collected.Aminoacid analysis of the collected fractions showed that the new peak contained all the amino acids in the same molar ratios as theET1 standard, with the important exception of tryptophan, which was missing. Consequently, because ET1 contains only a single COOH-terminal tryptophan (l),the deamidase cleavesthe COOH-terminal amino acid from ET1. The peak which coeluted with the ET1 standard was indeed ET1 since it contained all the amino acids, including Trp2',as determined by amino acid analysis. A third, less prominent peak was detected, but only with the higher concentration (35 p ~ of)ET1 (Fig. 1).The size of this peak also depended on incubation time and was reduced in the presence of inhibitors. The peak had the same elution time as a tryptophan standard and increased whenthe sample was spiked with authentic tryptophan. Thus, this peak contained the product, TrpZ1,resulting from the enzymatic hydrolysis of ET1 by deamidase. Kinetic constants were determined for purified deamidase using ET1 as substrate at pH 5.5 and 23 "C. The K , was 22 p ~ and , the Ifmax was 28 pmol/min/mg, giving a kcatof 1454 min" and a ket/Kmof 68 p"' min". RESULTS Because smooth muscle is a target tissue for endothelin, As established by HPLC, purified deamidase metabolized where it causes a long lasting contraction, we tested the final ET1 (8.6 p ~ at) a rate of 6.86 p m o l / m i n / ~at pH 5.5 and s u p e r n a ~ of t homogenized cultured rat vascular smooth 23 "C, The ratedecreased at higher pH values to 2.32 Fmol/ muscle cells for deamidase activity. The hydrolysis of Dnsmin/mg at pH 6.25 and 0.36 pmol/min/mg at pH 7.0. The Phe-Leu-Arg was measured fluorometrically, and ET1 inacreactions were run at 23 "C because the purified enzyme is tivation was determined by HPLC. The final supernatant (S,) somewhat unstable at pH values M.5 at 37 "6.The cleavage of vascular smooth muscle cells hydrolyzed Dns-Phe-Leu-Arg was not due to a putative c o n ~ i nin~ the t purified deam- at a rate of 3.1 ymol/h/mg at pH 5.5 and 0.8 pmol/h/mg at idase preparation, because it was inhibited by three inhibitors pH 7.0. ET1 was hydrolyzedat a rate of 2.1 pmol/h/mg at pH with different modes of action. We previously showed that 5.5 and 0.3 at pH 7.0. Inhibition studies indicated that the these compounds inhibit the enzyme with other substrates activity in thisfraction of the cells was due to deamidase. The (4). Thus, ET1 hydrolysis at pH 5.5 or 7.0 was inhibited 98 reaction was not inhibited by o-phenanthroline (1 mM), the or 100% with 1 mM DFP, 97 or 100% by 50 p~ Z-Gly-Leu- inhibitor of metalloproteases, or by the catheptic enzyme Phe-CH2C1,and 78 or 100% by 1 mM p-chloromercuriben- inhibitor E64 (100 p ~ )but , the hydrolysis was almost comzenesulfonate (PCMS). pletely abolished ( 9 5 - 1 ~ % by ) DFP (1mM) and Z-Gly-LeuHPLC analyses of the reaction mixtures revealed the ap- Phe-CH&l (100 PEA,Table I). The inhibition pattern was the pearance of a new peak increasing in size with the length of same with both ET1 and the short synthetic substrate Dnsincubation and proportional to the decrease in size of the Phe-Leu-Arg (Table I). substrate ET1 peak (Fig. 1).The metabolite peak did not The inactivation of the biological effects of ET1 by deamiappear or was greatly reduced in thepresence of the deamidase dase was observed in bioassay experiments on the isolated inhibitors mentioned above (DFP, Z-Gly-Leu-Phe-CHzC1,or guinea pig ileum.Here, incubation of 0.2 pmol of deamidase PCMS). In order to unequivocally identify this new product with 100nmol of ET1 at37 "C for 45 min at pH 5.5 abolished peak, in two experiments a higher concentration (35 PM) of 70% of the contractile effect of the peptide.

I

DISCUSSION

Des-TrD'l-ETl

20 min 20 rnin

+

1 mM

OFP \

15

20

25

30

35

40

Time,rnin

FIG. 1. HPLC tracing showing the metabolism of ET1 by

purified humandeamidase. Purified deamidase (14.4ng) was incubated with 35 ET1 at 25 "C (pH 5.5) for 5, 10, or 20 min as indicated onthe figure. In thetower tracing, enzyme was preincubated for 15 min with 1 mM DFP before the addition of substrate. Peaks were identified by coelution with authentic standards and/or amino acid analysis. For further details,see the text.

ET1 is released froma 38-amino acid proendothelin (or big endothelin) by the cleavage of the Trp2'-ValZ2 bond.Three enzymes have been described to activate ET1; two of the more active ones are intracellular aspartic proteases with an acidic pH optimum. The third is a neutral membrane metalloprotease. These converting enzymes are also present in vascular smooth muscle cells (12-18). In spite of numerous publications on the variety of biological actions of endothelins, such as the long lasting vasoconstriction (1,2, 18)and even on the cloning of its receptors (2, 19), there isa paucity of information on its enzymatic metabolism. Two recent publications reported the hydrolysis of ET1 by purified neutral endopeptidase 24.11 or enkephalinase in uitro (20, 21). This enzyme cleavesa variety of other peptide substrates including hypotensive peptides such as substance P and bradykinin, opioid peptides such as enkephalins, and atrialnatriuretic factor (22). Interestingly, the enzymatic conversion of big endothelin to endothelin is inhibited in uiuo

Inactivation of Endothelin

2874

TABLEI Inhibition of ET1 and Dm-Phe-Leu-Arg hydrolysis by the final supernatant of homogenized cultured rat vascular smooth muscle cells Inhibition Inhibitor

ET1

Concentration pH 5.5

Dns-Phe-Leu-Arg pH 7.0

a

pH 7.0

99

96

%

mM

DFP o-Phenanthroline Z-Gly-Leu-Phe-CH2C1 E64 EGTA

pH 5.5

1 1 100 0.1

100

0.5 ND 10

13 ND

3 100

100 10

8 99

8 95

8

ND" 9

ND 0

ND, not determined.

by theneutral endopeptidase inhibitor, phosphoramidon, probably by blocking another enzyme (12-18). The reported K,,, of ET1 with bovine kidney neutral endopeptidase is somewhat higher than theone we obtained with the human deamidase (30 uersus 22 p M ) , but theK,,, is lower (2.3p M ) with rat neutral endopeptidase (20,21). However, the turnover number (kcat) for ET1 with deamidase is much higher (1454 min-') than that reported for rat neutral endopeptidase (131 min-l) (21). Based on the kinetic constants, deamidase would hydrolyze 0.1m M ET1 ata rateof 23 pmol/min/mg, which is about 10-25 times faster than rates we found (at pH 5.5) for other biologically active substrates such as angiotensin I, bradykinin, oxytocin, or substance P (4). Although the deamidase used here ispresent in many tissues, it was purified from human platelets. The enzyme, as we showed, is identical with the so-called lysosomal protective protein (4). This protein, according to d'Azzo and colleagues (5, 6), forms high molecular weight complexes with P-galactosidase and neuraminidase in lysosomes. The lack of the mature protein causes a genetically determined disease, 6galactosialidosis. The properties of deamidase are quite similar to those of the structurally rather ill defined cathepsin A (4, 5). Deamidase belongs to the serine carboxypeptidase family of enzymes which includes yeast carboxypeptidase Y and the KEX 1 gene product. DFP and inhibitors of chymotrypsin-type enzymes (chymostatin, Z-Gly-Leu-Phe-CHzC1) block deamidase activity. Although it is also inhibited by the nonspecific sulflydryl-directed reagent PCMS, the more specific cathepsin inhibitor E64 is inactive (4). The carboxypeptidase activity of the enzyme has an acidic pH optimum, while its esterase and deamidase activities are optimal at neutrality. The dansylated peptide substrate (Dns-Phe-Leu-Arg),having hydrophobic residues in PI and Pz positions, was cleaved rapidly by removal of the COOH-terminal Arg. This is consistent with the results previously obtained with bradykinin, where deamidase readily cleaved the PheS-Ar$ bond (4). While deamidase will hydrolyze peptides with COOH-terminal Arg, it does not cleave them when arginine is the penultimate amino acid (e.g. vasopressin, metorphinamide, pancreatic polypeptide) (4).Besides Arg, the enzyme cleaves a variety of COOH-terminal free or protected amino acids, for example, Trp (asreported here), Phe, His, Leu, and Gly-NHz (4). Dansylated peptides have been employed before to assay diverse peptidases including prolylcarboxypeptidase (23), angiotensin I-converting enzyme (24), andcarboxypeptidases H (25-27) and M (28). Dns-Phe-Leu-Arg is also cleaved by carboxypeptidase B-type enzymes such as carboxypeptidases M, N, and H (26-28). At the assay pH of 5.5, only carboxypeptidase H would have significant activity, but this enzyme can be easilydistinguished from deamidase. Carboxypeptidase

H is a metalloenzyme and iscompletely inhibited by chelators such as o-phenanthroline andby arginine derivatives such as guanidinoethylmercaptosuccinicacid and 2-mercaptomethyl3-guanidinoethylthiopropanoic acid whichdo notinhibit deamidase (4, 25-27). Conversely, deamidase is inhibited by inhibitors of serine proteases which are inactive with carboxypeptidase H. In addition, carboxypeptidase H cleaves only basic COOH-terminal amino acids, while deamidase liberates basic, aliphatic, or aromatic amino acids (4). Although these studies have been carried out in uitro, deamidase could very well be involved in the inactivation of endothelin in uiuo. The long lasting pressor effects of injected endothelin indicate the peptide is not rapidly metabolized by blood-borne enzymes or plasma membrane-bound peptidases on endothelial cells. Indeed, a major endothelial cell peptidase, angiotensin I-converting enzyme, does not metabolize endothelin(20),and the only peptidase identified whichdoes inactivate it,neutral endopeptidase 24.11, has a very low activity on plasma membranes of endothelial cells or in blood plasma under normal circumstances (29, 30). Thus, endothelin actions may be regulated by receptor-mediated endocytosis as shown for other peptide hormones (31). In this pathway, peptide-receptor complexes are endocytosed into coated vesicles which fuse with prelysosomal or lysosomal vesicleswhere the peptide ligand is degraded. In some cases, the receptor is recycled whilein othercases it is also degraded. The lysosomal localization of deamidase and the rapid inactivation of ET1 by the enzyme make it an ideal candidate for this type of reaction. In addition, deamidase is released from platelets by thrombin (4)and could thereby regulate endothelin levels at sites of platelet activation and aggregation such as in inflammation or at atherosclerotic plaques. This ability to affect vasoconstrictor activity at these sites is of obvious importance. Furthermore, lysosomal enzymes, for example cathepsins B, G, and L (32-35), can become associated with cell membranes, and in this way deamidase may contribute to the regulation of endothelin levels both in asoluble, released form and as an extracellular, membrane-bound enzyme. Acknowledgments-We thank Dr. Sara Rabito, Department of Anesthesiology, for help in performing the bioassay for ET1 and the Protein Sequencing/Synthesis Laboratory of the Research Resources Center of the University of Illinois at Chicago for cooperation in performing the amino acid analyses. REFERENCES

1. Sokolovsky, M. (1991) Trends Biochem. Sci. 16,261-264 2. Webb, D. J. (1991) Trends Pharmacol. Sci. 12,43-46 3. Simonson, M. S., and Dunn, M. J. (1991) Hypertension 17,856863 4. Jackman, H. L., Tan, F., Tamei, H., Beurling-Harbury, C., Li,

Inactivation of Endothelin

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17.

X-Y., Skidgel, R.A., and Erdos, E. G. (1990) J. Biol. Chem. 265,11265-11272 Galjart, N. J., Morreau, H., Willemsen, R., Gillemans, N., Bonten, E. J., and d’Azzo, A. (1991) J. Biol. Chem. 2 6 6 , 14754-14762 Galjart, N. J., Gillemans, N., Harris, A., van der Horst, G. T. J., Verheijen, F. W., Galjaard, H., and d’Azzo, A. (1988) Cell 54, 755-764 Kimura, S., Kasuya, Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K., and Masaki, T. (1988) Biochem. Biophys. Res. Commun. 156,1182-1186 Nakajima, K., Kubo, S., Kumagaye, S.-I., Nishio, H., Tsunemi, M., Inui, T., Kuroda, H., Chino, N., Watanabe, T. X., Kimura, T., and Sakakibara, S. (1989) Biochem. Biophys. Res. Commun. 163,424-429 Konig, W., and Geiger, R. (1970) Chem. Ber. 1 0 3 , 788-798 Deddish, P. A., Skidgel, R. A., Kriho, V. B., Li, X-Y., Becker, R. P., and Erdos, E. G. (1990) J. Biol. Chem. 2 6 5 , 15083-15089 Erdos, E. G., Renfrew, A. G., Sloane, E. M., and Wohler, J. R. (1963) Ann. N. Y. Acad. Sci. 104,222-234 Ikegawa, R., Matsumura, Y., Tsukahara, Y., Takaoka, M., and 171, Morimoto, S. (1990) Biochem.Biophys.Res.Commun. 669-675 McMahon, E. G., Palomo, M.A., Moore, W. M., McDonald, J. F., and Stern, M. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 8 8 , 703-707 Hioki, Y., Okada, K., Ito, H., Matsuyama, K., and Yano, M. (1991) Biochem. Biophys. Res. Commun. 174,446-451 Takada, J., Okada, K., Ikenaga, T., Matsuyama, K., and Yano, M. (1991) Biochem. Biophys. Res. Commun. 176,860-865 Hisaki, K., Matsumura, Y., Ikegawa, R., Nishiguchi, S., Hayashi, K., Takaoka, M., and Morimoto, S. (1991) Biochem. Biophys Res. Commun. 177,1127-1132 Matsumura, Y., Ikegawa, R., Tsukahara, Y., Takaoka, M., and 178, Morimoto, S. (1991) Biochem.Biophys.Res.Commun. 899-905

2875

18. Pollock, D. M., and Opgenorth, T. J. (1991) Am. J. Physiol. 261, R257-R263 19. Lin, H. Y., Kaji, E. H., Winkel, G. K., Ives, H. E., and Lodish, H. F. (1991) Proc. Natl. Acad. Sci. U. S.A. 8 8 , 3185-3189 20. Sokolovsky, _ .M.,. Galron, R., Klooe, Y., Bdolah, A., Indie, F. E., Blumberg, S., and Fle.minger, GT(1990) Proc. Nutl. AGad. Sci: U.S. A. 87, 4702-4706 21. Vijayaraghavan, J., Scicli, A. G., Carretero, 0. A., Slaughter, C., Moomaw,C., and Hersh, L.B. (1990) J. Biol. Chem. 2 6 5 , 14150-14155 22. Erdos, E. G., and Skidgel, R. A. (1989) FASEB J. 3 , 145-151 23. Yang, H. Y. T., Erdos, E. G., Chiang, T. S., Jenssen, T. A., and Rodgers, J. G. (1970) Biochem. Pharmacol. 19, 1201-1211 24. Xgic, R., Erdos, E. G., Yeh, H. S. J., Sorrels, K., and Nakajima, T. (1972) Circ. Res. 31,11-51-11-61 25. Fricker, L. D. (1988) Annu. Rev. Physiol. 50,309-321 26. Fricker, L. D., and Snyder, S. H. (1982) Proc. Natl. Acad. Sci. U.S. A. 79,3886-3890 27. Fricker, L. D., Plummer, T. H., and Snyder, S. H. (1983) Biochem. Biophys. Res. Commun. 111,994-1000 28. Skidgel, R. A. (1991) in Methods in Neurosciences: Peptide Technology (Conn, P. M., ed) Vol. 6, pp. 373-385, Academic Press, Orlando, FL 29. Johnson, A.R., Coalson, J. J., Ashton, J., Larumbide, M., and Erdos, E. G. (1985) Am. Rev. Respir. Dis. 1 3 2 , 1262-1267 30. Johnson, A. R., Ashton, J., Schulz, W., and Erdos, E. G . (1985) Am. Rev. Respir. Dis. 132,564-568 31. Gammeltoft, S. (1991) in Degradation of Bioactiue Substances (Henriksen, J. H., ed) pp. 81-105, CRC Press, Inc., BocaRaton, FL 32. Rozhin, J., Wade, R. L., Honn, K. V., and Sloane, B. F. (1989) Biochem. Biophys. Res. Commun. 164,556-561 33. Skideel. R. A.. Jackman. H. L.. and Erdos. E. G. (1991) . , Biochem. PLrmacol. 41,1335-1344 ’ 34. Weiss. R. E.. Liu. B. C.-S.. Ahlerine. T.. Dubeau., L... and Droller. M. J. (1990) J.’Urol. 144, 798-854 ’ 35. Sloane, B. F., Rozhin, J., Robinson, D., and Honn, K.V. (1990) Biol. Chem. Hoppe-Seyler 3 7 1 , 193-198 ~

~~