Inhibition of Human Peptidyl Dipeptidase (Angiotensin I Converting Enzyme: Kininase II) by Human Serum Albumin and its Fragments RAINER J. KLAUSER, P H . D . , CAROL J. G.
ROBINSON, M.S.,
AND ERVIN G.
ERDOS,
D. V.
MARINKOVIC, P H . D .
M.D.
SUMMARY Purified peptidyl dipeptidase (angiotensln I converting enzyme or kininase II) from human lung or hog kidney is inhibited by commercially prepared plasma protein preparations, by human serum albumin and by the additive albumin stabilizer, acetyltryptophan. After the initial steps of purification, albumin was detected by iramunodiffusion as a component in human lung peptidyl dipeptidase preparation. Fragment C of albumin (sequence 124-298) is a more potent inhibitor than the parent molecule (K, = 1.7 X 10 8 M). Reduction and carboxymethylation of five of the six S-S bridges in Fragment C yield the most potent noncompetitive inhibitor (K, = 3 X 10 ' M). Reduction of the sixth bridge raises the K,. This indicates that maintenance of the tertiary structure in Fragment C is of importance for the inhibition. Neither albumin nor Fragment C are substrates of the enzyme. Fragment C and its derivative also inhibit the inactivation of bradykinin by the purified human enzyme and by the peptidyl dipeptidase on the surface of intact cultured human endotbeliai cells. (Hypertension 1: 281-286, 1979)
KEY WORDS • transfusion • shock • protein inhibitor • albumin • kininase inhibition angiotensin I conversion • bradykinin • converting enzyme * peptidyl dipeptidase
P
EPTIDYL DIPEPTIDASE (angiotensin I converting enzyme or kininase II, E.C. 3.4.15.1; CE) cleaves dipeptides from the Cterminal end of peptides such as angiotensin I or bradykinin.1's In addition converting enzyme (CE) hydrolyzes other biologically active peptides such as enkephalins.3 The enzyme is present in plasma of man and animals,4"* but plasma also contains an inhibitor or inhibitors of CE.2t 7 Because of the obvious importance of CE in the metabolism of vasoactive peptides,8'" we investigated the inhibition of the enzyme by protein preparations and by albumin and its fragments in vitro.
MO); cyanogen bromide (87%) and iodoacetic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Goat antiserum to human albumin was obtained from Miles Laboratories (Elkhart, IN). Hippuryl-glycyl-glycine (Hip-Gly-Gly) was from Vega-Fox Biochemicals (Tucson, AZ). Plasma protein preparations (Plasmatein, Protenate and Plasmanate) were obtained from Abbott Laboratories, Hyland Laboratories and Cutter Laboratories, respectively. Converting enzyme was prepared from swine kidney by the method of Oshima et al.7 Human lung CE was prepared by gel filtration of a Sephadex G-200 column followed by chromatography on DEAEcellulose and hydroxyapatite columns. Cultured human endothelial cells10 were also used as a source of enzyme.
Methods and Materials
Human serum albumin, essentially free of fatty acid, was obtained from Sigma Chemicals (St. Louis,
Assay of Converting Enzyme We assayed CE using te/7-butyloxycarbonyl-Phe (NO,)-Phe-Gly (BPPG) or Hip-Gly-Gly as substrate.2 With the former substrate the hydrolysis was followed at 310 nm and with the latter, at 254 nm in a Cary Model 118 spectrophotometer. In typical experiments, 0.1 ml enzyme solution in 5 mM tris pH 7.4 was added to 0.8 ml of 0.2 M tris buffer, pH 7.4, containing 0.2 M NaCl and preincubated at 37°C for
From the Departments of Pharmacology and Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas. Supported in part by the Department of the Navy contract N0O014-75-C-O807, and by NIH USPHS Grants HL16320 and HL14187. Dr. Rainer J. Klauser received a travel grant from Deutsche Forschungsgemeinschaft. Address for reprints: Ervin G. Erdos, M.D., Department of Pharmacology, University of Texas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
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VOL 1, No 3, MAY-JUNE
1979
Protein Determination
The concentration of protein in the solutions was either determined by the method of Lowry et al." or by measuring the absorption at 280 nm with Fragment C as a standard. Preparation of Albumin Fragments
10
2 0 3 0 4 0 5 0 6 0 7 0
80
90100
PLASMA FRACTION FIGURE I. Inhibition of purified hog kidney peptidyl dipeptidase by commercially prepared 5% plasma protein preparations. Four different batches of three manufacturers (see Methods section) were used. Substrate: t-Boc-Phe-p(NOt)-Phe-Gly. Abscissa: \d plasma fraction. Ordinate: changes in absorption at 310 nm.
10 minutes. Then 0.1 ml 10'2 M Hip-Gly-Gly was added and the increase in absorbance was registered at 254 nm. One unit of enzyme cleaves 1 /imoles of substrate per minute. When inhibitors were used, they were preincubated for 10-15 minutes with the enzyme. The best lung CE preparation contained 8 U/mg and showed a single protein band in polyacrylamide gel electrophoresis.
Human serum albumin was cleaved to Fragments A, B and C with cyenogen bromide and the fragments were separated and purified by the sequential use of gel filtration and column chromatography according to McMenamy et al.11 The reduction and carboxymethylation of the disulfide bridges of the fragment were performed according to Crestfield et al.14 with slight modifications. Fragment C (55.5 mg) was dissolved in 5 ml of 0.5 M tris buffer, pH 8.6. The solution (6.2 X 10"4 M Fragment C) was stirred under nitrogen for 1 hour. The mercaptoethanol was added in a concentration sufficient to give a 25-fold excess over the 12 possible sulfhydryl (SH) residues of Fragment C. The reaction was allowed to proceed for 90 minutes at room temperature. Iodoacetic acid (1.73 g) was then added dropwise, and the pH was simultaneously adjusted to 8.7 with 0.2 N NaOH. After stirring for 1 hour, the solution was dialyzed for 48 hours against distilled water and then lyophilized (Method 1). Alternatively, the reduction was carried out in 8 M urea (Method 2) or with only a fourfold excess of mercaptoethanol over the possible SH-groups (Method 3). ImmunodifTusion
Bioassay The inactivation of bradykinin by CE was followed by measuring contractions of isolated rat uterus. 1 - 7
Immunodiffusion was carried out on Ouchterlony plates using antiserum to human albumin and to purified human lung CE. The latter was produced in the goat by injecting purified enzyme.
Gel Electrophoresis Polyacrylamide gel electrophoresis was carried out in 7.5% polyacrylamide gels (120 X 6 mm) at pH 8.9 with a constant current of 3 mA per tube at 4°C for 3 hours. The sample was heated in 5% mercaptoethanol and 3% sodium dodecyl sulfate (SDS) for 1 hour at 60°C and the molecular weight was estimated by plotting log mobility against molecular weights of human serum albumin, ovalbumin, chymotrypsinogen and ribonuclease A protein standards. Amino Acid Analysis Amino acid analysis was performed in a Durrum Amino Acid Analyzer after hydrolyzing the peptides at 110°C for 16 hours with 6 N HC1 in sealed evacuated tubes. Mean values of three determinations were used. Values for residues of amino acids per mol fragment were calculated with amino acid determinations of McMenamy et al.11 and amino acid sequence data of Meloun et al. for comparison.12 A Beckman 121 automatic amino acid analyzer was used when attempts were made to release dipeptides from albumin and Fragment C with CE.
Results Commercially prepared plasma protein preparations inhibited purified CE of hog kidney. Four different preparations of three manufacturers were tested. Thirty-five n\ of the 5% protein solution inhibited the hydrolysis of BPPG by 50% (fig. 1). Because commercial plasma protein preparations contain 4 X 10~a M acetyltryptophan added as stabilizer, we tested this compound for inhibition of CE. The IM with Hip-Gly-Gly substrate and with either human or hog CE was 5 X 10"4 M. The other additive, sodium caprylate, had no effect on the activity of the enzyme. Related compounds such as tryptophan, acetylhistidine and histidine were also inactive at 10 s M concentration. Presence of Albumin in CE
During purification of human CE7> 16 crude preparations have low enzymic activity, so we examined human lung CE preparations during the various stages of purification for the presence of albumin. (The
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INHIBITION OF ANGIOTENSIN I CONVERTING ENZYME/Klauser et al. details of purification of human CE will be published elsewhere.) We found that CE of homogenized human lung extracted with detergent and partially purified by gel filtration on Sephadex G-200 column still contained albumin in the active peak (fig. 2), as shown by immunodiffusion. Figure 2 indicates that the preparation of CE reacted with antibody to human lung CE and also with antibody to human serum albumin. Albumin, which has a molecular weight of 66,500, eluted in gel filtration on Sephadex G-200 column with CE, which has a much higher molecular weight. In gel filtration, CE frequently shows a molecular weight of 200,000 or even higher,8 thus CE probably forms a complex with albumin. After additional steps of purification on DEAEcellulose and hydroxyapatite columns, CE. did not react with antibody to human albumin, indicating that albumin was removed from the preparation during purification (T. Stewart, to be published). CE, however, still formed a precipitin band with antibody to the human lung enzyme. Albumin
Because commercial plasma protein preparations contain over 83% albumin,1" we studied the effect of purified, fat-free albumin on the CE. Purified CE of human lung and hog kidney are equally inhibited by albumin. The IM values with Hip-Gly-Gly substrate were 2 X 10^ M and 1.9 X 10^ M. (The concentration of human enzyme was 42 mU/ml). The inhibition of human lung enzyme was noncompetitive. The Ki determined in the Dixon plot (fig. 3) was 3 X 10^ M. Fragments of Albumin Since the commercial plasma protein solutions inhibited CE relatively more than accounted for by albumin content, which is an order lower than the IM value of purified albumin, we investigated whether fragments of human albumin may be more potent inhibitors than the native protein. Human albumin was cleaved to three fragments at the methionine residues with CNBr and the fragments were separated by column chromatography. 11 ' 1J Following one of the several conflicting nomenclatures, we called Fragment B the residues 1-123 of albumin, Fragment C residues 124-298 and Fragment A residues 299-585. All three fragments inhibited CE. The IM values were 2.2 X 10^ M, 7.4 X 10"* M, 2.5 X 10"6 M for Fragments A, B and C, respectively.
283
LP
AA FIGURE 2. Immunodiffusion of peptidyl dipeptidase of human lung obtained after gelfiltration(LP) and after subsequent column chromatographies (HL). AB = antiserum to human lung enzyme, A A = antiserum to human albumin, SA = human serum albumin. After Sephadex G-200 filtration human lung enzyme (LP) reacts both with antibody to the enzyme and with antibody to human albumin, indicating the presence of albumin. After further purification of enzyme, HL does not cross-react with antibody to albumin.
calculated from the available data,11'12 indicating a high degree of purity of Fragment C. To establish whether the inhibition depends on the secondary and tertiary structure of the fragment or on free SH groups, we reduced and carboxymethylated the disulfide bridges in Fragment C (CMFC) formed
Fragment C Fragment C has most of the ligand binding properties of human plasma albumin,17-18 thus we studied the inhibition of CE by this protein. In electrophoresis, Fragment C in presence of SDS migrated as a single band of a molecular weight of 18,000 ± 800. The arnino acid composition of Fragment C is shown in table 1. The values for single amino acids obtained are in good agreement with the theoretical value
0
1
2
3
4
5
I[M]«1O~*
FIGURE 3. Dixon plot of inhibition ofpurified human lung peptidyl dipeptidase by human serum albumin. Solid circles = 2.5 X 10~' M Hip-Gly-Gly, solid triangles = 1 X 10' M Hip-Gly-Gly, AT, = 3 X 1Q-* M.
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TABLE 1. Amino Acid Composition of Fragment C of Human Serum Albumin and its Reduced and Carboxymelhylated Derivatives Fragment
Amino acid
C
ASP Thr
CMFC 1 CMFC 2 CMFC 3 (Method 1) (Method 2) (Method 3) Residues per mol Fragment C
15.8
16.7
5.8
5.3
Expected values
17.5
15.1
14
5.6
5.4
6
Ser
8.7
9.1
8.8
8.5
9
Glu
30.6
30.0
29.2
27.8
24
Pro
3.9
2.6
2.9
4.3
5
Gly
3.3
3.0
3.0
3.1
23.3
23.3
Ala
3 23
31.7*
25.3*
—t
Cys
~t
12
Val
4.6
4.8
5.0
5.6
5
He
2.7
3.0
2.9
3.2
4
Leu
18.6
17.7
17.6
18.4
19
Tyr
5.0
5.5
5.9
6.2
6
Phe
8.8
9.8
10.2
10.5
10
His
3.8
3.7
3.9
4.2
4
Lys
20.0
18.4
18.7
19
Arg
7.8
7.8
7.8
19.6 11.0
+
-t
—t
—t
1
1.0
1.0
1.0
1.0
1
—
12.lt
12.8
9.6
Trp
H-Ser CM-Cys
9
*Cys and Ala could not be separated under conditions applied. jTrp is destroyed upon acid hydrolysis. JUpon reduction and carboxymethylation of Fragment C (GMFC) cysteine residues are completely or partially converted into CM-Cys, depending on the conditions of reduction.
by 12 cysteine residues (CM-cysteinc). Three different conditions were used for reduction. Method 1: mercaptoethanol was used in 25-fold excess over the theoretically available SH groups to obtain Fragment CMFC 1. Method 2: mercaptoethanol was used in 25fold excess with the addition of 8 M urea (CMFC 2). Method 3: mercaptoethanol was used in fourfold excess (CMFC 3). Thus, with each method, the conditions of denaturing Fragment C differed. Different amounts of iodoacetic acid were incorporated into the
fragments when 4- or 25-fold excess of mercaptoethanol was applied for reduction of the disulfide bridges. With Methods 1 and 2 all disulfide bridges of Fragment C were derivatized in CMFC 1 and CMFC 2. Method 3 left one of the six bridges intact in CMFC 3 as shown in table 2. Accordingly, amino acid analysis of Fragment C indicated 12.1 and 12.8 CMcysteine residues obtained with Methods 1 or 2 but only 9.6 with Method 3, since two CM-cysteines are formed after reduction of each bridge. All three
TABLE 2. Inhibition of Human Lung Converting Enzyme by Fragment C and its Reduced and Carboxymelhylated (CM) Derivatives Inhibitor Fragment C CM Fragment C (Method 1; CMFC 1) CM Fragment C (Method 2; CMFC 2) CM Fragment C (Method 3; CMFC 3)
Cysteine content (mol/mol)*
CM-cysteine (mol/mol)
Ki (M)
8.7
—
1.7 X 10-'
_
12.1
7.5 X 10-'
_
12.8
8 X 10-'
9.6
3 X 10-«
2.3
'Determined by subtracting the theoretical value for alanine from the sum of alanine and cysteine obtained by amino acid analysis. Downloaded from http://hyper.ahajournals.org/ by guest on June 10, 2015
INHIBITION OF ANGIOTENSIN I CONVERTING ENZYME/Klauser et al. TABLE 3. Inhibilion by Albumin Fragments of the Inactivalion of Rradykinin by Cultured Human Endolhelial Cells
Fragment C Reduced and carboxymethylated Fragment C (CMFC 3)
Concentration (M)
Inhibition
10-"
49
10-'
58
Activity: 9.3 — 14.1 nmoles of bradykinin/hr/10"' cells.
reduced fragments inhibited purified human CE noncompetitively, but to a different degree. The calculated K, values for noncompetitive inhibition of CE by Fragment C and CMFC 1, CMFC 2 and CMFC 3 are shown in Table 2. Mild reduction, which leaves one disulfide bridge intact (Method 3), lowered the K] of Fragment C from 1.7 X 10"8 M to 3 X 10"8 M. Carboxy methylation of all cysteine residues (Method 1) decreased the inhibition by raising the K! to 7.5 X 10-* M. When Fragment C was completely denatured by urea and by reducing all the disulfide bridges (Method 2), the resulting protein inhibited less than Fragment C, and the K, was 8 X 10 8 M. The inhibition of Fragment C and CMFC 3 was not due to binding of a metal co-factor of human CE, because addition of 2 X 10~* M CoClj did not reverse the inhibition. Bioassay Fragment C inhibited the inactivation of bradykinin by human lung CE 60% at 7 X 10 8 M and CMFC 3 inhibited 73% at 4 X 10^ M. Fragment C also inhibited the enzyme on the surface of intact suspended cultured human endothelial cells as shown in Table 3. Here also CMFC 3 was a better inhibitor of the endothelial enzyme than was Fragment C. Albumin as Substrate of CE Purified human lung CE (0.01 U) was incubated with human serum albumin (4 X 10"* M) and Fragment C (3.6 X 10 8 M) at pH 7.4 for 1 and 6 hours, respectively. After the reaction was stopped by acid precipitation of proteins there was no increase in ninhydrin-positive material over the zero time blank. Similarly, when the reaction mixture was examined by the amino acid analyzer, no peaks corresponding to dipeptides could be detected. These experiments demonstrated that neither albumin nor Fragment C were substrates for CE. In addition, the experiments indicate that albumin and Fragment C, but not their breakdown products, are the inhibitors of CE. Discussion Various reports have described severe hypotensive reactions in patients, which followed the administra-
285
tion of plasma protein fractions manufactured by several companies."• ""M The reactions are particularly severe in patients with cardiopulmonary bypass. The plasma proteins are prepared by methods which include alcohol precipitation and heating over 10 hours at 6 0 o C . M ' u They contain over 83% albumin, plus acetyltryptophan and other additives. The plasma kallikrein-kinin system is believed to be involved in the hypotensive reaction since the preparations are frequently contaminated by bradykinin26 and by a prekallikrein activator, Hageman factor fragment.21 Inhibition of CE prolongs the hypotensive effects of kinins and abolishes the vasoconstrictor effect of renin by blocking the release of angiotensin I I . 8 ' ' Bradykinin is inactivated mainly by two enzymes, kininase I and kininase II. The former enzyme is carboxypeptidase N, which is mainly responsible for cleaving the peptide in blood plasma, while the latter enzyme is identical with the angiotensin I converting enzyme.1' M It is present in blood plasma although its more important location is on the surface of endothelial27 and epithelial cells.8 The low activity of CE in plasma may be due in part to its inhibition by albumin. Because the concentration of albumin is 5 to 8 X 10~* M in plasma, hypothetically it is high enough to inhibit sufficiently much of the activity of CE. Furthermore, albumin may interfere with assaying of CE activity in human plasma. Although in gel filtration CE exhibits a molecular weight about three times higher than albumin, albumin was eluted in the peak containing CE, indicating binding. Albumin present in partially purified human CE preparation may account for the low CE activity of these preparations. Digests of serum proteins can also inhibit other enzymes that inactivate bradykinin such as pancreatic carboxypeptidase B,28 a very potent kininase. Our experiments have shown that albumin and its fragments inhibit CE noncompetitively, without being substrates of the enzyme. This inhibition may be enhanced by the presence of acetyltryptophan, an additive in commercial plasma preparations. These preparations inhibit CE more than indicated by their albumin content alone. They also potentiate the effect of bradykinin on the isolated rat uterus (ErdSs and Robinson, unpublished data). A convenient way to obtain large fragments of proteins is to cleave them with CNBr at the methionine residues. All three fragments obtained by this method from human serum albumin inhibit CE more than albumin itself. The IM values were at least three times lower for the fragments than for serum albumin. Fragment C, which consists of the residues 124-298 of the polypeptide chain of albumin, is of particular interest because it has most of the ligand-binding potency of albumin.17 In addition to inhibiting soluble, purified human lung enzyme, Fragment C and its derivative also inhibit bradykinin hydrolysis by CE present on the surface of intact cultured human endothelial cells. Changes in the structure of Fragment C affect its inhibitory potency as shown by reduction and car-
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boxymethylation of this fragment. The Kf of CMFC 3, which has one disulfide bridge intact, is one sixth of that of Fragment C. Another protein, CMFC 1, is the product of reaction with a great excess of mercaptoethanol and all of the cysteine residues are derivatized (Method 1). Its K, is one-half of the value for underivatized Fragment C. These data suggest that introduction of carboxyl groups enhances the inhibitory properties of Fragment C, particularly when one intact disulfide bridge preserves the original conformation of a certain region within the polypeptide chain. As yet this bridge in the peptide chain has not been identified. In contrast, when derivatization is carried out under denaturing conditions, the CMFC 2 fragment obtained inhibits less than Fragment C itself. Since the CM-cysteine content of fragments obtained with Methods 1 and 2 is identical and CMFC 2 has a ten times higher Kf than CMFC 1, preservation of the original secondary structure seems to have greater influence on the inhibition constant than the incorporation of carboxyl groups. In conclusion, by inhibiting CE on vascular ertdothelial cells and in plasma, albumin, its fragments, and other plasma proteins may potentiate the hypotensive effects of bradykinin, which is present in or liberated by infused plasma protein preparations.21- 26 The presence of kinins, prekallikrein activator and kininase II inhibitors may explain the severe hypotension encountered in some cases after infusion of such preparations, especially when the lung is excluded from the circulation, since the pulmonary vascular bed is very active in cleaving kinins." This study of the inhibition of CE by albumin fragments should help to clarify the nature of complexing of the enzyme by albumin and possibly by other plasma proteins. Acknowledgments We are grateful for the advice of Dr. C. M. Herman of the University of Washington, Seattle, Washington, and for the assistance of Tess Stewart and Katy Hammon of the University of Texas Health Science Center. Endothelial cells were supplied by Dr. A. R. Johnson. Amino acid analysis was done by Dr. J. Capra and Miss P. Frank.
References 1. Yang HYT, ErdSs EG, Levin Y: A dipeptidyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochim Biophys Acta 214: 374, 1970 2. Yang HYT, Erdos EG, Levin Y: Characterization of a dipeptide hydrolase (kininase II; angiotensin I converting enzyme). J Pharmacol Exp Ther 177: 291, 1971 3. Erdos EG, Johnson AR, Boyden NT: Hydrolysis of enkephalin by cultured human endothelial cells and by purified peptidyl dipeptidase. Biochem Pharmacol 27: 843, 1978 4. Skeggs LT, Kahn JR, Shumway NP: Preparation and function of the hypertensin-converting enzyme. J Exp Med 103: 295, 1956
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1979
5. Yang HYT, Erdos EG: Second kininase in human blood plasma. Nature 215: 1402, 1967 6. Lanzillo JJ, Fanburg BL: Angiotensin I converting enzyme from human plasma. Biochemistry 16: 5491, 1977 7. Oshima G, Geese A, Erdos EG: Angiotensin I converting enzyme of the kidney cortex. Biochim Biophys Acta 350:26, 1974 8. ErdOs EG: The angiotensin I converting enzyme. Fed Proc 36: 1760, 1977 9. Oparil S, Katholi R: Angiotensin I conversion. In Renin, Vol. 2, edited by Hoirobin DF, Montreal, 1977, p 72 10. Johnson AR, Erdfls EG: Metabolism of vasoactivc peptides by human endothelial cells in culture: angiotensin I converting enzyme (kininase II) and angiotensinase. J Clin Invest 59: 684, 1977 11. McMenamy RH, Dintzis HM, Watson F: Cyanogen bromide fragments of human serum albumin. J Clin Invest 246: 4744, 1971 12. Meloun B, Moravek L, Kostka V: Complete amino acid sequence of human serum albumin. FEBS Lett 58: 134, 1975 13. Lowry OH, Rosebrough NJ, Fair AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265, 1951 14. Crestfield AM, Moore S, Stein WH: The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem 238: 622, 1963 15. Nishimura K, Yoshida N, Hiwada K, Ucda E, Kokubu T: Purification of angiotensin I-converting enzyme from human lung. Biochim Biophys Acta 483: 398, 1977 16. F.D.A. Bulletin: Adverse reactions to plasma protein fraction. Rockville, Md. 7: 20, 1977 17. Gambhir KK, McMenamy RH: Location of the indole binding site in human serum albumin. Characterization of major cyanogen bromide fragments with respect to affinity labeling positions. J Biol Chem 248: 1956, 1973 18. SjOdin T, Hansson R, Sjoholm I: Isolation and identification of a trypsin-resistant fragment of human serum albumin with bilimbin- and drug-binding properties. Biochim Biophys Acta 494: 61, 1977 19. Harrison GA, Robinson M, Stacey RV, McCulloch CH, Torda TA, Wright JS: Hypotensive effects of stable plasma protein solution (SPPS): a preliminary communication. Med J Aust 2: 1040, 1971 20. Bland JHL, Laver MB, Lowenstein E: Vasodilator effect of commercial 5% plasma protein fraction solutions. JAMA 224: 1721, 1973 21. Alving BM, Hojima Y, Pisano JJ, Mason BL, Buckingham RE Jr, Mozen MM, Finlayson JS: Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction. N Engl J Med 299: 66, 1978 22. Colman RW: Paradoxical hypotension after volume expansion with plasma protein fraction. N Engl J Med 299: 97, 1978 23. Mulford DJ, Mealey EH, Welton LD: Preparation of a stable human plasma protein solution. J Clin Invest 34: 983, 1955 24. Hink JH Jr, Hidalgo J, Seeberg VP, Johnson FF: Preparation and properties of a heat-treated human plasma protein fraction. Vox Sanguinis 2: 174, 1957 25. Izaka K, Tsutsui E, Mima Y, Hasegawa E: A bradykinin-like substance in heat-treated human plasma protein solution. Transfusion 14: 242, 1974 26. Igic R, Erdos EG, Yeh HSJ, SorTells K, Nakajima T: Angiotensin I converting enzyme of the lung. Circ Res 31 (suppl II): 11-51, 1972 27. Bakhle YS, Vane JR: Pharmacokinetic function of the pulmonary circulation. Physiol Rev 54: 1007, 1974 28. Hamberg U, Elg P, Stelwagen P: On the mechanism of bradykinin potcntiation. In Pharmacology of Hormonal Polypeptides and Proteins. Advances in Experimental Medicine and Biology, Vol. 2, edited by Back N, Martini L, Paoletti R, New York, Plenum Press, 1968, p 626
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Inhibition of human peptidyl dipeptidase (angiotensin I converting enzyme: kininase II) by human serum albumin and its fragments. R J Klauser, C J Robinson, D V Marinkovic and E G Erdös Hypertension. 1979;1:281-286 doi: 10.1161/01.HYP.1.3.281 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1979 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
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ROBINSON, M.S.,
AND ERVIN G.
ERDOS,
D. V.
MARINKOVIC, P H . D .
M.D.
SUMMARY Purified peptidyl dipeptidase (angiotensln I converting enzyme or kininase II) from human lung or hog kidney is inhibited by commercially prepared plasma protein preparations, by human serum albumin and by the additive albumin stabilizer, acetyltryptophan. After the initial steps of purification, albumin was detected by iramunodiffusion as a component in human lung peptidyl dipeptidase preparation. Fragment C of albumin (sequence 124-298) is a more potent inhibitor than the parent molecule (K, = 1.7 X 10 8 M). Reduction and carboxymethylation of five of the six S-S bridges in Fragment C yield the most potent noncompetitive inhibitor (K, = 3 X 10 ' M). Reduction of the sixth bridge raises the K,. This indicates that maintenance of the tertiary structure in Fragment C is of importance for the inhibition. Neither albumin nor Fragment C are substrates of the enzyme. Fragment C and its derivative also inhibit the inactivation of bradykinin by the purified human enzyme and by the peptidyl dipeptidase on the surface of intact cultured human endotbeliai cells. (Hypertension 1: 281-286, 1979)
KEY WORDS • transfusion • shock • protein inhibitor • albumin • kininase inhibition angiotensin I conversion • bradykinin • converting enzyme * peptidyl dipeptidase
P
EPTIDYL DIPEPTIDASE (angiotensin I converting enzyme or kininase II, E.C. 3.4.15.1; CE) cleaves dipeptides from the Cterminal end of peptides such as angiotensin I or bradykinin.1's In addition converting enzyme (CE) hydrolyzes other biologically active peptides such as enkephalins.3 The enzyme is present in plasma of man and animals,4"* but plasma also contains an inhibitor or inhibitors of CE.2t 7 Because of the obvious importance of CE in the metabolism of vasoactive peptides,8'" we investigated the inhibition of the enzyme by protein preparations and by albumin and its fragments in vitro.
MO); cyanogen bromide (87%) and iodoacetic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Goat antiserum to human albumin was obtained from Miles Laboratories (Elkhart, IN). Hippuryl-glycyl-glycine (Hip-Gly-Gly) was from Vega-Fox Biochemicals (Tucson, AZ). Plasma protein preparations (Plasmatein, Protenate and Plasmanate) were obtained from Abbott Laboratories, Hyland Laboratories and Cutter Laboratories, respectively. Converting enzyme was prepared from swine kidney by the method of Oshima et al.7 Human lung CE was prepared by gel filtration of a Sephadex G-200 column followed by chromatography on DEAEcellulose and hydroxyapatite columns. Cultured human endothelial cells10 were also used as a source of enzyme.
Methods and Materials
Human serum albumin, essentially free of fatty acid, was obtained from Sigma Chemicals (St. Louis,
Assay of Converting Enzyme We assayed CE using te/7-butyloxycarbonyl-Phe (NO,)-Phe-Gly (BPPG) or Hip-Gly-Gly as substrate.2 With the former substrate the hydrolysis was followed at 310 nm and with the latter, at 254 nm in a Cary Model 118 spectrophotometer. In typical experiments, 0.1 ml enzyme solution in 5 mM tris pH 7.4 was added to 0.8 ml of 0.2 M tris buffer, pH 7.4, containing 0.2 M NaCl and preincubated at 37°C for
From the Departments of Pharmacology and Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas. Supported in part by the Department of the Navy contract N0O014-75-C-O807, and by NIH USPHS Grants HL16320 and HL14187. Dr. Rainer J. Klauser received a travel grant from Deutsche Forschungsgemeinschaft. Address for reprints: Ervin G. Erdos, M.D., Department of Pharmacology, University of Texas, Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
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1979
Protein Determination
The concentration of protein in the solutions was either determined by the method of Lowry et al." or by measuring the absorption at 280 nm with Fragment C as a standard. Preparation of Albumin Fragments
10
2 0 3 0 4 0 5 0 6 0 7 0
80
90100
PLASMA FRACTION FIGURE I. Inhibition of purified hog kidney peptidyl dipeptidase by commercially prepared 5% plasma protein preparations. Four different batches of three manufacturers (see Methods section) were used. Substrate: t-Boc-Phe-p(NOt)-Phe-Gly. Abscissa: \d plasma fraction. Ordinate: changes in absorption at 310 nm.
10 minutes. Then 0.1 ml 10'2 M Hip-Gly-Gly was added and the increase in absorbance was registered at 254 nm. One unit of enzyme cleaves 1 /imoles of substrate per minute. When inhibitors were used, they were preincubated for 10-15 minutes with the enzyme. The best lung CE preparation contained 8 U/mg and showed a single protein band in polyacrylamide gel electrophoresis.
Human serum albumin was cleaved to Fragments A, B and C with cyenogen bromide and the fragments were separated and purified by the sequential use of gel filtration and column chromatography according to McMenamy et al.11 The reduction and carboxymethylation of the disulfide bridges of the fragment were performed according to Crestfield et al.14 with slight modifications. Fragment C (55.5 mg) was dissolved in 5 ml of 0.5 M tris buffer, pH 8.6. The solution (6.2 X 10"4 M Fragment C) was stirred under nitrogen for 1 hour. The mercaptoethanol was added in a concentration sufficient to give a 25-fold excess over the 12 possible sulfhydryl (SH) residues of Fragment C. The reaction was allowed to proceed for 90 minutes at room temperature. Iodoacetic acid (1.73 g) was then added dropwise, and the pH was simultaneously adjusted to 8.7 with 0.2 N NaOH. After stirring for 1 hour, the solution was dialyzed for 48 hours against distilled water and then lyophilized (Method 1). Alternatively, the reduction was carried out in 8 M urea (Method 2) or with only a fourfold excess of mercaptoethanol over the possible SH-groups (Method 3). ImmunodifTusion
Bioassay The inactivation of bradykinin by CE was followed by measuring contractions of isolated rat uterus. 1 - 7
Immunodiffusion was carried out on Ouchterlony plates using antiserum to human albumin and to purified human lung CE. The latter was produced in the goat by injecting purified enzyme.
Gel Electrophoresis Polyacrylamide gel electrophoresis was carried out in 7.5% polyacrylamide gels (120 X 6 mm) at pH 8.9 with a constant current of 3 mA per tube at 4°C for 3 hours. The sample was heated in 5% mercaptoethanol and 3% sodium dodecyl sulfate (SDS) for 1 hour at 60°C and the molecular weight was estimated by plotting log mobility against molecular weights of human serum albumin, ovalbumin, chymotrypsinogen and ribonuclease A protein standards. Amino Acid Analysis Amino acid analysis was performed in a Durrum Amino Acid Analyzer after hydrolyzing the peptides at 110°C for 16 hours with 6 N HC1 in sealed evacuated tubes. Mean values of three determinations were used. Values for residues of amino acids per mol fragment were calculated with amino acid determinations of McMenamy et al.11 and amino acid sequence data of Meloun et al. for comparison.12 A Beckman 121 automatic amino acid analyzer was used when attempts were made to release dipeptides from albumin and Fragment C with CE.
Results Commercially prepared plasma protein preparations inhibited purified CE of hog kidney. Four different preparations of three manufacturers were tested. Thirty-five n\ of the 5% protein solution inhibited the hydrolysis of BPPG by 50% (fig. 1). Because commercial plasma protein preparations contain 4 X 10~a M acetyltryptophan added as stabilizer, we tested this compound for inhibition of CE. The IM with Hip-Gly-Gly substrate and with either human or hog CE was 5 X 10"4 M. The other additive, sodium caprylate, had no effect on the activity of the enzyme. Related compounds such as tryptophan, acetylhistidine and histidine were also inactive at 10 s M concentration. Presence of Albumin in CE
During purification of human CE7> 16 crude preparations have low enzymic activity, so we examined human lung CE preparations during the various stages of purification for the presence of albumin. (The
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INHIBITION OF ANGIOTENSIN I CONVERTING ENZYME/Klauser et al. details of purification of human CE will be published elsewhere.) We found that CE of homogenized human lung extracted with detergent and partially purified by gel filtration on Sephadex G-200 column still contained albumin in the active peak (fig. 2), as shown by immunodiffusion. Figure 2 indicates that the preparation of CE reacted with antibody to human lung CE and also with antibody to human serum albumin. Albumin, which has a molecular weight of 66,500, eluted in gel filtration on Sephadex G-200 column with CE, which has a much higher molecular weight. In gel filtration, CE frequently shows a molecular weight of 200,000 or even higher,8 thus CE probably forms a complex with albumin. After additional steps of purification on DEAEcellulose and hydroxyapatite columns, CE. did not react with antibody to human albumin, indicating that albumin was removed from the preparation during purification (T. Stewart, to be published). CE, however, still formed a precipitin band with antibody to the human lung enzyme. Albumin
Because commercial plasma protein preparations contain over 83% albumin,1" we studied the effect of purified, fat-free albumin on the CE. Purified CE of human lung and hog kidney are equally inhibited by albumin. The IM values with Hip-Gly-Gly substrate were 2 X 10^ M and 1.9 X 10^ M. (The concentration of human enzyme was 42 mU/ml). The inhibition of human lung enzyme was noncompetitive. The Ki determined in the Dixon plot (fig. 3) was 3 X 10^ M. Fragments of Albumin Since the commercial plasma protein solutions inhibited CE relatively more than accounted for by albumin content, which is an order lower than the IM value of purified albumin, we investigated whether fragments of human albumin may be more potent inhibitors than the native protein. Human albumin was cleaved to three fragments at the methionine residues with CNBr and the fragments were separated by column chromatography. 11 ' 1J Following one of the several conflicting nomenclatures, we called Fragment B the residues 1-123 of albumin, Fragment C residues 124-298 and Fragment A residues 299-585. All three fragments inhibited CE. The IM values were 2.2 X 10^ M, 7.4 X 10"* M, 2.5 X 10"6 M for Fragments A, B and C, respectively.
283
LP
AA FIGURE 2. Immunodiffusion of peptidyl dipeptidase of human lung obtained after gelfiltration(LP) and after subsequent column chromatographies (HL). AB = antiserum to human lung enzyme, A A = antiserum to human albumin, SA = human serum albumin. After Sephadex G-200 filtration human lung enzyme (LP) reacts both with antibody to the enzyme and with antibody to human albumin, indicating the presence of albumin. After further purification of enzyme, HL does not cross-react with antibody to albumin.
calculated from the available data,11'12 indicating a high degree of purity of Fragment C. To establish whether the inhibition depends on the secondary and tertiary structure of the fragment or on free SH groups, we reduced and carboxymethylated the disulfide bridges in Fragment C (CMFC) formed
Fragment C Fragment C has most of the ligand binding properties of human plasma albumin,17-18 thus we studied the inhibition of CE by this protein. In electrophoresis, Fragment C in presence of SDS migrated as a single band of a molecular weight of 18,000 ± 800. The arnino acid composition of Fragment C is shown in table 1. The values for single amino acids obtained are in good agreement with the theoretical value
0
1
2
3
4
5
I[M]«1O~*
FIGURE 3. Dixon plot of inhibition ofpurified human lung peptidyl dipeptidase by human serum albumin. Solid circles = 2.5 X 10~' M Hip-Gly-Gly, solid triangles = 1 X 10' M Hip-Gly-Gly, AT, = 3 X 1Q-* M.
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TABLE 1. Amino Acid Composition of Fragment C of Human Serum Albumin and its Reduced and Carboxymelhylated Derivatives Fragment
Amino acid
C
ASP Thr
CMFC 1 CMFC 2 CMFC 3 (Method 1) (Method 2) (Method 3) Residues per mol Fragment C
15.8
16.7
5.8
5.3
Expected values
17.5
15.1
14
5.6
5.4
6
Ser
8.7
9.1
8.8
8.5
9
Glu
30.6
30.0
29.2
27.8
24
Pro
3.9
2.6
2.9
4.3
5
Gly
3.3
3.0
3.0
3.1
23.3
23.3
Ala
3 23
31.7*
25.3*
—t
Cys
~t
12
Val
4.6
4.8
5.0
5.6
5
He
2.7
3.0
2.9
3.2
4
Leu
18.6
17.7
17.6
18.4
19
Tyr
5.0
5.5
5.9
6.2
6
Phe
8.8
9.8
10.2
10.5
10
His
3.8
3.7
3.9
4.2
4
Lys
20.0
18.4
18.7
19
Arg
7.8
7.8
7.8
19.6 11.0
+
-t
—t
—t
1
1.0
1.0
1.0
1.0
1
—
12.lt
12.8
9.6
Trp
H-Ser CM-Cys
9
*Cys and Ala could not be separated under conditions applied. jTrp is destroyed upon acid hydrolysis. JUpon reduction and carboxymethylation of Fragment C (GMFC) cysteine residues are completely or partially converted into CM-Cys, depending on the conditions of reduction.
by 12 cysteine residues (CM-cysteinc). Three different conditions were used for reduction. Method 1: mercaptoethanol was used in 25-fold excess over the theoretically available SH groups to obtain Fragment CMFC 1. Method 2: mercaptoethanol was used in 25fold excess with the addition of 8 M urea (CMFC 2). Method 3: mercaptoethanol was used in fourfold excess (CMFC 3). Thus, with each method, the conditions of denaturing Fragment C differed. Different amounts of iodoacetic acid were incorporated into the
fragments when 4- or 25-fold excess of mercaptoethanol was applied for reduction of the disulfide bridges. With Methods 1 and 2 all disulfide bridges of Fragment C were derivatized in CMFC 1 and CMFC 2. Method 3 left one of the six bridges intact in CMFC 3 as shown in table 2. Accordingly, amino acid analysis of Fragment C indicated 12.1 and 12.8 CMcysteine residues obtained with Methods 1 or 2 but only 9.6 with Method 3, since two CM-cysteines are formed after reduction of each bridge. All three
TABLE 2. Inhibition of Human Lung Converting Enzyme by Fragment C and its Reduced and Carboxymelhylated (CM) Derivatives Inhibitor Fragment C CM Fragment C (Method 1; CMFC 1) CM Fragment C (Method 2; CMFC 2) CM Fragment C (Method 3; CMFC 3)
Cysteine content (mol/mol)*
CM-cysteine (mol/mol)
Ki (M)
8.7
—
1.7 X 10-'
_
12.1
7.5 X 10-'
_
12.8
8 X 10-'
9.6
3 X 10-«
2.3
'Determined by subtracting the theoretical value for alanine from the sum of alanine and cysteine obtained by amino acid analysis. Downloaded from http://hyper.ahajournals.org/ by guest on June 10, 2015
INHIBITION OF ANGIOTENSIN I CONVERTING ENZYME/Klauser et al. TABLE 3. Inhibilion by Albumin Fragments of the Inactivalion of Rradykinin by Cultured Human Endolhelial Cells
Fragment C Reduced and carboxymethylated Fragment C (CMFC 3)
Concentration (M)
Inhibition
10-"
49
10-'
58
Activity: 9.3 — 14.1 nmoles of bradykinin/hr/10"' cells.
reduced fragments inhibited purified human CE noncompetitively, but to a different degree. The calculated K, values for noncompetitive inhibition of CE by Fragment C and CMFC 1, CMFC 2 and CMFC 3 are shown in Table 2. Mild reduction, which leaves one disulfide bridge intact (Method 3), lowered the K] of Fragment C from 1.7 X 10"8 M to 3 X 10"8 M. Carboxy methylation of all cysteine residues (Method 1) decreased the inhibition by raising the K! to 7.5 X 10-* M. When Fragment C was completely denatured by urea and by reducing all the disulfide bridges (Method 2), the resulting protein inhibited less than Fragment C, and the K, was 8 X 10 8 M. The inhibition of Fragment C and CMFC 3 was not due to binding of a metal co-factor of human CE, because addition of 2 X 10~* M CoClj did not reverse the inhibition. Bioassay Fragment C inhibited the inactivation of bradykinin by human lung CE 60% at 7 X 10 8 M and CMFC 3 inhibited 73% at 4 X 10^ M. Fragment C also inhibited the enzyme on the surface of intact suspended cultured human endothelial cells as shown in Table 3. Here also CMFC 3 was a better inhibitor of the endothelial enzyme than was Fragment C. Albumin as Substrate of CE Purified human lung CE (0.01 U) was incubated with human serum albumin (4 X 10"* M) and Fragment C (3.6 X 10 8 M) at pH 7.4 for 1 and 6 hours, respectively. After the reaction was stopped by acid precipitation of proteins there was no increase in ninhydrin-positive material over the zero time blank. Similarly, when the reaction mixture was examined by the amino acid analyzer, no peaks corresponding to dipeptides could be detected. These experiments demonstrated that neither albumin nor Fragment C were substrates for CE. In addition, the experiments indicate that albumin and Fragment C, but not their breakdown products, are the inhibitors of CE. Discussion Various reports have described severe hypotensive reactions in patients, which followed the administra-
285
tion of plasma protein fractions manufactured by several companies."• ""M The reactions are particularly severe in patients with cardiopulmonary bypass. The plasma proteins are prepared by methods which include alcohol precipitation and heating over 10 hours at 6 0 o C . M ' u They contain over 83% albumin, plus acetyltryptophan and other additives. The plasma kallikrein-kinin system is believed to be involved in the hypotensive reaction since the preparations are frequently contaminated by bradykinin26 and by a prekallikrein activator, Hageman factor fragment.21 Inhibition of CE prolongs the hypotensive effects of kinins and abolishes the vasoconstrictor effect of renin by blocking the release of angiotensin I I . 8 ' ' Bradykinin is inactivated mainly by two enzymes, kininase I and kininase II. The former enzyme is carboxypeptidase N, which is mainly responsible for cleaving the peptide in blood plasma, while the latter enzyme is identical with the angiotensin I converting enzyme.1' M It is present in blood plasma although its more important location is on the surface of endothelial27 and epithelial cells.8 The low activity of CE in plasma may be due in part to its inhibition by albumin. Because the concentration of albumin is 5 to 8 X 10~* M in plasma, hypothetically it is high enough to inhibit sufficiently much of the activity of CE. Furthermore, albumin may interfere with assaying of CE activity in human plasma. Although in gel filtration CE exhibits a molecular weight about three times higher than albumin, albumin was eluted in the peak containing CE, indicating binding. Albumin present in partially purified human CE preparation may account for the low CE activity of these preparations. Digests of serum proteins can also inhibit other enzymes that inactivate bradykinin such as pancreatic carboxypeptidase B,28 a very potent kininase. Our experiments have shown that albumin and its fragments inhibit CE noncompetitively, without being substrates of the enzyme. This inhibition may be enhanced by the presence of acetyltryptophan, an additive in commercial plasma preparations. These preparations inhibit CE more than indicated by their albumin content alone. They also potentiate the effect of bradykinin on the isolated rat uterus (ErdSs and Robinson, unpublished data). A convenient way to obtain large fragments of proteins is to cleave them with CNBr at the methionine residues. All three fragments obtained by this method from human serum albumin inhibit CE more than albumin itself. The IM values were at least three times lower for the fragments than for serum albumin. Fragment C, which consists of the residues 124-298 of the polypeptide chain of albumin, is of particular interest because it has most of the ligand-binding potency of albumin.17 In addition to inhibiting soluble, purified human lung enzyme, Fragment C and its derivative also inhibit bradykinin hydrolysis by CE present on the surface of intact cultured human endothelial cells. Changes in the structure of Fragment C affect its inhibitory potency as shown by reduction and car-
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boxymethylation of this fragment. The Kf of CMFC 3, which has one disulfide bridge intact, is one sixth of that of Fragment C. Another protein, CMFC 1, is the product of reaction with a great excess of mercaptoethanol and all of the cysteine residues are derivatized (Method 1). Its K, is one-half of the value for underivatized Fragment C. These data suggest that introduction of carboxyl groups enhances the inhibitory properties of Fragment C, particularly when one intact disulfide bridge preserves the original conformation of a certain region within the polypeptide chain. As yet this bridge in the peptide chain has not been identified. In contrast, when derivatization is carried out under denaturing conditions, the CMFC 2 fragment obtained inhibits less than Fragment C itself. Since the CM-cysteine content of fragments obtained with Methods 1 and 2 is identical and CMFC 2 has a ten times higher Kf than CMFC 1, preservation of the original secondary structure seems to have greater influence on the inhibition constant than the incorporation of carboxyl groups. In conclusion, by inhibiting CE on vascular ertdothelial cells and in plasma, albumin, its fragments, and other plasma proteins may potentiate the hypotensive effects of bradykinin, which is present in or liberated by infused plasma protein preparations.21- 26 The presence of kinins, prekallikrein activator and kininase II inhibitors may explain the severe hypotension encountered in some cases after infusion of such preparations, especially when the lung is excluded from the circulation, since the pulmonary vascular bed is very active in cleaving kinins." This study of the inhibition of CE by albumin fragments should help to clarify the nature of complexing of the enzyme by albumin and possibly by other plasma proteins. Acknowledgments We are grateful for the advice of Dr. C. M. Herman of the University of Washington, Seattle, Washington, and for the assistance of Tess Stewart and Katy Hammon of the University of Texas Health Science Center. Endothelial cells were supplied by Dr. A. R. Johnson. Amino acid analysis was done by Dr. J. Capra and Miss P. Frank.
References 1. Yang HYT, ErdSs EG, Levin Y: A dipeptidyl carboxypeptidase that converts angiotensin I and inactivates bradykinin. Biochim Biophys Acta 214: 374, 1970 2. Yang HYT, Erdos EG, Levin Y: Characterization of a dipeptide hydrolase (kininase II; angiotensin I converting enzyme). J Pharmacol Exp Ther 177: 291, 1971 3. Erdos EG, Johnson AR, Boyden NT: Hydrolysis of enkephalin by cultured human endothelial cells and by purified peptidyl dipeptidase. Biochem Pharmacol 27: 843, 1978 4. Skeggs LT, Kahn JR, Shumway NP: Preparation and function of the hypertensin-converting enzyme. J Exp Med 103: 295, 1956
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5. Yang HYT, Erdos EG: Second kininase in human blood plasma. Nature 215: 1402, 1967 6. Lanzillo JJ, Fanburg BL: Angiotensin I converting enzyme from human plasma. Biochemistry 16: 5491, 1977 7. Oshima G, Geese A, Erdos EG: Angiotensin I converting enzyme of the kidney cortex. Biochim Biophys Acta 350:26, 1974 8. ErdOs EG: The angiotensin I converting enzyme. Fed Proc 36: 1760, 1977 9. Oparil S, Katholi R: Angiotensin I conversion. In Renin, Vol. 2, edited by Hoirobin DF, Montreal, 1977, p 72 10. Johnson AR, Erdfls EG: Metabolism of vasoactivc peptides by human endothelial cells in culture: angiotensin I converting enzyme (kininase II) and angiotensinase. J Clin Invest 59: 684, 1977 11. McMenamy RH, Dintzis HM, Watson F: Cyanogen bromide fragments of human serum albumin. J Clin Invest 246: 4744, 1971 12. Meloun B, Moravek L, Kostka V: Complete amino acid sequence of human serum albumin. FEBS Lett 58: 134, 1975 13. Lowry OH, Rosebrough NJ, Fair AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265, 1951 14. Crestfield AM, Moore S, Stein WH: The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem 238: 622, 1963 15. Nishimura K, Yoshida N, Hiwada K, Ucda E, Kokubu T: Purification of angiotensin I-converting enzyme from human lung. Biochim Biophys Acta 483: 398, 1977 16. F.D.A. Bulletin: Adverse reactions to plasma protein fraction. Rockville, Md. 7: 20, 1977 17. Gambhir KK, McMenamy RH: Location of the indole binding site in human serum albumin. Characterization of major cyanogen bromide fragments with respect to affinity labeling positions. J Biol Chem 248: 1956, 1973 18. SjOdin T, Hansson R, Sjoholm I: Isolation and identification of a trypsin-resistant fragment of human serum albumin with bilimbin- and drug-binding properties. Biochim Biophys Acta 494: 61, 1977 19. Harrison GA, Robinson M, Stacey RV, McCulloch CH, Torda TA, Wright JS: Hypotensive effects of stable plasma protein solution (SPPS): a preliminary communication. Med J Aust 2: 1040, 1971 20. Bland JHL, Laver MB, Lowenstein E: Vasodilator effect of commercial 5% plasma protein fraction solutions. JAMA 224: 1721, 1973 21. Alving BM, Hojima Y, Pisano JJ, Mason BL, Buckingham RE Jr, Mozen MM, Finlayson JS: Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction. N Engl J Med 299: 66, 1978 22. Colman RW: Paradoxical hypotension after volume expansion with plasma protein fraction. N Engl J Med 299: 97, 1978 23. Mulford DJ, Mealey EH, Welton LD: Preparation of a stable human plasma protein solution. J Clin Invest 34: 983, 1955 24. Hink JH Jr, Hidalgo J, Seeberg VP, Johnson FF: Preparation and properties of a heat-treated human plasma protein fraction. Vox Sanguinis 2: 174, 1957 25. Izaka K, Tsutsui E, Mima Y, Hasegawa E: A bradykinin-like substance in heat-treated human plasma protein solution. Transfusion 14: 242, 1974 26. Igic R, Erdos EG, Yeh HSJ, SorTells K, Nakajima T: Angiotensin I converting enzyme of the lung. Circ Res 31 (suppl II): 11-51, 1972 27. Bakhle YS, Vane JR: Pharmacokinetic function of the pulmonary circulation. Physiol Rev 54: 1007, 1974 28. Hamberg U, Elg P, Stelwagen P: On the mechanism of bradykinin potcntiation. In Pharmacology of Hormonal Polypeptides and Proteins. Advances in Experimental Medicine and Biology, Vol. 2, edited by Back N, Martini L, Paoletti R, New York, Plenum Press, 1968, p 626
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Inhibition of human peptidyl dipeptidase (angiotensin I converting enzyme: kininase II) by human serum albumin and its fragments. R J Klauser, C J Robinson, D V Marinkovic and E G Erdös Hypertension. 1979;1:281-286 doi: 10.1161/01.HYP.1.3.281 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1979 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
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