BIOCHEMICAL
Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1230-1236
PURIFICATION AND CHARACTERIZATION OF PUTATWE ENDOTHELIN CONVERTING ENZYME IN BOVINE ADRENAL MEDULLAi EVIDENCE FOR A CATHEPSIN D-LIKE ENZYME Tatsuya Sawamura, Sadp Kimura ‘, 0-2 Masashi Yanagisawa , Katsutoshi Goto
Shkuni, Yoshild Su$ta, and Tomoh Masald
Department of Biochemistry and #Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsuhuba, Tsukuba, Ibarald 305, Japan
Received
April
10,
1990
SUMMARY: A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules. ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,080,30,000 and 15,088 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease. 0 1990
Academic
Prass,
Endothelin endothelial
Inc.
(ET) is a novel vasoconstrictor peptide purified from a culture medium of porcine aortic
cells (1). From the analyses of the genomic libraries of human, rat and mouse, it was found that
there are three isopeptides of ET,
named ET-l, ET-Z (VIC) and ET-3 (2-4). By the analysis of cDNA of
ET-l, it was suggested that ET-I is generated from a 39-(porcine) called big endothelin-1
(big ET-l)
by unusual processing at the Trp-Val
dibasic pairs in approximately UK)-residue preproendothelin boxy1 terminal peptide
ET-1(22-39)
medium of porcine aortic endothelial
or a 38-(human)
residue intermediate
bond after processing at positions of
(1,5). In fact, bii ET-l (ET-1(1-39))
beginning with Val in addition to ET-1(1-21)
and its car-
were found in a culture
cells (6). Big ET-1 is considerably less active than mature ET-l on the
isolated porcine coronary artery and the conversion from big ET-l to ET-l increases the contractile activity about two orders of magnitude (7-9). Although it is still uncertain whether vessel endothelium source producing ET-l and big ET-l in circulating blood (see the hypothalamus-pituitary
system in Ref. 10)
and where the conversion from big ET-1 to ET-l occurs (inside or outside of ET-l producing concentration
Copyright AU rights
cells), the
of big ET-l was found to be approximately equimolar compared to that of ET-1 in circulating
’ To whom all correspondence should be addressed. 0006-291X/90
is the only
$1.58
Q 1990 by Academic Press, Inc. of reproduction in any form reserved.
1230
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
blood (11,X!). On the other hand, as for intracellular ET related peptides, ET-1 from porcine spinal cord and ET-l and ET-3 from porcine brain were isolated but big ET-I was very scarceIy identified in both tissues (l3,14). Until now, no one shows the characteristics of ECE which specifically cleaves between Tr~~l-Val~. Here we report the assay system for ECE activity, and purification and characterization
of an aspartic
protease from bovine adrenal medulla which has ECE activity. MATERIALS
AND METHODS
Chemicals: Porcine big ET-l, ET-l, leupeptin, phosphoramidon, E-64, pepstatin and phenyhnethylsulfonylfluoride (PMSF) were obtained from Peptide Institute Inc. (Osaka, Japan). Bovine spleen cathepsin D was obtained from Sigma Chemical Co.. Protein assay: Protein concentration was determined by Bio-Rad protein assay kit (Bio-Rad) using gamma globulin as a,standard. ECE assay: ECE activity was assayed by measuring ET-l generated from porcine big ET-l with an ET C-terminal specific radioimmunoassay. 330 l.~l of 0.1 M sodium acetate buffer (pH 5.5) containing various amounts of the enzyme (or samples) was mixed with 10 l.~l of 0.35 M CaC12 and 10 p,l of 10 PM porcine big ET-l. After incubation at 37’C for 6 h, the reaction mixture was boiled for 5 min to stop the enzyme reaction. ET C-terminal specific radioimmunoassay using As-ETC was described previously (13). For protease inhibitor study, specified concentrations of the inhibitors (see Table 2) were added to the reaction buffers without CaC12 and the reaction mixtures were incubated and assayed as described above. Purification of ECE Bovine adrenal glands were taken out immediately after killing and stored at 4’C until used (l-3 h). The medullas (550 g) were dissected out from 200 glands. Chromaffin granules were prepared by Smith and Wiier’s method (15). Chromaffm granules were suspended in 5 mM Tris-HCl buffer (pH 7.4), and lysed by freeze-thawing three times. After centrifugation at 100,OOOgfor 60 min, ammonium sulfate was directly added to the supernatant to a final concentration of 80% saturation. The resulting precipitate was dissolved in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and dialysed against the same buffer. The dialysate was applied to a column of Toyopearl DEAE 650s (22 x 200 mm) equilibrated with the same buffer. The enzyme activity was eluted by NaCl up to 0.5 M with a 95 min linear gradient at a flow rate of 4 ml/mm. To ECE active fractions (fr. 15-22), ammonium sulfate was directly added to a final concentration of 1 M. After removing the precipitate by filtrating with 0.22 pm pore size (Minisart NML, Sartorius), the solution was applied to a column of Toyopearl butyl650M (22 x 200 mm) equilibrated with 1 M ammonium sulfate in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA. ECE activity was eluted by reducing ammonium sulfate concentration to 0 M with a 60 min linear gradient at a flow rate of 4 mlfmin. The ECE active fractions in this step were used for inhibitor study. The same fractions were further separated by a gel filtration HPLC column (TSKgel G3000swxl7.8 x 300 mm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 M sodium sulfate and 0.05% sodium azide at a flow rate of 1 ml/mm. To calibrate the molecular weight, MW-Marker (HPLC) (Oriental Yeast Co., Ltd.) was applied and eluted with the same condition. All chromatographies were performed at 4’C. SDS-oolvacr&mide ael electroohoresis: SDS-polyacrylamide (12.5%) gel electrophoresis was carried out according to Laemmli (16). Before SDS gel electrophoresis, the protein sam les were dissolved inTrisHCl buffer containing 0.1% SDS and 4% 2-mercaptoethanol, and heated at 100 B C for 3 min. The gel were stained according to Oakley’s silver stain method. Analysis of ECE activitv of catheosin D: 1 nmole of porcine big ET-l was digested by 1.56 kg of cathepsin D for 2 h in the same buffers described in Fig. 3. After stopping reaction, the reaction mixture was applied to a column of Cosmosil5C18 (4.6 x 250 mm) and eluted by a gradient of acetonitrile: from 15% to 30% over 30 mm and then 30% isocratic. Each amount of big ET-l, ET-l(l-21) or ET-1(22-39) was estimated by the area of a peak with the identical retention time with standard big ET-l, ET-1(1-21) or ET-1(22-39), respectively. RESULTS AND DISCUSSION We chose adrenal medulla to purify and characterize
ECE as a starting material because adrenal
medulla is one of the organs producing ET-l and ET-3 (17 , 0. S. unpublished results and M.Y. et al. submitted) and it has been established to isolate chromaftin granules which are expected to contain ECE.
1231
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4 D.25; ?O., P N uu0.25 c 0 9 o-
_________--
---
z”
____---0
---------
~Pudi~cmofEfmmbovinea~
duomafhgan~
(a) DEAE anion exchange chromatography of the supernatant fraction of the adrenal chromaffin granule lyxateby Toyopearl DJZAE 650s. The column was preequilibrated with 1 mM BDTA in 5 n&4 TrisHCI (pH 7.4). Elutions were carried out by a gradient of NaCl in 5 r&i Tris-HCI (pH 7.4) and 1 mM EDTA indicated by a broken line at a flow rate of 4 mUmin. (b) Hydrophobic chromatography of the ECE active fractions by Toyopearl butyl 650M. The column was pre-equilibrated with 1 M ammonium sulfate in 5 mM Tris-HCI (pH 7.4) and 1 n&l EDTA. Elutions were carried out by a decreasing gradient of ammonium sulfate indicated by a broken line at a flow rate of 4 mbin. To measure ECE activity, we used the following method. Porcine big ET-1 was used as a substrate and ET-1 generated by ECE was measured by an ET C-terminal specific radioimmunoassay
using As-ETC.
The
specificity of the antisera has been confirmed in our previous report that AS-ETC does not crossreact with big ET-1 (13). The minimum detection limit of ET-1 was about 50 fmole/tube. The chromaffin granules, prepared from bovine adrenal medullas, were lysed by three times of freezethawing and the lysate was separated into soluble and membrane fractions by ultracentrifugation.
Since the
soluble fraction contained approximately 80% of total ECE activity in the chromaffin granules, we used it for further purification.
The soluble fraction was concentrated by ammonium sulfate precipitation
and the pre-
cipitate was applied to a column of Toyopearl DEAE 650s after dialysis. Elution was performed by a gradient of NaCl at
(Fig.
la).
A major
ECE
activity
was eluted
between
20-40
mM
NaCI
and a minor
activity
was eluted
45 mM. By this purification step, a great increase in total ECE activity was observed (Table l), which may
be due to the removal of other proteases.
Table 1. PmiGation of emdathelin comm6gmuymehbovineadrenal~gramdea Step
Chromaffin
Protein
granules
DEAE ion exchange Butyl hydrophobic Gel filtration
Total activity
( ql (pmol/h) 173.Y6 0.704 0.122 0.019
302.6 855.3 460.6 104.3
Specific activity
(-fold)
tpmol/h/mqj 1.75 1214.9 3775.4 5489.5
Purification steps of the enzyme from 550 g bovine adrenal medulla are presented. described in “Materials and Methods”.
1232
Purification
69-i 2157 3139 Details
of procedures are
Vol.
168, No. 3, 1990 A I
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
q CDE vvvv I
6
02
0
Tim~%nin)
3”
0 3
--
--
1
2
3
I
4
PH 5
6
7
0
9
w
Gel~~~~p~oftheECEactive~a~byamlmrmofTSggelG300aFwd The column were equilibrated and &ted at a flow rate of 0.5 m&n with 0.1 M sodium phosphate (pH 7.4) containing 0.1 M sodium sulfate and 0.05% sodium azide. Arrows indicate the elution points of molecuter weight standar&,A:ghrtamate dehydrogenase290 kD, B: lactate dehydrogenase 142 kD, C: enolase 67 IQ D: adenylate kinase 32 kD, E: cytochrome c 12.4 kD. w
pH dependency of EXE xtivity. The pH dependence of ECE was determined by using the following buffers: 0.1 M glycine-HC1 for pH 2.0, 3.0 and 35, 0.1 M sodium acetate for pH 4.0, 4.5, 5.0 and 5.5; 0.1 M potassium phosphate for pH 6.0 and 7.0.0.1 M Tris-HCl for pH 8.0 and 9.0. The incubations were carried out as described in “Materials and methods”.
Major active fractions (fr. 15-22) were further separated by Toyopearl butyl650M eluted by reducing the concentration
and the column was
of ammonium sulfate (Fig. lb). The ECE activity was eluted at almost
no ammonium sulfate in the buffer. The active fractions were further separated by a gel filtration column of TSKgcl G3000swxI. The molecular weight of ECE was estimated to be 30K dalton by this chromatography (Fig. 2). The summary of the purification steps was shown in Table 1. As shown in Fig. 3, ECE showed a bimodal pH dependency pattern with a major pH optimum at 3.5 and a minor pH optimum at 4.5 in its enzymatic activity, suggesting that ECE may be an acid protease. As shown in Table 2, pepstatin, an aspartic protease inhibitor, concentration
of 1 rig/ml. Inhibitors
showed a strong inhibitory
effect even at the
of other types of category for proteases, E-64, phosphoramidon
and
PMSF did not show marked inhibitory effects at the concentrations tested up to 50 nmol/ml, 50 p&ml and 50 @ml, respectively. EDTA also did not show an inhibitory effect. The acidic pH optimum and the inhibitory effect of pepstatin on the purified enzyme prompted us to compare it with a well-known
aspartic protease, cathepsin D. The purified ECE and the commercial bovine
1233
BIOCHEMICAL
Vol. 168, No. 3, 1990
Table 2 El&h
Inhibitors
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of protease inhibitors
on ECE activity
Concentration
Activity
pmol/h E-64
1.87 2.37 2.32 2.13 2.27
5 MM 50 pM
Phosphoramidon
5 w/ml 50 l.tg/ml 5 t.tg/ml 50 pg/ml 100 pg/ml 1 rig/ml 10 rig/ml 3mM
PMSF Pepstatin EDTA Control
3.11 1.81 0.02 0.07 1.26
1.49
spleen cathepsiu D were applied to SDS polyacryhuuide gel electrophoresis.
ECE contained the same protein
components with the molecular weights 45,000,30,000 and 15,000 as cathepsin D (Fig. 4). Reverse phase HPLC analyses of the cathepsin D digests at various pHs showed that big ET-l was cleaved into ET-1(1-21) and ET-1(22-39), and the pH optimum of cathepsiu D was at pH 3.5 and very similar to that of ECE (Pig. 5). These data strongly suggest that ECE is au aspartic protease and identical or very similar to cathepsiu D. The fact that the purified enzyme has a bimodal pH optimum was observed. However, the similar bimodal pH dependencies were also reported in the case of cathepsiu D; porcine spleen cathepsin D (pH 3.4 and 3.8) (17) human and chicken liver cathepsin D (18) and rat kidney cathepsin D (pH 3.0 and 4.5) (19).
ECE Cath D - 66.2 kDa - 45.0 - 31.0
600 3 !3
4400 5 z! 200
- 21.5 2
3
4
5
PH *
SDS-PAGE for puritied ECE and cat&sin Lane 1: pwiiied ECE. Lane 2 bovine spleen catbepsin D (Sigma).
w
pH~~~m6~~~-lto~-l~~D.
D.
Porcine big ET-I was incubated with catbepsiu D and then applied to revemephase HPLC. The amounts of the peptides were estimated by the peak areas. Diagonal bars, t%d bars and blank bars huticate ET-1(2.2-39),
ET-l(l-21)
and bii ET-l,
respectively.
1234
Vol.
168,
3, 1990
No.
Cathepsin
D
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is known as a major lysosomal proteolytic enzyme and also exists in endosome as reported
recently (20). Existence of cathepsin D-like ECE activity proposes the following two questions. First, is the purified enzyme derived from whether contaminated lysosomedendosomes selves? Secondly, do
or chromaffin granules them-
the lysosomal cathepsins have another function which converts prohormone
hormone,as in the present report on ET,other than the catabolic digestion of intracellular ble that
ECE
is
proteins? It is possi-
derived from lysosomes or endosomes, because the method used in the present study is a
method to isolate chromaffin
preparative
to
granules for large quantity.
However, the evidence has been
accumulating that the typical lysosomal cathepsins (cathepsins B, H, Y. Uchiyama, personal communication) localize in granules other than lysosomes and may exert the different role other than catabolism. Watanabe et al. have recently reported that a lysosomal enzyme cathepsin B colocalized with atrial natriuretic peptides in secretory granules of rat atria1 endocrine cells using double immunostaining showed the colocalization
method (21). Taugner et al.
of cathepsin D with renin in granules of the renin producing cells (22). Therefore,
alternatively, it is possible that the cathepsin D is present with endothelins in chromaffin granules. It should be noted that the puriBed enzyme, cathepsin D, is a major enzyme having the converting activity of big ET in adrenal medulla.
In respect of the converting activity, cathepsin D can be classified into a
group of ECE and seems to have a different role of generating ET from big ET. van Noort et al. recently reported that bovine spleen cathepsin D preferentially cleaves between an aromatic amino acid residue and a hydrophobic amino acid residue by the product analysis of limited digestion of proteins with cathepsin D (23). Its characteristicsare tween
consistent with those of ECE converting from big ETs into ETs through processing be,
Tryptophan and Valine (ET-3 and -2)Asoleucine (ET-3). As examples for different roles other than catabolism, cathepsin D is involved in the conversion from
PTH(l-84)
IO PTH(l-34)
and PTH(l-34)
has specific effects on bone calcium metabolism (24,25). Even if
ECE is cathepsin D itself derived from lysosomes, there is a possibility that bii ET-l is endocytosed into cells, fused with lysosomes and cleaved to ET-l in them. Further studies are needed to characterize where the purilied ECE localizes in adrenal medulla. In endothelial
cells, originally thought to be the oniy source of ET, the mechanism of big ET conver-
sion may have some differences since they do not have stored secretory granules like chromaffin granules of adrenal medulla. It is not known whether a common processing mechanism of big ET among endothelial and non-endothelial
cells exists or not. We are now investigating how big ET conversion is occurring in endothe-
lial cells. We have
observed
that
endothelial
cells also have a cathepsin D-like ECE activity which generates
ET-1 from big ET-1 (TS. et al., in preparation).
1235
Vol.
168,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by grants from University of Tsukuba Special Project Research on Metabolism, the Ministries of Education, Science and Culture of Japan and Japan Ciba Geigy Foundation.
REFERENCES
1. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature, 332,411-415. 2. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Ma&i, T. (1989) Proc. Natl. Acad. Sci. USA, 86,2863-2867. 3. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T.X., Sakakibara, S., Goto, K., and Ma&i, T. (1988) Proc. Natl. Acad. Sci. USA 85,6964-6967 4. Saida, K., Mitsui, Y., and I&da, N. (1989) J. Bioi. Chem. 264,14613-14616 5. Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A., Ishida, N., Mitsui, Y., Onda, H., Fujino, M. and Ma&i, T. (1988) FEBS Lett. 231,440-444. 6. Sawamura, T., Kimura, S., Shinmi, O., Sugita, Y., Yanagisawa, M. and Masaki, T. (1989) Biochem. Biophys. Res. Commun., 162,1287-1294. 7. 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. 8. Kimura, S., Kasuya, Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K. and Masaki, T. (1989) J. Cardiovasc. Pharmacol. 13 (Suppl. 5), S5-S8. 9. Kashiwabara, T., Inagaki, Y., Ohta, H., Iwamatsu, A., Nom&u, M., Morita, A., and Nishikori, K. (1989) FEBS Lett. 247, 73-76 10. Yoshizawa, T., Shinmi, O., Giaid, A., Yanagisawa, M., Gibson, S. J., Kimura, S., Uchiyama, Y., Polak, J. M., Masaki, T. and Kanazawa, I. (1990) Science 247,462~464. 11. Miyauchi, T., Yanagisawa, M., Tomizawa, T., Sugishita, Y., Suzuki, N., Fujino, M., Ajisaka, R., Goto, K. and Masaki, T. (1989) Lancet ii, 53-54. 12. Suzuki, N., Matsumoto, H., Kitada, C., Kimura, S., Miyauchi, T., and Fujino, M. (1990) J. Immunol. Methods, in press. 13. Shinmi, O., Kimura, S., Yoshizawa, T., Sawamura, T., Uchiyama, Y., Sugita, Y., Kanazawa, I., Yanagisawa, M., Goto, K. and Ma&i, T. (1989) Biochem. Biophys. Res. Commun., 162,340-346. 14. Shinmi, O., Kimura, S., Sawamura, T., Sugita, Y., Yoshizawa, T., Uchiyama, Y., Yanagisawa, M., Goto, K., Masaki, T. and Kanazawa, I. (1989) Biochem. Biophys. Res. Commun., 164,587-593 15. Smith, A. D. and Wiier, H. (1967) Biochem. J. 103,480~482. 16. Laemmh, U. K. (1970) Nature, 227,680~685. 17. Cunningham, M. and Tang, J. (1976) J. Biol. Chem., 251,4528-4534. 18. Barret, A. J. (1971) in Tissue Proteinases (Barret, A. J. and DingIe, J. T., Eds.) pp 109-133, North-Holland Publishing Co., Amsterdam. 19. Figueiredo, A. F. S., Takii, Y., Tsuji, H., Kato, K. and Inagami, T. (1983) Biochemistry, 22, 5476-5481. 20. Diment, S., Leech, M. S. and Stahl, P. D. (1988) J. Biol. Chem., 263,6901-6907. 21. Watanabe, T., Watanabe, M., I&ii, Y., Matsuba, H., Kimura, S., Fujita, T., Kominami, E., Katsunuma, N. and Uchiyama, Y. (1989) J. Histochem. Cytochem., 37,347-351. 22. Taugner, R., Yokota, S., Buhrle, C.P. and Hackenthal, E. (1986) Hiitochemistry 84,19-22. 23. van Noort, J. M. and van der Drift, A. C. M. (1989) J. Biol. Chem., 264, 14159-14164. 24. Martin, K. J., Hruska, K. A., Freitag, J. J., KIahr, S. and Slatopolsky, E. (1979) New End. J. Med., 301, 1092-1018. 25. Pillai, S., Botti, R. Jr. and Zull, J. E. (1983) J. Biol. Chem., 258,9724-9728.
1236
Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1230-1236
PURIFICATION AND CHARACTERIZATION OF PUTATWE ENDOTHELIN CONVERTING ENZYME IN BOVINE ADRENAL MEDULLAi EVIDENCE FOR A CATHEPSIN D-LIKE ENZYME Tatsuya Sawamura, Sadp Kimura ‘, 0-2 Masashi Yanagisawa , Katsutoshi Goto
Shkuni, Yoshild Su$ta, and Tomoh Masald
Department of Biochemistry and #Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsuhuba, Tsukuba, Ibarald 305, Japan
Received
April
10,
1990
SUMMARY: A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules. ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,080,30,000 and 15,088 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease. 0 1990
Academic
Prass,
Endothelin endothelial
Inc.
(ET) is a novel vasoconstrictor peptide purified from a culture medium of porcine aortic
cells (1). From the analyses of the genomic libraries of human, rat and mouse, it was found that
there are three isopeptides of ET,
named ET-l, ET-Z (VIC) and ET-3 (2-4). By the analysis of cDNA of
ET-l, it was suggested that ET-I is generated from a 39-(porcine) called big endothelin-1
(big ET-l)
by unusual processing at the Trp-Val
dibasic pairs in approximately UK)-residue preproendothelin boxy1 terminal peptide
ET-1(22-39)
medium of porcine aortic endothelial
or a 38-(human)
residue intermediate
bond after processing at positions of
(1,5). In fact, bii ET-l (ET-1(1-39))
beginning with Val in addition to ET-1(1-21)
and its car-
were found in a culture
cells (6). Big ET-1 is considerably less active than mature ET-l on the
isolated porcine coronary artery and the conversion from big ET-l to ET-l increases the contractile activity about two orders of magnitude (7-9). Although it is still uncertain whether vessel endothelium source producing ET-l and big ET-l in circulating blood (see the hypothalamus-pituitary
system in Ref. 10)
and where the conversion from big ET-1 to ET-l occurs (inside or outside of ET-l producing concentration
Copyright AU rights
cells), the
of big ET-l was found to be approximately equimolar compared to that of ET-1 in circulating
’ To whom all correspondence should be addressed. 0006-291X/90
is the only
$1.58
Q 1990 by Academic Press, Inc. of reproduction in any form reserved.
1230
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
blood (11,X!). On the other hand, as for intracellular ET related peptides, ET-1 from porcine spinal cord and ET-l and ET-3 from porcine brain were isolated but big ET-I was very scarceIy identified in both tissues (l3,14). Until now, no one shows the characteristics of ECE which specifically cleaves between Tr~~l-Val~. Here we report the assay system for ECE activity, and purification and characterization
of an aspartic
protease from bovine adrenal medulla which has ECE activity. MATERIALS
AND METHODS
Chemicals: Porcine big ET-l, ET-l, leupeptin, phosphoramidon, E-64, pepstatin and phenyhnethylsulfonylfluoride (PMSF) were obtained from Peptide Institute Inc. (Osaka, Japan). Bovine spleen cathepsin D was obtained from Sigma Chemical Co.. Protein assay: Protein concentration was determined by Bio-Rad protein assay kit (Bio-Rad) using gamma globulin as a,standard. ECE assay: ECE activity was assayed by measuring ET-l generated from porcine big ET-l with an ET C-terminal specific radioimmunoassay. 330 l.~l of 0.1 M sodium acetate buffer (pH 5.5) containing various amounts of the enzyme (or samples) was mixed with 10 l.~l of 0.35 M CaC12 and 10 p,l of 10 PM porcine big ET-l. After incubation at 37’C for 6 h, the reaction mixture was boiled for 5 min to stop the enzyme reaction. ET C-terminal specific radioimmunoassay using As-ETC was described previously (13). For protease inhibitor study, specified concentrations of the inhibitors (see Table 2) were added to the reaction buffers without CaC12 and the reaction mixtures were incubated and assayed as described above. Purification of ECE Bovine adrenal glands were taken out immediately after killing and stored at 4’C until used (l-3 h). The medullas (550 g) were dissected out from 200 glands. Chromaffin granules were prepared by Smith and Wiier’s method (15). Chromaffm granules were suspended in 5 mM Tris-HCl buffer (pH 7.4), and lysed by freeze-thawing three times. After centrifugation at 100,OOOgfor 60 min, ammonium sulfate was directly added to the supernatant to a final concentration of 80% saturation. The resulting precipitate was dissolved in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and dialysed against the same buffer. The dialysate was applied to a column of Toyopearl DEAE 650s (22 x 200 mm) equilibrated with the same buffer. The enzyme activity was eluted by NaCl up to 0.5 M with a 95 min linear gradient at a flow rate of 4 ml/mm. To ECE active fractions (fr. 15-22), ammonium sulfate was directly added to a final concentration of 1 M. After removing the precipitate by filtrating with 0.22 pm pore size (Minisart NML, Sartorius), the solution was applied to a column of Toyopearl butyl650M (22 x 200 mm) equilibrated with 1 M ammonium sulfate in 5 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA. ECE activity was eluted by reducing ammonium sulfate concentration to 0 M with a 60 min linear gradient at a flow rate of 4 mlfmin. The ECE active fractions in this step were used for inhibitor study. The same fractions were further separated by a gel filtration HPLC column (TSKgel G3000swxl7.8 x 300 mm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 M sodium sulfate and 0.05% sodium azide at a flow rate of 1 ml/mm. To calibrate the molecular weight, MW-Marker (HPLC) (Oriental Yeast Co., Ltd.) was applied and eluted with the same condition. All chromatographies were performed at 4’C. SDS-oolvacr&mide ael electroohoresis: SDS-polyacrylamide (12.5%) gel electrophoresis was carried out according to Laemmli (16). Before SDS gel electrophoresis, the protein sam les were dissolved inTrisHCl buffer containing 0.1% SDS and 4% 2-mercaptoethanol, and heated at 100 B C for 3 min. The gel were stained according to Oakley’s silver stain method. Analysis of ECE activitv of catheosin D: 1 nmole of porcine big ET-l was digested by 1.56 kg of cathepsin D for 2 h in the same buffers described in Fig. 3. After stopping reaction, the reaction mixture was applied to a column of Cosmosil5C18 (4.6 x 250 mm) and eluted by a gradient of acetonitrile: from 15% to 30% over 30 mm and then 30% isocratic. Each amount of big ET-l, ET-l(l-21) or ET-1(22-39) was estimated by the area of a peak with the identical retention time with standard big ET-l, ET-1(1-21) or ET-1(22-39), respectively. RESULTS AND DISCUSSION We chose adrenal medulla to purify and characterize
ECE as a starting material because adrenal
medulla is one of the organs producing ET-l and ET-3 (17 , 0. S. unpublished results and M.Y. et al. submitted) and it has been established to isolate chromaftin granules which are expected to contain ECE.
1231
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4 D.25; ?O., P N uu0.25 c 0 9 o-
_________--
---
z”
____---0
---------
~Pudi~cmofEfmmbovinea~
duomafhgan~
(a) DEAE anion exchange chromatography of the supernatant fraction of the adrenal chromaffin granule lyxateby Toyopearl DJZAE 650s. The column was preequilibrated with 1 mM BDTA in 5 n&4 TrisHCI (pH 7.4). Elutions were carried out by a gradient of NaCl in 5 r&i Tris-HCI (pH 7.4) and 1 mM EDTA indicated by a broken line at a flow rate of 4 mUmin. (b) Hydrophobic chromatography of the ECE active fractions by Toyopearl butyl 650M. The column was pre-equilibrated with 1 M ammonium sulfate in 5 mM Tris-HCI (pH 7.4) and 1 n&l EDTA. Elutions were carried out by a decreasing gradient of ammonium sulfate indicated by a broken line at a flow rate of 4 mbin. To measure ECE activity, we used the following method. Porcine big ET-1 was used as a substrate and ET-1 generated by ECE was measured by an ET C-terminal specific radioimmunoassay
using As-ETC.
The
specificity of the antisera has been confirmed in our previous report that AS-ETC does not crossreact with big ET-1 (13). The minimum detection limit of ET-1 was about 50 fmole/tube. The chromaffin granules, prepared from bovine adrenal medullas, were lysed by three times of freezethawing and the lysate was separated into soluble and membrane fractions by ultracentrifugation.
Since the
soluble fraction contained approximately 80% of total ECE activity in the chromaffin granules, we used it for further purification.
The soluble fraction was concentrated by ammonium sulfate precipitation
and the pre-
cipitate was applied to a column of Toyopearl DEAE 650s after dialysis. Elution was performed by a gradient of NaCl at
(Fig.
la).
A major
ECE
activity
was eluted
between
20-40
mM
NaCI
and a minor
activity
was eluted
45 mM. By this purification step, a great increase in total ECE activity was observed (Table l), which may
be due to the removal of other proteases.
Table 1. PmiGation of emdathelin comm6gmuymehbovineadrenal~gramdea Step
Chromaffin
Protein
granules
DEAE ion exchange Butyl hydrophobic Gel filtration
Total activity
( ql (pmol/h) 173.Y6 0.704 0.122 0.019
302.6 855.3 460.6 104.3
Specific activity
(-fold)
tpmol/h/mqj 1.75 1214.9 3775.4 5489.5
Purification steps of the enzyme from 550 g bovine adrenal medulla are presented. described in “Materials and Methods”.
1232
Purification
69-i 2157 3139 Details
of procedures are
Vol.
168, No. 3, 1990 A I
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
q CDE vvvv I
6
02
0
Tim~%nin)
3”
0 3
--
--
1
2
3
I
4
PH 5
6
7
0
9
w
Gel~~~~p~oftheECEactive~a~byamlmrmofTSggelG300aFwd The column were equilibrated and &ted at a flow rate of 0.5 m&n with 0.1 M sodium phosphate (pH 7.4) containing 0.1 M sodium sulfate and 0.05% sodium azide. Arrows indicate the elution points of molecuter weight standar&,A:ghrtamate dehydrogenase290 kD, B: lactate dehydrogenase 142 kD, C: enolase 67 IQ D: adenylate kinase 32 kD, E: cytochrome c 12.4 kD. w
pH dependency of EXE xtivity. The pH dependence of ECE was determined by using the following buffers: 0.1 M glycine-HC1 for pH 2.0, 3.0 and 35, 0.1 M sodium acetate for pH 4.0, 4.5, 5.0 and 5.5; 0.1 M potassium phosphate for pH 6.0 and 7.0.0.1 M Tris-HCl for pH 8.0 and 9.0. The incubations were carried out as described in “Materials and methods”.
Major active fractions (fr. 15-22) were further separated by Toyopearl butyl650M eluted by reducing the concentration
and the column was
of ammonium sulfate (Fig. lb). The ECE activity was eluted at almost
no ammonium sulfate in the buffer. The active fractions were further separated by a gel filtration column of TSKgcl G3000swxI. The molecular weight of ECE was estimated to be 30K dalton by this chromatography (Fig. 2). The summary of the purification steps was shown in Table 1. As shown in Fig. 3, ECE showed a bimodal pH dependency pattern with a major pH optimum at 3.5 and a minor pH optimum at 4.5 in its enzymatic activity, suggesting that ECE may be an acid protease. As shown in Table 2, pepstatin, an aspartic protease inhibitor, concentration
of 1 rig/ml. Inhibitors
showed a strong inhibitory
effect even at the
of other types of category for proteases, E-64, phosphoramidon
and
PMSF did not show marked inhibitory effects at the concentrations tested up to 50 nmol/ml, 50 p&ml and 50 @ml, respectively. EDTA also did not show an inhibitory effect. The acidic pH optimum and the inhibitory effect of pepstatin on the purified enzyme prompted us to compare it with a well-known
aspartic protease, cathepsin D. The purified ECE and the commercial bovine
1233
BIOCHEMICAL
Vol. 168, No. 3, 1990
Table 2 El&h
Inhibitors
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of protease inhibitors
on ECE activity
Concentration
Activity
pmol/h E-64
1.87 2.37 2.32 2.13 2.27
5 MM 50 pM
Phosphoramidon
5 w/ml 50 l.tg/ml 5 t.tg/ml 50 pg/ml 100 pg/ml 1 rig/ml 10 rig/ml 3mM
PMSF Pepstatin EDTA Control
3.11 1.81 0.02 0.07 1.26
1.49
spleen cathepsiu D were applied to SDS polyacryhuuide gel electrophoresis.
ECE contained the same protein
components with the molecular weights 45,000,30,000 and 15,000 as cathepsin D (Fig. 4). Reverse phase HPLC analyses of the cathepsin D digests at various pHs showed that big ET-l was cleaved into ET-1(1-21) and ET-1(22-39), and the pH optimum of cathepsiu D was at pH 3.5 and very similar to that of ECE (Pig. 5). These data strongly suggest that ECE is au aspartic protease and identical or very similar to cathepsiu D. The fact that the purified enzyme has a bimodal pH optimum was observed. However, the similar bimodal pH dependencies were also reported in the case of cathepsiu D; porcine spleen cathepsin D (pH 3.4 and 3.8) (17) human and chicken liver cathepsin D (18) and rat kidney cathepsin D (pH 3.0 and 4.5) (19).
ECE Cath D - 66.2 kDa - 45.0 - 31.0
600 3 !3
4400 5 z! 200
- 21.5 2
3
4
5
PH *
SDS-PAGE for puritied ECE and cat&sin Lane 1: pwiiied ECE. Lane 2 bovine spleen catbepsin D (Sigma).
w
pH~~~m6~~~-lto~-l~~D.
D.
Porcine big ET-I was incubated with catbepsiu D and then applied to revemephase HPLC. The amounts of the peptides were estimated by the peak areas. Diagonal bars, t%d bars and blank bars huticate ET-1(2.2-39),
ET-l(l-21)
and bii ET-l,
respectively.
1234
Vol.
168,
3, 1990
No.
Cathepsin
D
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is known as a major lysosomal proteolytic enzyme and also exists in endosome as reported
recently (20). Existence of cathepsin D-like ECE activity proposes the following two questions. First, is the purified enzyme derived from whether contaminated lysosomedendosomes selves? Secondly, do
or chromaffin granules them-
the lysosomal cathepsins have another function which converts prohormone
hormone,as in the present report on ET,other than the catabolic digestion of intracellular ble that
ECE
is
proteins? It is possi-
derived from lysosomes or endosomes, because the method used in the present study is a
method to isolate chromaffin
preparative
to
granules for large quantity.
However, the evidence has been
accumulating that the typical lysosomal cathepsins (cathepsins B, H, Y. Uchiyama, personal communication) localize in granules other than lysosomes and may exert the different role other than catabolism. Watanabe et al. have recently reported that a lysosomal enzyme cathepsin B colocalized with atrial natriuretic peptides in secretory granules of rat atria1 endocrine cells using double immunostaining showed the colocalization
method (21). Taugner et al.
of cathepsin D with renin in granules of the renin producing cells (22). Therefore,
alternatively, it is possible that the cathepsin D is present with endothelins in chromaffin granules. It should be noted that the puriBed enzyme, cathepsin D, is a major enzyme having the converting activity of big ET in adrenal medulla.
In respect of the converting activity, cathepsin D can be classified into a
group of ECE and seems to have a different role of generating ET from big ET. van Noort et al. recently reported that bovine spleen cathepsin D preferentially cleaves between an aromatic amino acid residue and a hydrophobic amino acid residue by the product analysis of limited digestion of proteins with cathepsin D (23). Its characteristicsare tween
consistent with those of ECE converting from big ETs into ETs through processing be,
Tryptophan and Valine (ET-3 and -2)Asoleucine (ET-3). As examples for different roles other than catabolism, cathepsin D is involved in the conversion from
PTH(l-84)
IO PTH(l-34)
and PTH(l-34)
has specific effects on bone calcium metabolism (24,25). Even if
ECE is cathepsin D itself derived from lysosomes, there is a possibility that bii ET-l is endocytosed into cells, fused with lysosomes and cleaved to ET-l in them. Further studies are needed to characterize where the purilied ECE localizes in adrenal medulla. In endothelial
cells, originally thought to be the oniy source of ET, the mechanism of big ET conver-
sion may have some differences since they do not have stored secretory granules like chromaffin granules of adrenal medulla. It is not known whether a common processing mechanism of big ET among endothelial and non-endothelial
cells exists or not. We are now investigating how big ET conversion is occurring in endothe-
lial cells. We have
observed
that
endothelial
cells also have a cathepsin D-like ECE activity which generates
ET-1 from big ET-1 (TS. et al., in preparation).
1235
Vol.
168,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by grants from University of Tsukuba Special Project Research on Metabolism, the Ministries of Education, Science and Culture of Japan and Japan Ciba Geigy Foundation.
REFERENCES
1. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature, 332,411-415. 2. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Ma&i, T. (1989) Proc. Natl. Acad. Sci. USA, 86,2863-2867. 3. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T.X., Sakakibara, S., Goto, K., and Ma&i, T. (1988) Proc. Natl. Acad. Sci. USA 85,6964-6967 4. Saida, K., Mitsui, Y., and I&da, N. (1989) J. Bioi. Chem. 264,14613-14616 5. Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A., Ishida, N., Mitsui, Y., Onda, H., Fujino, M. and Ma&i, T. (1988) FEBS Lett. 231,440-444. 6. Sawamura, T., Kimura, S., Shinmi, O., Sugita, Y., Yanagisawa, M. and Masaki, T. (1989) Biochem. Biophys. Res. Commun., 162,1287-1294. 7. 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. 8. Kimura, S., Kasuya, Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K. and Masaki, T. (1989) J. Cardiovasc. Pharmacol. 13 (Suppl. 5), S5-S8. 9. Kashiwabara, T., Inagaki, Y., Ohta, H., Iwamatsu, A., Nom&u, M., Morita, A., and Nishikori, K. (1989) FEBS Lett. 247, 73-76 10. Yoshizawa, T., Shinmi, O., Giaid, A., Yanagisawa, M., Gibson, S. J., Kimura, S., Uchiyama, Y., Polak, J. M., Masaki, T. and Kanazawa, I. (1990) Science 247,462~464. 11. Miyauchi, T., Yanagisawa, M., Tomizawa, T., Sugishita, Y., Suzuki, N., Fujino, M., Ajisaka, R., Goto, K. and Masaki, T. (1989) Lancet ii, 53-54. 12. Suzuki, N., Matsumoto, H., Kitada, C., Kimura, S., Miyauchi, T., and Fujino, M. (1990) J. Immunol. Methods, in press. 13. Shinmi, O., Kimura, S., Yoshizawa, T., Sawamura, T., Uchiyama, Y., Sugita, Y., Kanazawa, I., Yanagisawa, M., Goto, K. and Ma&i, T. (1989) Biochem. Biophys. Res. Commun., 162,340-346. 14. Shinmi, O., Kimura, S., Sawamura, T., Sugita, Y., Yoshizawa, T., Uchiyama, Y., Yanagisawa, M., Goto, K., Masaki, T. and Kanazawa, I. (1989) Biochem. Biophys. Res. Commun., 164,587-593 15. Smith, A. D. and Wiier, H. (1967) Biochem. J. 103,480~482. 16. Laemmh, U. K. (1970) Nature, 227,680~685. 17. Cunningham, M. and Tang, J. (1976) J. Biol. Chem., 251,4528-4534. 18. Barret, A. J. (1971) in Tissue Proteinases (Barret, A. J. and DingIe, J. T., Eds.) pp 109-133, North-Holland Publishing Co., Amsterdam. 19. Figueiredo, A. F. S., Takii, Y., Tsuji, H., Kato, K. and Inagami, T. (1983) Biochemistry, 22, 5476-5481. 20. Diment, S., Leech, M. S. and Stahl, P. D. (1988) J. Biol. Chem., 263,6901-6907. 21. Watanabe, T., Watanabe, M., I&ii, Y., Matsuba, H., Kimura, S., Fujita, T., Kominami, E., Katsunuma, N. and Uchiyama, Y. (1989) J. Histochem. Cytochem., 37,347-351. 22. Taugner, R., Yokota, S., Buhrle, C.P. and Hackenthal, E. (1986) Hiitochemistry 84,19-22. 23. van Noort, J. M. and van der Drift, A. C. M. (1989) J. Biol. Chem., 264, 14159-14164. 24. Martin, K. J., Hruska, K. A., Freitag, J. J., KIahr, S. and Slatopolsky, E. (1979) New End. J. Med., 301, 1092-1018. 25. Pillai, S., Botti, R. Jr. and Zull, J. E. (1983) J. Biol. Chem., 258,9724-9728.
1236
