Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

ANALYTICAL BIOCHEMISTRY 140, 520-53 1 (1984)

Purification of a Human Urinary Carboxypeptidase (Kininase) Distinct from Carboxypeptidases A, B, or N’ RANDAL

A. SIUDGEL, RICHARD

M. DAVIS, AND ERVIN G. ERD~S

Departments of Pharmacology and Internal Medicine, The University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235 Received January 16, 1984 A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, a&tine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSKG3OOOSW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 rmol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of M, 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the M, 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a K, of 46 gM and a K,, of 32 min-‘. Its activity was enhanced by CoClr and inhibited by cadmium acetate, o-phenanthroline, or DL-2mercaptomethyl-3guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37°C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the “Western blot” technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid. KEY WORDS:protein/enzyme purification; peptide hormones; proteases; kinins; HPLC, proteins; urinary enzymes.

Bradykinin, kallidin, and Met-Lys-bradykinin are excreted in human urine (l), although the presence of Met-Lys-bradykinin in urine under physiological conditions has been questioned (2). Kinins release prostaglandins and may affect sodium and water metabolism and renal vascular resistance (3). Kinins are vulnerable to enzymatic cleavage. For example, bradykinin, a nonapeptide, is inactivated by the hydrolysis of any one of its eight peptide bonds and kallidin can be con-

vetted to bradykinin by the removal of its Nterminal lysine (4,5). A peptidyl dipeptidase-type kininase (kininase II or angiotensin I converting enzyme) is highly concentrated on the brush border of the proximal tubules (6) but its level in urine is low (7,8). A carboxypeptidase-type kininase is also present in the kidney (8) and in urine (9), but the relationship of this enzyme to plasma carboxypeptidase N (kininase I) has not been explored. Kininases in urine, through the inactivation of kinins, may play an important role in the control of salt and water balance. We therefore decided to purify and characterize the urinary carboxypeptidase-type

’ This work was supported by Grants HL 16320, HL 20594, and HL 28813 from the National Institutes of Health (NHLBI) and by a Grant-In-Aid (82782) from the American Heart Association. 0003-2697/84 $3.00 Copyright Q 1984 by Academic PRS, Inc. All rights of reproduction in any fOtm re~~‘~ed.

520

PURIFICATION

OF HUMAN

URINARY

kininase which cleaves basic C-terminal amino acids. Because carboxypeptidase N of blood plasma is an enzyme of M, 280,000 consisting of two I%&48,000 and two it& 83,000 subunits (lo), it would not be filtered intact through the glomerulus. We therefore also investigated the possible relationship of the urinary enzyme to the active M, 48,000 subunit of carboxypeptidase N and to human pancreatic carboxypeptidase B (Mr 34,000) (11). We found that the homogeneous urinary carboxypep tidase is different from the plasma enzyme, its M, 48,000 subunit, and pancreatic carboxypeptidase B. MATERIALS

AND METHODS

Benzoyl-alanyl-lysine (Bz-Ala-Lys)2, BzGly-argininic acid, Bz-Phe-Lys, and guanidinoethylmercaptosuccinic acid (GEMSA) were kindly provided by Dr. Yehuda Levin of the Weizmann Institute of Science, Rehovot, Israel. Aprotinin was donated by Dr. G. Haberland of Bayer AG. Sephacryl S-300 and Sepharose 6B were purchased from Pharmacia Fine Chemicals, Piscataway, N. J. and Affi-Gel Blue was from Bio-Rad, Richmond, Calif. L-Arginine-Sepharose was prepared from epichlorohydrin-activated Sepharose 6B ( 12). Bradykinin, pchloromercuriphenylsulfonate (PCMS), phenylmethylsulfonylfluoride (PMSF), and Bz-Gly-Lys were purchased from Sigma Chemical Company, St. Louis, MO. DL - 2 - Mercaptomethyl - 3 - guanidinoethylthiopropanoic acid (MGTA) was from Calbiochem-Behring, La Jolla, Calif. and Bz-Gly-Arg was from Bachem, Torrance, Calif. Human pancreatic carboxypeptidase B was purified from autopsy samples as reported ( 11). Enzyme assays. Urinary carboxypeptidase activity was routinely measured during pu’ Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonylfluoride; MGTA, DL2-mercaptomethyl-3guanidinoethyhhiopropanoic acid; PCMS, gchloromercuriphenylsulfonate; Bz, benzoyl; GEMSA, guanidinoethylmercaptosuccinic acid.

CARBOXYPEPTIDASE

521

rification with a continuous spectrophotometric assay which monitors the hydrolysis of 1 mM Bz-Gly-argininic acid (the ester substrate) at 254 nm in 0.1 M Tris-HCI buffer, pH 8.5, at 37°C (13,14). The esterase activities of carboxypeptidase N, its M, 48,000 subunit, and pancreatic carboxypeptidase B were also measured in this manner. The peptidase activities of the various carboxypeptidases (using Bz-Ala-Lys or Bz-Phe-Lys as substrates) were similarly measured in the spectrophotometer except the buffer was 0.1 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), pH 7.0 (10,13,14). Carboxypeptidase activity was also assayed using Bz-Gly-Arg or Bz-Gly-Lys as substrate by extracting and spectrophotometrically measuring the Bz-Gly released. This assay is a modification of the method of Koheil and Forstner ( 15) and has recently been described in detail (16). Briefly, enzyme sample (up to 100 pl), CoC12 (20 ~1 of 10 mM), and inhibitor (if any), in 0.1 M Tris-HCl, pH 7.5 (final volume, 200 pl), were preincubated for 2 h at 4°C. The reaction was initiated by the addition of 50 ~1 of 25 mM Bz-Gly-Lys or Bz-GlyArg (final concentration = 5 mM) and the samples were incubated at 37°C for 2 h. The reaction was terminated by the addition of 250 ~1 of 1 N HCl. The product, Bz-Gly, was extracted by vigorously mixing the reaction sample with 1.5 ml ethyl acetate followed by centrifugation (500g for 5 min) to separate the phases. A l.O-ml aliquot of the ethyl acetate layer was transferred to a clean tube, evaporated, reconstituted in 1 ml H20, and the absorbance at 228 nm measured in a Varian Cary 2 19 uv spectrophotometer. Zerotime-reaction blanks were run for each sample by adding 250 ~1 of 1 N HCl to the tube before the addition of substrate. Reactions were routinely run in the presence and absence of CoC12 since cobalt is known to stimulate the peptidase activity of carboxypeptidases ( 17,18). In a typical assay with pure enzyme, 0.2-0.5 pg of carboxypeptidase N or 0.5-l pg of urinary carboxypeptidase was used. To determine whether urinary carboxypep-

522

SKIDGEL,

DAVIS,

tidase had any carboxypeptidase A-like activity, 0.54 pg of the purified enzyme was incubated at 37°C for 17 h with 1 mM Bz-GlyPhe in 0.05 M Hepes, pH 7.0 (final volume = 0.1 ml). The reaction was stopped with 20 ~1 of 5% trifluoroacetic acid and aliquots of 40 ~1 were analyzed on a Waters gradient HPLC system consisting of a Z-Module with a Radial-Pak PBondapak C, s cartridge (8 X 100 mm), an M-6000A pump, an M-45 pump, a WISP 710B automatic injector, a Model 720 system controller, an M730 data module, and a Model 44 1 absorbance detector. The solvents used were H20/0.02% trifluoroacetic acid (solvent A) and CHJCN/0.02% trifluoroacetic acid (solvent B). Products were eluted with an increasing linear gradient of solvent B, starting at 18% B and ending at 48% B in 15 min at a flow rate of 2 ml/min. The column was equilibrated at initial conditions for 8 min before each run. The products were detected at 2 14 nm and, under these conditions, Bz-Gly-Phe eluted at 12.2 min and the expected product, Bz-Gly, at 5.0 min.

AND

ERDiiS

concentrated to 100 ml on an Amicon DC-2 hollow-fiber concentrator equipped with an H 1PlO cartridge ( 10,000 molecular weight cutoff). Low-molecular-weight (< 10,000) material was removed by diluting the concentrate to 1 liter with 0.05 M Tris-HCl, pH 7.2, and concentrating to 100 ml on the hollow-fiber apparatus. The dilution and concentration was repeated and the final concentrate reduced to 45 ml on an Amicon ultrafiltration apparatus with a YM- 10 membrane. The urine concentrate was applied to a column (2.5 X 20 cm) of Affi-Gel Blue (agarose-bound CibacronBlue F3GA) equilibrated with 0.05 M TrisHCl, pH 7.2. The column was washed with 500 ml of equilibration buffer (25 ml/h) to remove weakly bound proteins and the urinary carboxypeptidase eluted with 175 ml of the same buffer containing 0.25 M NaCl. Tightly bound protein (e.g., albumin) was eluted with 200 ml of equilibration buffer containing 1.4 M NaCI. The active fractions were pooled (113 ml) and concentrated in an Amicon ultrafiltration cell with a YM-10 membrane to 7.6 PuriJication of carboxypeptidase N and its ml. The concentrate was dialyzed overnight active subunit. Carboxypeptidase N was pu- against two changes (2 liters each) of 0.05 M rified to homogeneity from outdated human Tris-HCl, pH 7.2, and then applied to a colplasma (obtained from the Parkland Memorial umn (2.5 X 20 cm) of arginine-Sepharose Hospital blood bank) by ion-exchange and L- which had been equilibrated with the dialysis arginine-Sepharose affmity chromatography as buffer. The column was washed with 190 ml previously described (10). The enzyme was of the same buffer (25 ml/h) and the enzyme purified 2665-fold with a 20-30% yield and eluted with 170 ml of buffer containing 2 mM had a specific activity of 144 ~mol/min/mg GEMSA. Contaminant proteins were removed with Bz-Ala-Lys as substrate. Carboxypeptiwith 150 ml of buffer containing 0.5 M NaCl. dase N was treated with 3 M guanidine-HCl The active fractions from the arginine-sepharose affinity column were pooled and dito dissociate the subunits. An inactive, M, 83,000 subunit and an active, M, 48,000 sub- alyzed overnight against 2 changes (4 liters unit were obtained following separation by gel each) of 0.1 M NH4HC03. The dialyzed enfiltration on Sephadex G-75 superfine (10). zyme (46 ml) was concentrated to 0.75 ml in Purification of human urinary carboxypepan Amicon ultrafiltration cell with a YM-10 tidase. Human urine ( 13.5 liters) was collected membrane. The final step in purification was from healthy volunteers into bottles containgel filtration with a 0.75 X 30-cm TSKing 0.1 M NH4HC03 (100 ml/liter urine) and G3000SW column (Varian, Walnut Creek, aprotinin (5000 units/liter urine) over a period Calif.) connected to the HPLC system deof 3 days during which it was stored at 4°C. scribed above. Proteins were separated on the All subsequent procedures were carried out at TSK column at room temperature with 0.07 4°C unless otherwise stated. Urine was filtered M sodium phosphate buffer, pH 7.2, containthrough filter paper (Whatman No. 1) and ing 0.4 M KC1 at a flow rate of 0.3 ml/min

PURIFICATION

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HUMAN

URINARY

and were detected at 2 14 nm. The outlet was connected to a fraction collector and fractions were collected at 0.7-min intervals (0.2 1 ml). Several injections were made and the corresponding fractions collected into the same test tubes. The tubes were kept on ice except during the collection period. The last four fractions of the major protein peak gave the highest specific activity and were pooled for use as the source of the pure urinary carboxypeptidase. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was done in 9% slab gels (14 X 14 X 0.15 cm) according to Laemmli (19). Native polyacrylamide gel electrophoresis was done in a similar fashion except 7% gels were used without SDS or mercaptoethanol in the buffers or the gel. Proteins were detected using the silver-staining procedure of Wray et al. (20). Electroblotting. Proteins were transferred from SDS-polyacrylamide gels to nitrocellulose paper similar to reported procedures (2 1,22) and detected immunologically with the following modifications. Transfer was carried out for 24 h at 30-40 V (180 mA) using a Trans-Blot Cell (Bio-Rad, Richmond, Calif.). The nitrocellulose paper was washed for 30 min at 37°C in 30 ml of 10 mM Tris-HCl, pH 7.4, with 0.15 M NaCl, 5% bovine serum albumin, and 0.2% Nonidet P-40 detergent (buffer A). The nitrocellulose was then incubated for 2.5 h at room temperature in buffer A containing antisera (raised in rabbits) to carboxypeptidase N at a final dilution of 1:2000. The buffer A-antisera mixture was removed and the nitrocellulose washed 3 times (15 min each) with 30 ml of 10 ITIM TrisHCl, pH 7.4, with 0.15 M NaCl, 0.2% Nonidet P-40, 0.1% SDS, and 0.25% sodium deoxycholate (buffer B). The nitrocellulose was quickly rinsed with buffer B, water, and then incubated for 45 min at room temperature with 15 ml of buffer A containing about lo6 cpm/ml ‘251-labeled goat anti-rabbit immunoglobulin G. The radioactive solution was removed, the nitrocellulose paper rinsed with

CARBOXYPEPTIDASE

523

buffer B and then washed 3 times (15 min each) with 30 ml of buffer B. The nitrocellulose was rinsed again with buffer B, water, and then air-dried. It was wrapped in Hand&Wrap and exposed to Kodak X-Omat XAR-2 film at -70°C for l-4 h. Kinetic studies. Kinetic studies of the hydrolysis of bradykinin were carried out by measuring product release in a high-pressure liquid chromatograph. Substrate, in 0.1 M Hepes buffer, pH 7.0, and enzyme were incubated in a final volume of 100 ~1 at 37°C for 15-30 min. The reaction was terminated by adding 20 ~1 of 5% trifluoroacetic acid and aliquots of 40 ~1 were analyzed on the HPLC system described above equipped with a PBondapak Cl8 reverse-phase column (3.9 mm X 30 cm). Products were eluted with an increasing linear gradient of CH3CN/0.05% trifluoroacetic acid (solvent D) in H,0/0.05% trifluoroacetic acid (solvent C) at a flow rate of 1.0 ml/min. The column was equilibrated for 10 min at initial conditions before injection of sample and the products separated with a linear gradient of 25 to 35% D in 10 min. Under these conditions, bradykinin eluted in 6.5 min and des-Arg’-bradykinin in 10.2 min. Peptides were detected by absorbance at 214 nm. The amount of product released was calculated by comparing the integrated peak area of product to the peak area of a known amount of authentic standard. Standards were injected 2-4 times during each analysis. Kinetic constants were obtained by initial velocity measurements of product formation at seven substrate concentrations, ranging from 5 to 250 pM. Data were plotted according to Hanes ([S] vs [S]/V) and fit to the best straight line by linear regression (23). Correlation coefficients of r = 0.97 or better were always obtained. Acid stability studies. Enzymes were incubated at room temperature for 1 h in 0.05 M sodium acetate (pH 4.0 to 5.0) and then tested for activity with Bz-Gly-argininic acid at pH 8.5 as stated above. Controls were diluted in 0.1 M Tris-HCl buffer, pH 8.0, and treated as above.

SKIDGEL,

524 0.7

-

0.6

0.05M

I

0.5J

% g

0.4

-

f

0.3

-

z!i

0.2-.

TRIS.

pH

DAVIS, AND ERDijS

7.2

0.1-

FIG. 1. Chromatography of concentrated human urine on an Affi-Gel blue column. Carboxypeptidase activity was assayedwith Bz-Gly-argininic acid. Column fractions were assayed for protein according to Bradford (24). The urinary carboxypeptidase was eluted from the column with 0.05 M Tris-HCl, pH 7.2, containing 0.25 M NaCI.

Protein determinations. Individual column fractions were assayed for protein according to the method of Bradford (24). All other protein values were obtained with the method of Lowry (25) using bovine serum albumin as a standard. RESULTS

PuriJication of human urinary carboxypeptidase. Gel filtration of concentrated human urine on Sephacryl S-300 revealed the presence of carboxypeptidase activity with an apparent

molecular weight of 73,000. When gel filtration was used as a first purification step, it yielded only a twofold purification. A 12-fold purification was achieved when the crude urine was first chromatographed on a column of Al&Gel Blue (Fig. 1, Table 1). The enzyme was retained on the column when applied in 0.05 M Tris-HCl, pH 7.2, and was eluted with 0.25 M NaCl in the same buffer. Initial attempts at affinity chromatography on an arginine-Sepharose column yielded variable results when, after sample was applied, the column was washed with 0.05 M Tris-HCl, pH 7.2, containing 0.1 M NaCl. Under these conditions, the enzyme sometimes coeluted with the nonspecifically bound protein (not shown). However, when the active fractions from the Al&Gel Blue column were applied to the affinity column in 0.05 M Tris-HCl, pH 7.2, without added NaCl, most of the protein and the activity were retained. Addition of 2 mM GEMSA, a competitive inhibitor, to the buffer eluted most of the carboxypeptidase activity with very little protein (Fig. 2). The contaminating proteins were eluted with 0.5 M NaCl in the same buffer (Fig. 2). The final purification step consisted of gel filtration in a highpressure liquid chromatograph on a TSKG3000SW column (Fig. 3). The last half of the major peak eluting at 26.4 min contained the highest specific activity and was pooled (Fig. 3). With this scheme, the urinary car-

TABLE PURIFICATION

Purification step Urine concentrate’ Affi-Gel Blue column Arginine-Sepharose column Gel filtration-TSK-G3000SW

columnd

OF HUMAN

1

URINARY

CARBOXYPEPTIDASE

Volume (ml)

Protein” bat)

Activity b (units)

Sp. act. (Units/m&

Yield @)

45 7.6 0.75 4.58

584 35.6 0.49 0.16

68.8 50.3 12.8 7.8

0.12 1.41 26.1 48.7

100 73.1

Purification (x-fold) 1 12

18.6

217

11.3

406

a Protein was determined according to the method of Lowry et al. (25). b One unit equals 1 pmol of Bz-Gly-argininic acid hydrolyzed per min at 37°C. ‘Thirteen liters of human urine were concentrated on an Amicon hollow-fiber concentrator; See Materials and Methods. d Sixty percent of the protein from the arginine-Sepharose step was applied to the gel filtration column. The values given for this step were increased by a factor of 1.67 to compensate for this fact.

PURIFICATION

OF HUMAN

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CARBOXYPEPTIDASE

525

Fraction FIG. 2. Arginine-Sepharose affinity chromatography of human urinary carboxypeptidase. The carboxypeptidase was eluted with 0.05 M Tris-HCl, pH 7.2, containing 2 mM guanidinoethylmercaptosuccinic acid (GEMSA). See Fig. 1 and Materials and Methods.

boxypeptidase was purified 406-fold with a yield of 11.3% compared to the crude concentrated urine (Table 1). The purification was monitored by polyacrylamide gel electrophor

1.00

0.75 d FJ 4. 0.50

0.25

n :: II II

-

resis in the presence or absence of SDS (Figs. 4 and 5). The purified enzyme migrated as a single band and was homogeneous as determined by silver staining the native or SDSpolyacrylamide gels. The apparent molecular weight of the carboxypeptidase on SDS-gel electrophoresis was 75,700 (Fig. 4) in good agreement with the k& value of 73,000 found by gel filtration on a Sephacryl S-300 column. A single band with the same molecular weight was also found when the enzyme was boiled in the presence of 2-mercaptoethanol and analyzed by SDSgel electrophoresis, indicating the enzyme exists as a single polypeptide chain (not shown). Characterization of human urinary carboxypeptidase activity. In order to study the similarities and differences among human carboxypeptidases, the hydrolysis of peptide and ester substrates by urinary, pancreatic, and plasma carboxypeptidases was compared. As shown in Table 2, the urinary carboxypeptidase cleaved the ester substrate (Bz-Glyargininic acid) relatively faster than either peptide substrate (Bz-Ala-Lys or Bz-Phe-Lys). Pancreatic carboxypeptidase B also had higher esterase activity, but the ratio was not as high as with the urinary enzyme (Table 2). Car-

:;Jd -

-

-

0

10

20

30

40

TIME,min

FIG. 3. Gel filtration of human urinary carboxypeptidase by HPLC on a TSK-G30OOSW column, The column effluent was monitored for absorbance at 2 14 nm (I .O absorbance unit full scale) and fractions of 0.21 ml were collected. The peaks which eluted at 32 and 33 min contained no protein. Fractions were assayed with Bz-Glyargininic acid and the peak at 26 min contained all the activity.

SKIDGEL,

526

DAVIS, AND ERDijS

94k+ 67k +

43k+ 30kl,

1234567

presence of 0.1 mM cadmium acetate while the activity of human pancreatic carboxypeptidase B was increased by 100% and the urinary carboxypeptidase was not significantly affected (3% inhibition). To determine whether the purified urinary carboxypeptidase could cleave substrates with nonbasic C-terminal amino acids, 0.54 pg of enzyme was incubated with the carboxypeptidase A substrate, Bz-Gly-Phe (1 mM) at pH 7.0 for 17 h at 37°C. Analysis of the products by HPLC showed that the substrate was not cleaved by urinary carboxypeptidase during the incubation, indicating a lack of carboxypeptidase A-like activity. The effects of various enzyme inhibitors and activators on the peptidase activity of urinary

FIG. 4. Polyacrylamide gel electrophoresis in the presence of SDS of human urinary carboxypeptidase during purification. Samples were run in 9.5% gels and stained with silver. Lane 1, molecular weight standards; Lane 2, concentrated human urine (32 gg); Lane 3, pooled fractions from the A&Gel Blue column (19 pgh Lane 4, pooled fractions from the arginine-Sepharose column (3 pg); Lane 5, blank; Lane 6, pooled fractions from the TSKG3OOOSW gel filtration column (1 pg); Lane 7, same as lane 6 (3 4.

boxypeptidase N and its M, 48,000 subunit were different because they cleaved Bz-AlaLys faster than Bz-Gly-argininic acid which was cleaved only 1.3 (carboxypeptidase N) or 1.4 (M, 48,000 subunit) times faster than BzPhe-Lys (Table 2). Both carboxypeptidase N and its M, 48,000 subunit cleaved Bz-GlyLys faster than Bz-Gly-Arg (six- and fivefold, respectively) while the urinary enzyme and carboxypeptidase B showed only a 1.6-fold higher activity with Bz-Gly-Lys (Table 2). The urinary enzyme was further distinguished from both carboxypeptidases B and N in experiments designed to study the effect of 0.1 mM cadmium acetate on the esterase activity with 1 mM Bz-Gly-argininic acid as substrate. Cadmium inhibits the esterase activity of carboxypeptidase N ( 14) while it enhances the esterase activity of pancreatic carboxypeptidase B (17). The activity of carboxypeptidase N was inhibited by 93% in the

1

234

5

FIG. 5. Native polyacrylamide gel electrophoresis of human urinary carboxypeptidase during purification and after trypsin treatment. Samples were. run in 7% gels without SDS and stained with silver. Lane I, concentrated human urine (32 Gg); Lane 2, pooled fractions from the A&Gel Blue column (19 pg); Lane 3, pooled fractions from the arginineSepharose column (3 fig); Lane 4, pooled fractions from the TSK-G3OOOSW gel filtration column (I pg); Lane 5, 3 pg of pure urinary carboxypeptidase incubated for 1 hat room temperature with 0.3 pg trypsin.

PURIFICATION

OF HUMAN

URINARY

CARBOXYPEPTIDASE

527

TABLE 2 COMPARISON OF THE ACTIVITIES OF HUMAN URINARY CARBOXYPEPTIDASE, PANCREATIC CARBGXYPEPTIDASE B, CARBGXYPEPTIDASE N, AND ITS M, 48,000 SUBUNIT Activity ratio0

Enzyme ’

Bz-Gly-Arg.Ac. b Bz-Ala-Lys

Bz-Gly-Arg. AC. Bz-Phe-Lys

Bz-Gly-Lys Bz-Gly-Arg

Carboxypeptidase N (M, 280,000) Active subunit (M, 48,000) Urinary carboxypeptidase Pancreatic carboxypeptidase B

0.5 0.7 3.4 1.5

1.3 1.4 5.7 3.3

6.0 5.0 1.6 1.6

’ Enzymes were assayed with substrates as described under Materials and Methods. Results are expressed as the ratio of activity (pmol/min/mg) of one substrate compared to the other. b Abbreviations: Bz, benzoyl; Arg.Ac., argininic acid. c Enzymes were purified from human sources as described under Materials and Methods.

absence of added CoC12. Under these conditions, cadmium acetate (0.1 mM), MGTA ( 10 PM), and o-phenanthroline ( 1 mM) inhibited the urinary enzyme 7 1, 93, and 78%, respectively, and the plasma enzyme 56, 100, and 97% (Table 3). With both enzymes, the addition of 1 IYtM CoC12 enhanced the activity about fivefold. In the presence of 1 mrvt Co&, TABLE 3 10 pM MGTA inhibited the urinary enzyme ACTIVATION AND INHIBITION OF CARBOXYPEPTIDASE 85% and the plasma enzyme 93% while aproN AND URINARY CARBOXYPEPTIDASE tinin ( 1300 U/ml), PMSF ( 1 m&Q, and PCMS Activity” (W) (0.1 InM) did not significantly inhibit either carboxypeptidase (Table 3). Utina~ carboxyThe pH activity profile of the urinary enCarbOXy- peptidase Additionb Cont. COCl*’ pePnd= N zyme with Bz-Ala-Lys as substrate was quite different from that of carboxypeptidase N or None 100 100 its active M, 48,000 subunit (Fig. 6). Although CdOAc 0.1 mM 29 44 all three enzymes had maximal peptidase acMGTA 10 NM 7 0 o-Phentivity at pH 7.0, the urinary carboxypeptidase anthroline 1.0 mM 22 3 activity decreased by 60% at pH 6.0, while the COCI, 1.0 mM + 523 565 plasma enzyme and its active subunit retained MGTA 10 plu + 18 40 Aprotinin 1300 U/ml + 544 514 80 and 100% of their activity at pH 6.0 (Fig. PMSF 1.0 mM + 659 531 6). In addition, the activity of urinary carPCMS 0.1 mM + 570 548 boxypeptidase fell less sharply above pH 7.0 EUrinary carhoxypeptidaseand carhoxypeptidaseN were pu- than did the activities of carboxypeptidase N rified as describedand their activitiesdetermined using Bz-Glyor the kf, 48,000 subunit (Fig. 6). Lys as substrate.For details seeMaterials and Methods. Hydrolysis of bradykinin. The kinetic values bActivatorsor inhibitors were preincuhated with enzymefor 2 h on ice at the concentration given ( 18).Addition of substrateto for the hydrolysis of bradykinin by the urinary startthe reactiondecmasedthe final concentrationby 20%.CdOAc, carboxypeptidase are summarized in Table 4. cadmium acetate. ’ The preincuhationand reactionswere carriedout in the presence Turnover numbers (k& are expressed as mol(+) or absence(-) of I mM CoC12. ecules of substrate hydrolyzed per min per carboxypeptidase and carboxypeptidase N were determined using Bz-Gly-Lys as substrate. The results obtained with both the urinary carboxypeptidase and carboxypeptidase N were similar (Table 3). The effect of chelating agents and metal ions was tested in the

SKIDGEL,

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DAVIS, AND ERDiiS

the reaction was carried out with urinary carboxypeptidase that had been preincubated (2 h at 4°C) with 1 mM CoC&, the K, decreased aoto 32 PM and the &t increased to 47 min-‘, increasing the specificity constant to 1.5. With E i.= socarboxypeptidase N, addition of cobalt in3 creased the specificity constant about twofold; :: 40however, this was due to a fivefold increase in the k,, as the K, actually increased about 2.5-fold (Skidgel and ErdGs, unpublished 20 work). Stability. The stability of urinary carboxy.t. 4 5 6 7 8 9 10 peptidase was compared to that of carboxyPH peptidase N and its M, 48,000 subunit at 37°C FIG. 6. The effect of pH on the peptidase activity of human plasma carboxypeptidase N, its active subunit, I%& and at acid pH. At 37°C both the urinary enzyme and carboxypeptidase N were stable 48,000, and urinary carboxypeptidase. Carboxypeptidase for 2 h, retaining over 90% of their initial activity was assayedwith 1 mM Bz-Ala-Lys in the following buffers: 0.1 M sodium acetate (pH 5.0-5.5); 0.1 M 2-(Nactivity, while the M, 48,000 subunit lost 75% morpholino)ethanesulfonic acid (pH 6.0-7.0); 0. I M Trisof its activity (Fig. 7). The same enzymes were HCI (pH 7.5-9.5). Abbreviations: Carboxy, carboxypep incubated at room temperature for 1 h at pH tidase; 280K, 280,000 &f, holoenzyme; 48K, 48,000 M, lOO-

active subunit.

active site, assuming one active site per enzyme molecule of M, 74,350 (an average of the values obtained in gel filtration and SDSgel electrophoresis). The K,,, was 46 PM with a k,,, of 32, giving a specificity constant (k,J K,,,) of 0.7 PM-‘, min-’ (Table 4). In comparison, human plasma carboxypeptidase N has a K, of 19 PM, a kt of 58, and a specificity constant of 3 with bradykinin, calculated on the basis of two active sites per molecule (Skidgel and Erdiis, unpublished work). When TABLE 4 HYDROLYSISOFBRADYKININ BY HUMAN URINARY CARB~XYPEPTIDASE

COCl2”

&I

kat

k2.t

(0.1 mM)

(PM)

(min-‘)

K,

+

46 32

32 47

0.7 1.5

a Enzyme was preincubated for 2 h on ice in the presence (+) or absence (-) of 0.1 mM Co& Kinetic constants were determined as described under Materials and Methods.

30

60

SO

120

Time, min FIG. 7. Stability of carboxypeptidase N, its active subunit, 1%4,48,000, and urinary carboxypeptidase at 37°C. Enzymes were incubated at 37°C in 0.1 M Tris-HCI, pH 8.0, and, at the time points indicated, ahquots were removed and assayed with Bz-Gly-argininic acid. See Fig. 6 for abbreviations.

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CARBOXYPEPTIDASE

529

values between 4 and 5, and then tested for susceptibility of carboxypeptidase N to proactivity with Bz-Gly-argininic acid at pH 8.5. teolytic enzymes ( 10). At pH 4.0, carboxypeptidase N was 92% as Immunological studies. The crossreactivity active as a control sample incubated at pH of the urinary enzyme with antisera raised in 8.0, the urinary enzyme was only 42% as ac- rabbits to carboxypeptidase N was tested with tive, and the &fr 48,000 subunit only 5% as the “Western blot” technique. Urinary caractive (Fig. 8). boxypeptidase (1 pg) was run on polyacrylTo test its susceptibility to proteolytic deg- amide gels in the presence of SDS, transferred radation, purified urinary carboxypeptidase (3 electrophoretically to nitrocellulose, incubated pg) was incubated for 1 h at room temperature with antisera to carboxypeptidase N, and fiwith 0.3 Kg trypsin and then analyzed by poly- nally with 1251-labeled goat anti-rabbit IgG. acrylamide gel electrophoresis. There was no When the nitrocellulose was exposed to Xapparent hydrolysis of the urinary carboxy- ray film, no crossreacting bands were seen (not peptidase by trypsin as the enzyme still shown). In control experiments, 250 ng of carboxypeptidase N gave intense bands corremigrated as a single band in native polyacrylamide gel electrophoresis (Fig. 5, sponding to the M, 83,000 and M, 48,000 lane 5) and SDS-gel electrophoresis (not subunits (not shown). Thus, the homogeneous urinary carboxypeptidase did not crossreact shown). When assayed with Bz-Gly-argininic acid, the activity of urinary carboxypeptidase with antisera to human plasma carboxypeptidase N, indicating a lack of immunological was also not affected by trypsin treatment. identity between the two enzymes. These findings are in contrast to the known DISCUSSION 100

1

l --.

90 60 I

O5:o PH

FIG. 8. Activity of carboxypeptidase N, its active subunit, M, 48,000, and urinary carboxypeptidase exposed to pH 4.0-5.0 for 1 h at room temperature then assayed at pH 8.5. Control activity, exposed to pH 8.0 for 1 h = 100%. Substrate: Bz-Gly-argininic acid. See Fig. 6 for abbreviations.

In this report we have described for the first time the complete purification of a basic carboxypeptidase from human urine. The properties of this enzyme differ from those of other known carboxypeptidases. The molecular weight of the urinary carboxypeptidase was found to be 73,000 by gel filtration and 75,700 by SDS-gel electrophoresis. It consists of a single chain (as determined by SDS-gel electrophoresis) and is not susceptible to cleavage by trypsin in its native conformation. Its substrate specificity, pH activity profile, molecular weight, resistance to trypsin, stability at 37°C and acid pH, immunological reactivity, and activation/inhibition in the presence of divalent cations clearly distinguish it from human plasma carboxypeptidase N, the active M, 48,000 subunit of carboxypeptidase N and human pancreatic carboxypeptidase B. In addition, while it readily cleaved C-terminal lysine or arginine from peptide substrates, it did not cleave Bz-Gly-Phe, a substrate of carboxypeptidase A. Earlier studies reported the presence of kininase activity in urine (4,8). More recent in-

530

SKIDGEL,

DAVIS, AND ERDiiS

vestigations with partially purified preparations revealed the presence of both kininase I- and II-type activities (8,26,27). Kininase II (converting enzyme or peptidyl dipeptidase) was recently purified from human urine (7). In an earlier study, a kininase I-like enzyme was partially purified from human urine (9). Although the activity of this enzyme was similar to that of the enzyme we report here, it had a molecular weight of only 40,000 (9). This discrepancy may be explained by the fact that the purification techniques used in the earlier study yielded an unstable preparation with a final yield of only 0.8%. It is thus possible that partial degradation of the enzyme occurred during purification, yielding a lower molecular weight than that of the enzyme we report here. Kinins which enter the nephron after glomerular filtration are hydrolyzed by kininase II concentrated on the brush border of proximal tubules (6,28). This is a protective mechanism because if intrarenal kinins play a role in autoregulation of the kidney, then the kinins liberated in plasma must be inactivated after entering the nephron. Kallikrein is released at the level of the distal tubules into the urine from the luminal side of the cells and presumably also into the circulation from the basal membrane of renal tubular epithelial cells (29,30). Thus, urinary kinins originate at the level of the distal tubules, possibly in the collecting ducts, where kininase II activity is very low (3 1). The concentration of kinins in the urine is therefore probably regulated, in part, by the urinary kininases. Studies on the pH activity profile of the urinary carboxypeptidase revealed that it was very sensitive to changes in pH between 6 and 7. Thus, while it was fully active at pH 7, at pH 6, its activity had dropped by 60%. The pH of the urine could therefore significantly affect the activity of this carboxypeptidase. Two recent studies showed that changes in urinary pH could affect urinary kinin levels (32,33). Because the changes in urinary kinin levels did not correlate with kallikrein excretion, the authors concluded that pH, among

other factors, may influence urinary kininase activity (32,33). The source of the urinary carboxypeptidase has not yet been determined. Many of the properties of urinary carboxypeptidase and carboxypeptidase N differ, including a lack of immunological crossreactivity. Urinary carboxypeptidase is thus not derived from plasma carboxypeptidase N but very likely comes from the kidney, possibly from the brush border of the proximal tubules, where many other peptidases are concentrated (34). Indeed, carboxypeptidase-type kininase activity has been extracted from a particulate fraction of human cadaver kidneys (9) and more recently was detected in a plasma membrane-enriched fraction of a perfused, blood-free human kidney (Skidgel and Erdos, unpublished work). Many peptides derived from larger precursor proteins are released through the action of a trypsin-like enzyme and thus contain a C-terminal basic amino acid (e.g., kinins, Lys6or Arg6-enkephalins, insulin, anaphylatoxins, etc.). Carboxypeptidases capable of cleaving these basic amino acids may thus be involved in either the further processing or in the inactivation of these peptides. The urinary carboxypeptidase reported here may participate in this process in the urine and possibly in the kidney. ACKNOWLEDGMENTS We thank Youngsook Kim and Mary Fields for their expert assistance and Brenda Moore for help in preparing the manuscript.

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