©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Extracellular Conversion of Epidermal Growth Factor (EGF) to des-Arg-EGF by Carboxypeptidase M (*)

(Received for publication, March 21, 1995)

Gerd B. McGwire , Randal A. Skidgel (§)

From the Departments of Pharmacology and Anesthesiology, Laboratory of Peptide Research, University of Illinois College of Medicine, Chicago, Illinois 60612

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Epidermal growth factor (EGF) is a 53-amino-acid mitogenic polypeptide present in a variety of tissues and fluids including kidney, urine, and amniotic fluid. An EGF isoform, des-Arg-EGF, has been identified in urine and is the earliest metabolite generated in target cells upon EGF binding. In this study, purified carboxypeptidase M efficiently released the COOH-terminal arginine residue from EGF with a K = 56 µM, k = 388 min, and k/K = 6.9 µM min. When EGF was incubated with urine or amniotic fluid, des-Arg-EGF was the only metabolite detected. This conversion was blocked by immunoprecipitation with specific antiserum to carboxypeptidase M or by 10 µMDL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (a carboxypeptidase M inhibitor), indicating that the major EGF metabolizing enzyme in these fluids is carboxypeptidase M. When incubated on a confluent monolayer of Madin-Darby canine kidney (MDCK) cells, EGF was readily converted to a single metabolite, des-Arg-EGF, by carboxypeptidase M. To investigate one possible functional consequence of this conversion, mitogenic activities of EGF and des-Arg-EGF were tested. Both peptides were equipotent in stimulating [H]thymidine incorporation in MDCK cells at all doses tested. In addition, inhibition of the conversion of EGF to des-Arg-EGF by the carboxypeptidase M inhibitor did not affect the mitogenic potency of EGF. These data indicate that carboxypeptidase M, present in a variety of cells and biological fluids, can convert EGF to des-Arg-EGF. However, in contrast to many other peptide hormones whose activity depends on a final carboxypeptidase processing step, removal of Arg of EGF is not required for its mitogenic activity.


INTRODUCTION

Epidermal growth factor (EGF)()is a 53-amino-acid polypeptide originally isolated from the mouse submandibular gland(1) . EGF was subsequently shown to be synthesized in high amounts in the kidney, whereas most other tissues generally contain low amounts(1) . Biological fluids also contain EGF. One of the highest concentrations is in urine from which two forms were originally isolated; native EGF and EGF (or des-Arg-EGF), lacking the COOH-terminal arginine residue (2) . Studies of EGF metabolism have shown that COOH-terminal processing of EGF occurs rapidly in target cells (3, 4, 5, 6) with the earliest metabolite being identified as des-Arg-EGF(4) . The carboxypeptidase(s) involved in the initial metabolism of EGF has not been identified, but it is likely to be a B-type carboxypeptidase that specifically releases COOH-terminal Arg or Lys residues from peptides and proteins(7) . We have previously identified and characterized a membrane-bound member of this group of peptidases, carboxypeptidase M (CPM)(7, 8) . CPM was originally purified, cloned, and sequenced from human placenta (8, 9) and was subsequently found to be widely distributed. Relatively rich sites include: organs such as lung, kidney, intestine, brain, and peripheral nerves; biological fluids such as seminal plasma and amniotic fluid; cultured cells including Madin-Darby canine kidney (MDCK) cells, fibroblasts, endothelial cells, and amnion-derived cell lines(7, 10, 11, 12, 13, 14, 15) . Naturally occurring substrates for CPM include biologically active peptides such as kinins, dynorphin A, and enkephalin hexapeptides(8) . The physiological role of CPM in tissues and nonserous fluids has not been clearly defined, but may be comparable to that of carboxypeptidase N in blood(7) . For example, CPM in kidney and urine can inactivate kinins which affect sodium and water excretion. CPM in placenta and amniotic fluid could either activate, alter the properties of, or inactivate a peptide substrate which is important in fetal development, fetal-maternal exchange, or parturition. One peptide that has been implicated in all of these processes is EGF.

The significance of the conversion of EGF to des-Arg-EGF in biological fluids or on target cells is not clear. In cases where receptor binding and biological activity have been investigated, des-Arg-EGF is as potent or more potent than the parent EGF(5, 16, 17) . However, in no studies was this conversion blocked to determine whether it is a necessary processing step for biological activity, as has been found in other peptide hormone systems. For example, most neuropeptides are synthesized as a larger precursor protein which must be enzymatically processed in intracellular secretory granules; first, by endoproteolytic cleavage at basic residues by a prohormone convertase (KEX2/subtilisin-like serine protease) and, next, by a B-type carboxypeptidase (carboxypeptidase E or H) to remove the remaining COOH-terminal Arg or Lys residue(7, 18) . In comparison, EGF is synthesized as a 1207-amino-acid, membrane-bound extracellular protein that must be processed, by still undefined steps, to release the 53-amino-acid EGF(1) . Because of the relatively ubiquitous distribution of CPM, the extracellular conversion of EGF to des-Arg-EGF could potentially be a normal second processing step in many or most tissues.

The goals of the present study were 3-fold: 1) to determine whether EGF is a substrate for CPM; 2) to investigate whether the enzyme on cells and in nonserous biological fluids that converts EGF to des-Arg-EGF is CPM; 3) to determine whether the conversion is required for the mitogenic activity of EGF. MDCK cells were used as a model system to investigate these questions for two reasons. First, we have previously shown that CPM is highly expressed on the membrane of these cells as the only neutral pH-optimum B-type carboxypeptidase(13) . Second, MDCK cells, being derived from distal tubular epithelial cells, would be representative of cells exposed to EGF synthesized in the kidney and secreted into urine in vivo.


EXPERIMENTAL PROCEDURES

Materials

EGF was from Boehringer Mannheim Biochemica. des-Arg-EGF was from Upstate Biotechnology Inc. or, alternatively, was generated by hydrolysis of EGF (0.15 mg/ml) with carboxypeptidase B (1.5 µg/ml) in Tris buffer, pH 8, for 30-60 min at 37 °C(4) . Fetal bovine serum was from Atlanta Biologicals, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) was from Calbiochem, and [H]thymidine was from Amersham. Dulbecco's modified Eagle's medium (DMEM), nutrient mixture F-12 Ham, Hanks' balanced salt solution (HBSS), carboxypeptidase B, Triton X-100, and protein A-Sepharose 4B were from Sigma. Trifluoroacetic acid was from Pierce. All other chemicals were from Fisher Scientific.

Purification and Assay of CPM

CPM was solubilized from placental microvilli with phosphatidylinositol-specific phospholipase C and purified as described(8, 19) . Routine assays of CPM activity were carried out using dansyl-Ala-Arg substrate as described(8, 19) .

Metabolism of EGF by Purified CPM

In initial studies, EGF (7.5 µM) was incubated for 0-60 min with 62.6 ng/ml pure CPM in a 50 mM Hepes buffer, pH 7.5 containing 1 mg/ml bovine serum albumin. Bovine serum albumin was included to prevent the nonspecific loss of EGF, and the potential for introducing contaminating protease activity was eliminated by heat inactivation for 30 min at 60 °C. The products were analyzed by HPLC using a Waters µBondapak C18 reverse phase column (0.39 30 cm) and an increasing linear gradient of acetonitrile/0.05% trifluoroacetic acid in water/0.05% trifluoroacetic acid(12, 15) . Peptides were detected at 214 nm, and the peaks were quantitated by integration of the peak area and comparison to the area obtained with a known quantity of authentic standard treated identically, except lacking CPM. For kinetic studies, triplicate reactions were run at each of 5 substrate concentrations ranging from 5 to 100 µM with 31.3 ng/ml pure CPM in the same buffer as above. The initial rates of product formation (v) (between 5% and 13% of substrate hydrolysis) were measured at each substrate concentration ([S]), and kinetic constants were determined by plotting [S] versus [S]/v(15) .

Metabolism of EGF by MDCK Cells

MDCK cells were grown to confluency in 96-well plates in DMEM supplemented with 2.2 g/liter sodium bicarbonate, 25 mM Hepes, 100 units/liter penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum. The monolayers were incubated for 24 h in serum-free DMEM:nutrient mixture F-12 Ham (1:1), followed by a 1-h preincubation in HBSS supplemented with 2.2 g/liter sodium bicarbonate, 100 units/liter penicillin, 0.1 mg/ml streptomycin, and 25 mM Hepes with or without 10 µM MGTA. EGF was added to the cells (final concentration = 5 µM) and incubated at 37 °C for 2 or 4 h, at which time the medium was collected and the products were analyzed by HPLC as above.

Metabolism of EGF in Biological Fluids

Amniotic fluid samples (clinical discards drawn for other purposes) were obtained from Dr. M. Fejgin of the Dept. of Obstetrics and Gynecology at the University of Illinois. Human urine was obtained from normal volunteers and was concentrated 15-fold with Immersible CX-10 ultrafiltration units (10,000-dalton cutoff, Millipore). EGF (7.5 µM final) was incubated with 17-22 µl of either amniotic fluid or concentrated urine in 50 mM Hepes buffer, pH 7.4 (final volume = 50 µl) with or without 10 µM MGTA for 1-2 h at 37 °C. The products were then analyzed by HPLC as described above.

Immunoprecipitation

Polyclonal antiserum to purified human placental CPM was raised in rabbits(8) . Amniotic fluid or urine (150 µl) was mixed with 70 µl of 50 mM Tris, pH 7.4, containing 0.75 mM NaCl and 5% Triton X-100, and 120 µl HO, and then preadsorbed with 50 µl of a protein A-Sepharose suspension (10%) for 30 min at 4 °C. The protein A-Sepharose was removed by centrifugation for 4 min in a microcentrifuge, and the supernatant was incubated with 10 µl of antiserum against CPM (diluted 1:2) overnight at 4 °C. Protein A-Sepharose (50 µl of a 10% suspension) was added, and the incubation was continued for 30 min at 4 °C followed by centrifugation for 4 min to pellet the immune complexes. Supernatants were removed and assayed for remaining CPM activity. Control experiments were conducted in the same way, except either normal rabbit serum or HO was used instead of the CPM antiserum.

Mitogenic Effect of EGF and des-Arg-EGF on MDCK Cells

MDCK cells were grown to confluency in 24-well plates, incubated in serum-free DMEM:F-12 Ham (1:1) for 24 h, and then pre-incubated for 1 h in HBSS with or without 10 µM MGTA as above. EGF or des-Arg-EGF (1-100 ng/ml) was added for 20-24 h followed by 2 µCi/ml [H]thymidine for an additional 4 h. The buffer was then removed, and the cells were washed sequentially with phosphate-buffered saline (0.5 ml/well), 10% trichloroacetic acid (0.25 ml/well twice), methanol (0.25 ml/well) and then solubilized in 0.5 M NaOH (0.25 ml/well) for 20 min followed by neutralization with 0.5 M HCl (0.25 ml/well). The radioactivity in each well was counted in a Beckman LS 2800 scintillation counter.


RESULTS

Metabolism of EGF by CPM

Incubation of EGF with CPM resulted in a time-dependent decrease in EGF and a corresponding increase in a single product that coeluted with authentic des-Arg-EGF. The metabolism was blocked in the presence of 10 µM MGTA, a CPM inhibitor (Fig. 1). The production of only des-Arg-EGF is consistent with the known specificity of CPM, which cleaves only COOH-terminal Arg or Lys from peptide substrates(8) . An identical product was observed when EGF was incubated with pancreatic carboxypeptidase B (not shown) which others have shown to yield only des-Arg-EGF(4) . These initial studies indicated that EGF was a relatively good substrate for CPM, compared with other biologically active peptides. This was confirmed by determination of the kinetic constants for EGF hydrolysis (Table 1). Thus, the specificity constant (k/K of 6.9 µM min) of EGF is similar to that of bradykinin and about one-third lower than the best substrate, [Arg-Met]enkephalin (Table 1). Interestingly, whereas EGF has a K similar to that of [Arg-Leu]enkephalin (which both have a COOH-terminal -Leu-Arg sequence), the turnover number is about 3-fold higher for EGF (Table 1).


Figure 1: Hydrolysis of EGF by purified carboxypeptidase M. EGF (7.5 µM) was incubated with CPM (62.6 ng/ml) at 37 °C for varying times, and the reaction products were analyzed by HPLC as described under ``Experimental Procedures.'' A, control, EGF incubated alone in buffer for 60 min; B, EGF + CPM, 20 min; C, EGF + CPM, 40 min; D, EGF + CPM, 60 min; E, EGF + CPM, 60 min in the presence of 10 µM MGTA.





Metabolism of EGF by MDCK Cells

To investigate whether des-Arg-EGF can be generated by renal cells or if other metabolic pathways predominate, EGF was incubated with confluent monolayers of MDCK cells. A single metabolite, des-Arg-EGF, appeared in the medium, with 59.8 ± 1.0% (± S.E., n = 4) converted after 2 h and 84.6 ± 0.3% (n = 4) after 4 h. The metabolism was completely blocked by 10 µM MGTA, the CPM inhibitor (Fig. 2). Because we previously determined that CPM is the only B-type carboxypeptidase on the plasma membrane of MDCK cells active at physiological pH(13) , these data show that CPM is the primary EGF metabolizing enzyme on these cells.


Figure 2: Metabolism of EGF by MDCK cells. EGF (5 µM) was incubated with confluent monolayers of MDCK cells at 37 °C for 2 or 4 h, the medium was collected, and the products were analyzed by HPLC as described under ``Experimental Procedures.'' A, EGF + MDCK cells, 2 h; B, EGF + MDCK cells, 4 h; C, EGF + MDCK cells in the presence of 10 µM MGTA, 4 h.



Metabolism of EGF in Biological Fluids

When measured with dansyl-Ala-Arg substrate, the CPM-like activity in urine was 16.6 ± 2.0 nmol/h/ml (n = 7). Specific antiserum to human CPM precipitated 95.7 ± 0.9% (n = 3) of the activity, showing that the B-type carboxypeptidase activity in urine is indeed a soluble form of CPM. When EGF was incubated with three different samples of urine, a single product, des-Arg-EGF, was formed in all cases, an example of which is shown in Fig. 3. The rate of conversion of EGF ranged from 0.41-0.48 nmol/h/ml and was reduced by 85% ± 8% (n = 3) after immunoprecipitation with anti-CPM antiserum and by 67 ± 9% (n = 3) with 10 µM MGTA (Fig. 3). These data indicate that des-Arg-EGF can be generated in urine and that CPM is the primary EGF metabolizing enzyme here, although another enzyme or enzymes might play a minor role.


Figure 3: Metabolism of EGF by human urine. EGF (7.5 µM) was incubated with 22 µl of urine (5-fold concentrated) in a final volume of 50 µl for 1 h at 37 °C, and products were analyzed by HPLC as described under ``Experimental Procedures.'' A, control, EGF incubated alone in buffer; B, control, urine incubated alone in buffer; C, EGF + urine; D, EGF + urine after immunoprecipitation with anti-CPM antiserum; E, EGF + urine in the presence of 10 µM MGTA.



Amniotic fluid hydrolyzed EGF at a much higher rate, 4.19-8.82 nmol/h/ml (n = 4), but again, only a single product, des-Arg-EGF, was produced (Fig. 4). The higher rate of EGF conversion is consistent with the (on average) 13-fold higher activity of CPM found in amniotic fluid compared with urine when measured with dansyl-Ala-Arg substrate(14) . Proof that CPM in the amniotic fluid was responsible for the hydrolysis of EGF was obtained by immunoprecipitation with specific antiserum to CPM which reduced the EGF hydrolysis by 98 ± 1% (n = 3) and by the use of 10 µM MGTA which completely (100%; n = 3) inhibited hydrolysis (Fig. 4). Thus, in amniotic fluid under these conditions, CPM is essentially the only EGF metabolizing enzyme.


Figure 4: Metabolism of EGF by human amniotic fluid. EGF (7.5 µM) was incubated with 17 µl of amniotic fluid for 1 h (A-C) or 22 µl of amniotic fluid for 2 h (D-F) at 37 °C in a final volume of 50 µl. A, EGF + amniotic fluid in the presence of 10 µM MGTA; B, EGF + amniotic fluid; C, amniotic fluid alone, no EGF added; D, EGF + amniotic fluid pretreated by immunoprecipitation with specific anti-CPM antiserum; E, EGF + amniotic fluid; F, amniotic fluid alone, no EGF added.



Effect of Conversion of EGF to des-Arg-EGF on Mitogenic Potency

Incubation of EGF with MDCK cells resulted in a dose-dependent increase in [H]thymidine incorporation, to a maximum of 160 ± 11% (n = 11) at 100 ng/ml (Fig. 5). The response to EGF is similar to that reported previously for MDCK cells(20) . des-Arg-EGF also stimulated [H]thymidine incorporation in MDCK cells, with a dose-response relationship that was indistinguishable from that of EGF (Fig. 5). These results do not rule out the possibility that the metabolism of EGF is required for mitogenic activity (i.e. that des-Arg-EGF is the active form). In order to investigate this, [H]thymidine incorporation was measured in the presence of MGTA (Fig. 6), at a concentration (10 µM) that completely blocks the conversion of EGF to des-Arg-EGF in MDCK cells (Fig. 2). Under these conditions, EGF maintained its mitogenic effect and was still equally as potent as des-Arg-EGF, which was used to control for any possible nonspecific effects of MGTA (Fig. 6).


Figure 5: Mitogenic effects of EGF and des-Arg-EGF on MDCK cells. Bars represent percent of [H]thymidine incorporation compared to control cells incubated in buffer alone under the same conditions. Values are given as the mean ± S.E. (n = 10-12). Open bars, des-Arg-EGF; dark bars, EGF.




Figure 6: Effect of a carboxypeptidase inhibitor on the mitogenic effects of EGF and des-Arg-EGF on MDCK cells. Bars represent percent of [H]thymidine incorporation compared to control cells incubated in buffer alone under the same conditions. Values are given as mean ± S.E.; n = 11-13 for the 10-ng dose; n = 5 for the 100-ng dose. Dark bars, EGF; dark slashed bars, EGF in the presence of 10 µM MGTA; open bars, des-Arg-EGF; light slashed bars, des-Arg-EGF in the presence of 10 µM MGTA.




DISCUSSION

The enzymes responsible for the proteolytic processing of the 1207-amino-acid EGF precursor have not been identified, but recent data suggest that kallikrein-like enzymes may be involved in some tissues (21, 22) . EGF concentrations in the kidney are relatively high compared to most other tissues, but 2000-fold lower than submandibular gland from which the peptide was first isolated(23) . Nevertheless, the prepro-EGF mRNA level in kidney is about 50% of that found in submandibular gland and 100- to 1000-fold higher than in other tissues, indicating EGF is synthesized at a high rate, but is secreted rather than stored there(23, 24) . This is consistent with the finding that urine contains high levels of EGF and that urinary EGF is derived primarily from renal tubules, most likely secreted from cells of the thick ascending limb of Henle or distal convoluted tubule(23) . The function of renal and urinary EGF is unclear, but it inhibits sodium absorption in the cortical collecting duct(25, 26) , and it may exert a mitogenic effect on tubular cells and maintain the epithelial surface (23, 27) .

In one of the first purifications of EGF from human urine, des-Arg-EGF was identified as an active, naturally occurring metabolite(2) . However, whether EGF was converted by an enzyme on renal tubular cells or in urine was unknown. The present investigation shows that exogenous EGF is converted to des-Arg-EGF by renal tubular epithelial cells as well as in urine, indicting that this conversion may take place in both locations in vivo. We previously purified and characterized the major B-type carboxypeptidase in human urine, prior to the discovery of CPM(12) , and later noted that the two enzymes had similarities. The present studies show that this urinary enzyme is indeed a soluble form of CPM. CPM protein and mRNA are also present in human kidney, showing that the protein is synthesized there(9, 10) . It can thus be concluded that CPM, both in its membrane-bound and soluble forms, is likely responsible for the appearance of des-Arg-EGF in urine.

Another biological fluid that contains EGF is amniotic fluid(1) . In the present study, exogenously added EGF was metabolized to only des-Arg-EGF, attributable to hydrolysis by CPM in this fluid. Whether this conversion influences the physiological effects of EGF during gestation has not been investigated, although the specific binding of I-des-Arg-EGF was reported to be 18-20% higher than that of I-EGF to placental membrane receptors(16) . Although the present data would argue against an effect on mitogenesis, EGF has other effects that might be influenced by conversion to des-Arg-EGF, such as its ability to increase the level of prostaglandin H synthetase and prostaglandin E production in amnion cells or to stimulate the production of placental lactogen, human chorionic gonadotropin, and progesterone in placental cells(1) .

A membrane-bound carboxypeptidase is involved in the initial processing/proteolysis of EGF during or after receptor binding and internalization(3, 4, 5, 6) . Studies on early processing of mature EGF in fibroblasts (3, 4, 5) and isolated perfused rat livers (6) revealed that EGF is first cleaved by a basic carboxypeptidase to remove the COOH-terminal Arg, producing des-Arg-EGF at the cell surface or in early endosomes. The second and third steps, occurring in late endosomes, involve endoprotease cleavage after Lys followed by basic carboxypeptidase hydrolysis to produce EGF(3, 4, 5, 6) . Subsequent to these steps, EGF is completely degraded to trichloroacetic acid-soluble products in lysosomes. The enzymes carrying out the initial processing steps were not identified and, although the processing is sequential in cells, it is not known whether removal of the COOH-terminal Arg of EGF is required before further cleavage at Lys can occur. The present study shows that CPM in MDCK cells efficiently converts EGF to des-Arg-EGF. Because CPM is widely distributed(7, 10, 11, 13, 15) , it is likely involved in this conversion on other cell types as well. Studies on truncated EGF indicate that receptor binding is decreased only after removal of Lys and especially Leu (100-fold decrease in affinity)(16, 28, 29) . However, it has never been determined whether the initial processing is required for the mitogenic activity of EGF. Because EGF is originally released from a larger precursor protein, it is not unreasonable to speculate that the release of the COOH-terminal arginine is a final prohormone processing step as is the case for most neuropeptide hormones(7, 18) . Some support for this hypothesis is provided by the finding that des-Arg-EGF binds to the EGF receptor with an equal or greater affinity than native EGF(5, 16, 17) . However, as demonstrated here, the mitogenic potency of des-Arg-EGF is equal to that of EGF on MDCK cells, and the conversion of EGF to des-Arg-EGF is not a required step for this activity to be expressed.

Whether or not removal of the COOH-terminal Arg could alter other activities or intracellular transport of EGF is not known. For example, in MDCK cells, 5-30% of the EGF bound to basolateral receptors is transcytosed to the apical side without the receptor(30, 31) , during which time (90-120 min) it would be expected that one or more of the initial processing steps would have taken place(3, 4, 5, 6) . Although it was claimed that transcytosed EGF is intact, the methods used to assess degradation, SDS-polyacrylamide gel electrophoresis (31) or gel filtration(30) , would not have been able to differentiate EGF from a metabolite lacking one or a few amino acids. Thus, the initial processing of EGF may serve as a signal or, alternatively, remove a signal for targeting of EGF to different extra- or intracellular locations where it could either exhibit additional activities or be further degraded. The COOH-terminal Arg does have one known important function in both EGF and nerve growth factor (NGF). In both cases, the COOH-terminal Arg is required for association with their respective binding proteins, as removal of this residue completely abolishes the ability of EGF and NGF to form high molecular weight complexes(32, 33) . The functional importance of the growth factor complexes is not clear, but both EGF and NGF are protected from hydrolysis by carboxypeptidase B in this form(34) .


FOOTNOTES

*
These studies were supported by National Institutes of Health Grant DK41431. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology (m/c 868), University of Illinois College of Medicine, 835 S. Wolcott, Chicago, IL 60612. Tel.: 312-996-9179; Fax: 312-996-1648.

The abbreviations used are: EGF, epidermal growth factor; NGF, nerve growth factor; MGTA, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid; MDCK, Madin-Darby canine kidney; CPM, carboxypeptidase M; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks' balanced salt solution; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We gratefully acknowledge the cooperation of Dr. Bruno Michel in purifying human placental carboxypeptidase M and Dr. M. Fejgin of the Dept. of Obstetrics and Gynecology in supplying the amniotic fluid samples.


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