Study of Interaction of Ceruloplasmin with Serprocidins - Springer Link

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Ceruloplasmin (CP; ferro:O2 oxidoreductase, EC. 1.16.3.1) is a copper containing protein of blood plasma with molecular mass ~132 kDa. An increase of its ...
ISSN 00062979, Biochemistry (Moscow), 2010, Vol. 75, No. 11, pp. 13611367. © Pleiades Publishing, Ltd., 2010. Published in Russian in Biokhimiya, 2010, Vol. 75, No. 11, pp. 15441552.

Study of Interaction of Ceruloplasmin with Serprocidins A. V. Sokolov*, K. V. Ageeva, V. A. Kostevich, M. N. Berlov, O. L. Runova, E. T. Zakharova, and V. B. Vasilyev Research Institute of Experimental Medicine, ul. Akademika Pavlova 12, 197376 St. Petersburg, Russia; fax: (812) 2349489; Email: [email protected] Received March 31, 2010 Revision received April 24, 2010 Abstract—This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1 : 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDSfree disc electrophoresis. CP affected the activi ties of NE, CG, and PR3 as a competitive inhibitor with Ki ~ 1 µM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards pphenylenediamine. CAP37 displayed the strongest inhibitory effect (Ki ~ 20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (513, 6163 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among ser procidins CAP37 can be regarded as the specific partner of CP. DOI: 10.1134/S0006297910110076 Key words: ceruloplasmin, azurocidin, neutrophil elastase, cathepsin G, proteinase 3, protein–protein interactions

Ceruloplasmin (CP; ferro:O2oxidoreductase, EC 1.16.3.1) is a coppercontaining protein of blood plasma with molecular mass ~132 kDa. An increase of its expres sion in inflammatory reactions allows regarding CP as one of the proteins of the acute phase of inflammation [1]. As an enzyme it is a universal scavenger of free radicals due to its activities of ferroxidase [2], cuproxidase [3], superoxide dismutase [4], glutathionedependent peroxidase [5], and NOoxidase [6]. We have studied the formation of com plexes of CP with such proteins of neutrophilic leukocytes as lactoferrin (LF) and myeloperoxidase (MPO) [7]. Interaction of anionic CP with cationic proteins seems to be its typical feature. Indeed, in vitro experiments show that CP readily forms a complex with protamine contain ing polyarginine clusters [8], while synthetic cationic pep tides RRRR (LF fragment 25) and KRYKQRVKNK (fragment 2938 of neuropeptide PACAP38) displace LF from its complex with CP [9]. Studying the interaction of Abbreviations: CAP37, azurocidin; CP, ceruloplasmin; CG, cathepsin G; LF, lactoferrin; MPO, myeloperoxidase; NE, neutrophilic elastase; PR3, proteinase 3. * To whom correspondence should be addressed.

CP with protamine allowed spotting at least six binding sites for cationic peptides in the CP molecule [8]. This feature allows CP forming both in vitro and in vivo multi meric complexes that include LF and MPO [7, 10]. LF increases the ferroxidase activity of CP upon forming a complex with the latter [11]. Interaction of CP with MPO results in inhibiting the prooxidant activity of the leuko cytic enzyme [12]. As shown in our latest study, the inhibitory effect of CP depends on the integrity of its mol ecule, whereas proteolyzed CP is inefficient as an inhibitor of chlorinating activity of MPO [13]. Screening the proteins of leukocytes revealed that, along with LF and MPO, four members of the serpro cidin family are partners of CP [14]. This family includes cationic proteins of neutrophilic leukocytes such as azurocidin (CAP37), elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). All these are structural homologs having antimicrobial properties. Serprocidins display in vitro activity against Gramnegative and Grampositive bacteria, Phycomycetes, and protozoa. Their antimicro bial activity is suppressed by moderate concentrations of NaCl (0.10.2 M), cations Ca2+, Mg2+, and also by serum, which speaks in favor of a qualitative similarity

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among the mechanisms of action of serprocidins. Proteolytic activity of serprocidins is not a prerequisite feature for displaying their antimicrobial functions as these are not lost when enzymes are inhibited [15]. It was shown that by splitting the Nterminal domain of throm bin receptor NE, CG, and PR3 inhibit the thrombin induced activation of endothelial cells and platelet aggre gation [16]. Blood clotting factor IX becomes bereft of its coagulative activity, once proteolyzed by NE [17]. Processing of antimicrobial peptides cathelicidins also depends on NE activity [18]. Bacteria lose their virulence when their flagellin is proteolyzed by NE [19]. It is accepted that NE and CG decrease activity of the cell surface proteolytic cascade by cleaving urokinase/CD 87 receptor on monocytes and thus releasing the chemotac tic derivatives of that protein [20]. Proteolytic cleavage by NE of the second type receptor on fibroblasts induces production of such cytokines as interleukin8 and chemotactic protein1 of monocytes [21]. NE performs its negative feedback regulation of granulopoiesis by cleaving the granulocytic colonystimulating factor [22]. Neutrophils treated with NE or CG loose ability to bind to Pselectin on endothelial cells, which prevents adhe sion [23]. Chemotactic activity of CAP37 towards monocytes reaches its maximum at concentration 110 nM [24]. Adhesion molecules (ICAM1, VCAM1, and E adhesin) appear upon CAP37 treatment of endothelium, which results in fixation of monocytes and neutrophils on it [25]. Monocytes are activated and start producing interleukin6 when CAP37 reacts with calreticulin on their surface [26]. Molecules of MHC (major histocom patibility complex) of class II, TNFα, and interleukin 1β appear upon treatment of microglia with CAP37 [27]. Its binding to bacterial lipopolysaccharides prevents microbial activation of peritoneal macrophages [28]. We accomplished a comparative study in which specificity of interaction of CP with proteins of the ser procidin family and features of complexes formed were investigated taking into account the presence of anti and proinflammatory properties in both parties. This paper demonstrates specificity of interaction between CP and CAP37 resulting from the contact of the former with cationic cluster (513, 6163) in the latter. This cluster endows CAP37 with its antimicrobial properties and allows binding to heparin.

MATERIALS AND METHODS The following reagents were used: dry defatted milk (BioRad, USA); triethylamine and EDTA (Merck, Germany); Sepharose 4B, phenylSepharose, CM Sephadex C50, DEAESephadex A50, QAESephadex A50, Sephadex G75 Superfine, and Sephacryl S200 HR (Pharmacia, Sweden); complete and incomplete

Freund’s adjuvant, sodium azide, arginine, glycerol, Coomassie R250, molecular mass markers for gel filtra tion, mercaptoethanol, ammonium persulfate, and Tris (Serva, Germany); aprotinin, NbenzoylLtyrosine ethyl ether (BTE), NtBOCLalanine pnitrophenyl ether (NBA), glycine, odianisidine, SDS, salmon prot amine, ophenylenediamine, pphenylenediamine dihy drochloride (pPD), phenylmethylsulfonyl fluoride (PMSF), and 4chloro1naphtol (Sigma, USA); CM Toyopearl (Toyo Soda, Japan); acrylamide, N,N′meth ylenebisacrylamide, and N,N,N′,N′tetramethylethyl ene diamine (MEDIGEN Laboratories, Russia); heparin (SPOFA, Poland). Cyanogen bromide was obtained by bromination of KCN in biphasic medium water– dichloroethane. The solution of BrCN in dichloroethane was used to activate Sepharose for subsequent immobi lization on the resin of heparin, protamine, arginine, aprotinin and CP [8]. Buffers used were phosphate buffer saline (PBS; 0.15 M NaCl, 1.9 mM Na2HPO4/8.1 mM NaH2PO4, pH 7.4); 0.1 M sodium acetate buffer (0.089 M AcONa/0.011 M AcOH, pH 5.5); 0.1 M sodi um acetate buffer (0.07 M AcONa/0.03 M AcOH, pH 5.0); 0.1 M sodium acetate buffer (0.054 M AcONa/0.046 M AcOH, pH 4.7); 0.1 M sodium citrate buffer (0.059 M citric acid/0.041 M sodium citrate, pH 4.0). Peptide RKARPRQFPRRR was synthesized in solid phase (Institute of Extrapure Biopreparations, St. Petersburg, Russia), and its purity was 99.5% as assayed by HPLC, amino acid, and mass spectrum analyses. Isolation of proteins. Stable preparation of mono meric CP having A610/A280 > 0.049 was obtained using affinity chromatography on protamineSepharose [29]. Homogeneous LF was purified from breast milk on CM Sephadex [7]. Leukocytic MPO with RZ ~ 0.85 was puri fied by affinity chromatography on heparinSepharose, phenylSepharose, and subsequent gel filtration [7]. Serprocidins from human leukocytes were purified by consecutive isolation of CAP37 on heparinSepharose, separation of endogenous inhibitors of proteinases (NE, CG, and PR3) by gel filtration on Sephadex G75, sepa ration of PR3 on aprotininSepharose, and final purifica tion of the proteins on CMToyopearl. Precipitate of leukocytes (40 g) was suspended in 100 ml of 100 mM sodium acetate buffer, pH 4.7, then frozen and thawed, after which three 30 sec sonications (44 kHz) followed with 1 min intervals in ice. The leukocytic extract was centrifuged for 30 min at 15,000g (4°C). The supernatant was loaded on a column (5 × 15 cm) with heparin Sepharose equilibrated with 100 mM sodium acetate buffer, pH 4.7. The heparinSepharose was washed twice by 100 ml of gradient 02 M NaCl in 100 mM sodium acetate buffer, pH 4.7. CAP37 was eluted at the end of the gradient. It was diluted 1/20 with water and applied on a column with CMToyopearl (1.5 × 10 cm) equilibrated with 100 mM sodium acetate buffer, pH 4.7. Proteins were eluted with 2 × 75 ml linear gradient 00.5 M NaCl BIOCHEMISTRY (Moscow) Vol. 75 No. 11 2010

CERULOPLASMIN AND SERPROCIDINS in 100 mM sodium acetate buffer, pH 4.7. Protein frac tion eluted in the void volume from the column with heparinSepharose was used to isolate NE, CG, and PR3. The fraction was lyophilized and dissolved in 25 ml of 50 mM sodium acetate buffer, pH 4.7, containing 1 M NaCl. Insoluble admixtures were eliminated by centrifu gation for 30 min at 15,000g (4°C). Proteins were subject ed to gel filtration on a column (5 × 100 cm) with Sephadex G75 Superfine equilibrated with 50 mM sodi um acetate buffer, pH 4.7, containing 1 M NaCl. Chromatographic fractions were assayed for NE, PR3 [30], and CG [31] activities. All three proteinases were eluted concurrently, so 1 M Tris was added to the pro teinasescontaining fraction to reach pH 7.0, after which the latter was loaded on a column with aprotinin Sepharose (5 × 10 cm). The column was washed with 50 mM TrisHCl, pH 7.4, containing 1 M NaCl till A280 < 0.005 of the effluent was achieved. This resulted in elution of PR3 that, like CAP37, was purified on CMToyopearl. Elution of NE and CG was achieved by 200 mM sodium acetate buffer, pH 4.3, after which the proteins were iso lated by ionexchange chromatography on CM Toyopearl equilibrated with 50 mM sodium acetate buffer, pH 4.7. Protein solution diluted to the final con centration of the buffer 50 mM was applied on the col umn. NE and CG were eluted with 2 × 50 ml linear gra dient 01 M NaCl (50 mM sodium acetate buffer, pH 4.7). Homogeneity of proteins was tested in SDS PAGE [32]. Preparations of proteinases and CAP37 con tained 99% of 30kDa protein bands and had neither extrinsic proteolytic activities nor fragments of structural homologs as judged by mass spectrometry of their tryptic fragments. All in all we obtained 32 mg of CAP37, 28 mg of NE, 24 mg of CG, and 12 mg of PR3. Analytical gel filtration was performed on a column (1.5 × 150 cm) with Sephacryl S200 HR equilibrated either with PBS or with 10 mM sodium phosphate buffer, pH 7.4, containing 0.5 M NaCl. The elution rate was 0.5 ml/min. Samples containing CP, serprocidins, or a mixture of CP with serprocidins (1 mg of CP + 0.5 mg of a serprocidin) were applied on the column. In course of subsequent elution the content of a serprocidin in the CPcontaining peak and in that with CPfree serprocidin was assayed. Catalase, aldolase, rat CP, transferrin, albu min, ovalbumin, and myoglobin were used for the column calibration. Disc electrophoresis at alkaline pH. PAGE at alkaline pH was run to analyze the formation of complexes of CP with serprocidins [33]. Samples of CP (1 µg), serpro cidins (0.25 µg), mammalian serum (210 µl), heparin (0.2 µg), peptide CAP37 (0.1 µg), and PMSF (0.01 µg) were brought to 20 µl by adding PBS and mixed with 5 µl of 50% glycerol and 0.5% bromophenol blue. After 10 min of incubation, 25 µl of each sample was loaded in a well of polyacrylamide gel slab and run in electrophore sis till the leading bromophenol blue reached the end of BIOCHEMISTRY (Moscow) Vol. 75 No. 11 2010

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the gel. Activity of CP in polyacrylamide gel was detected by oxidation of chromogenic substrate odianisidine [34]. Peroxidase activity of MPO was detected after incubating the gel in 4chloro1naphtol and H2O2 [7]. Measuring the kinetic parameters of enzymatic reac tions. Effect of 0.75 µM CP on the activity of NE and PR3 (30 nM) towards of NtBOCLalanine pnitro phenyl ether (NBA) was evaluated by measuring the rate of pnitrophenol formation (A400/min) in 50 mM Tris HCl, pH 7.4, at various concentrations of substrate (0.1 0.5 mM). Hanes–Wolf graphs (S, S/V) were plotted accounting for the presence and the absence of CP, after which Km, kcat, and Ki were calculated. Similar measure ments were made to study the effect of 0.75 µM CP on the activity of CG (30 nM) towards NbenzoylLtyrosine ethyl ether (BTE) taken in various concentrations (0.5 2.5 mM). In this case the rate of ethanol formation (A256/min) in 50 mM sodium phosphate buffer, pH 7.4, was determined. Effect of NaCl concentration on inhibi tion by CP (3 µM) of NE, PR3, and CG (30 nM) was evaluated by measuring the rate of hydrolysis of 0.1 mM NBA or 0.5 mM BTE in 50 mM sodium phosphate buffer, pH 7.4. Effect of serprocidins (100 nM) on oxidation of p phenylenediamine (pPD, 0.251.25 mM) catalyzed by 50 nM CP was evaluated by measuring the rate of forma tion of oxidized substrate (A530/min) in 0.1 M sodium acetate buffer, pH 5.0. Measuring dissociation constant for the complex of CAP37 with immobilized CP. CP was immobilized on BrCNactivated Sepharose (~1 mg CP/ml wet gel). In an additional portion of activated Sepharose active groups were blocked like in case with CPSepharose [8] and used to control any nonspecific adsorption of CAP37. The set for measuring dissociation constant contained CPSepharose (or the control resin) and PBS to which portions of CAP37 (50200 nM) were added. Upon equi libration Sepharose was precipitated by centrifugation at 500g for 5 min and nonbound protein concentration was assayed. The difference between CAP37 concentra tions allowed determining the amount of protein bound to CP ([B]) and the relation between bound and free CAP37 ([B]/[F]). Then the Scatchard plot was drawn. Such testing procedure was repeated thrice with varying amounts of CPSepharose (~0.5 and 1 µM of immobi lized CP). CAP37 concentration was assayed by ELISA. With that purpose rabbit antibodies to CAP37 were obtained. Animals were three times immunized with suspension 1/10 (v/v) of Freund’s adjuvant (complete for the first injection and incomplete for the two subsequent) and 100 µg of CAP37 (per injection) in SDSfree polyacry lamide gel where it had been resolved by electrophoresis. On the 10th day after the third immunization blood por tions were taken from rabbits and IgG fraction was isolat ed from serum. The antibodies were used in ELISA.

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Polystyrene plates were activated upon overnight incubation with 0.1 ml of 0.5% glutaric aldehyde in PBS. After thricerepeated washing with PBS either 0.1 ml of samples under investigation or standard dilutions of CAP37 (0.8120 nM) were dropped into the wells and kept for 1 h. This was followed by one after another 1h incu bations with 0.2 ml of BLOTTO (proteins of dry skimmed milk diluted to 3% in PBS with 0.05% Tween 20), 0.1 ml of rabbit antiCAP37 IgG solution (250 µg/ml), 0.1 ml horseradish peroxidaselabeled (1 : 1000) goat antibodies against rabbit immunoglobulins. Removing every solution was followed by thricerepeated washing of the plate with PBS. Peroxidase reaction was visualized by treating the wells with 10 mg of ophenylenediamine in 1 ml of ethanol mixed with 11 ml of 5 mM H2O2 in 0.1 M sodium citrate buffer, pH 4.0. The chromogenic reaction was stopped by adding 0.05 ml of 6 M H2SO4, after which A492 in the wells was determined in a StatFax (USA) plate photometer. The dependence of A492 on log[CAP37] was approximated by a straight line obtained by the leastsquares method. Determinancy coefficient (R2) was no less than 0.99 (Microsoft Excel 2002). The obtained equation, A492 = k·log[CAP37] + b, allowed calculating concentration of CAP37 in the samples.

trophoresis. However, coupling of NE, PR3, CG, and CAP37 with CP caused a decrease of its electrophoretic mobility (Fig. 1a). It should be noted that upon mixing equimolar amounts of CP with serprocidins the former entirely entered the complex only with CAP37. In con trast, when NE, PR3, and CG were added, more than half of the amount of CP in the mixture did not interact with serprocidins. Preincubation of serprocidins with PMSF did not affect formation of the complex. Synthetic peptide RKARPRQFPRRR (513, 6163 CAP37) mim icking the cationic motif of CAP37 needed to display its antimicrobial properties and to bind to heparin [35] dis placed CAP37 from the complex with CP, while adding excess amounts of heparin resulted in dissociation of the CP–CAP37 complex and release of nonbound CP (Fig. 1b). Adding CAP37 to CP–LF–MPO or to CP–MPO complex would displace MPO from either of these com plexes and cause formation of new ones including, respectively, CP, LF, and CAP37 or CP and CAP37 (data not shown). Therefore, CAP37 successfully competes with MPO for the common binding site in CP. Adding

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RESULTS AND DISCUSSION Formation of complexes of CP with serprocidins in course of gel filtration. When subjected to gel filtration nonbound CP was eluted by 0.15 M NaCl in front of its complexes with serprocidins. This phenomenon prevented plain calculation of molecular mass of the complexes using the calibration curve. It should be noted that 1 mg of CP mixed with 0.5 mg of a serprocidin was always loaded on the gel filtration column, which corresponds to 1 : 2 ratio of CP to serprocidin. However, every time the first to elute was their 1 : 1 complex (1 mg of 132kDa CP and 0.23 mg of 30kDa serprocidin) followed by nonbound serprocidin (0.27 mg). This indicates that complexes of CP with ser procidins have 1 : 1 stoichiometry. A higher elution volume needed for CP–serprocidin complexes is likely to be caused by interaction of cationic serprocidins with the resin that we observed subjecting those separately to gel fil tration (data not shown). Gel filtration in the presence of 0.5 M NaCl resulted in dissociation of cationic proteins from the complexes with CP, so that the proteins were elut ed separately. Such an effect of ionic strength on interac tion of CP with serprocidins is in line with the data on elu tion of serprocidins by 0.5 M NaCl from CPSepharose in the course of affinity chromatography [14] and favors the notion of electrostatic character of that interaction. Formation of complexes of CP with serprocidins in detergentfree electrophoresis. We had shown in control experiments that positively charged serprocidins without CP do not enter polyacrylamide gel in alkaline disc elec

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Fig. 1. Electrophoretic analysis of interaction between CP and ser procidins. Staining with odianisidine. a) Interaction of CP (1 µg) with serprocidins (0.25 µg) in the presence of PMSF (0.01 µg): 1) CP + NE + PMSF; 2) CP + CG + PMSF; 3) CP + PR3 + PMSF; 4) CP; 5) CP + CAP37; 6) CP + NE; 7) CP + CG; 8) CP + PR3. b: 1) CP (1 µg); 2) CP (1 µg) + CAP37 (0.25 µg) + RKARPRQF PRRR (0.1 µg); 3) CP (1 µg) + CAP37 (0.25 µg); 4) CP (1 µg) + CAP37 (0.25 µg) + heparin (0.2 µg); 5) CP (1 µg) + RKARPRQF PRRR (0.1 µg). c) Blood sera: 1) human (2 µl); 2) mouse (10 µl); 3) dolphin (10 µl); 4) horse (10 µl); 5) rabbit (2 µl); 6) rat (1 µl); 7) dog (5 µl). CAP37 (0.1 µg) was added in series 1′7′.

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CERULOPLASMIN AND SERPROCIDINS

[NBA]/V, M·sec

0.08 0.06 0.04 PR3 + CP NE + CP PR3 NE

0.02 0.00 0.1

0.2

b [ВТЕ]/V, mM·sec

0.3

0.4

0.5

NBA, mM

2

1 CG + CP CG 0 1.0

2.0

3.0

ВТЕ, mM Fig. 2. Graphs plotted in Hanes–Woolf coordinates reflecting the inhibitory effect of 0.75 µM CP on activity of NE and PR3 (30 nM) towards NBA (0.10.5 mM) in 50 mM TrisHCl, pH 7.4 (a), and activity of CG (30 nM) towards BTE (0.52.5 mM) in 50 mM sodiumphosphate buffer, pH 7.4 (b).

each separate serprocidin under study to blood plasma of humans, rat, mouse, rabbit, dolphin, dog, and horse changed the electrophoretic mobility of oxidasepositive bands only in case of CAP37 (Fig. 1c; data shown only for CAP37). It seems likely that the preferred partners for serprocidins with proteinase activity (NE, CG, and PR3) are serpins, their plasma concentration and affinity to proteinases exceeding those of CP. Formation of inter specific complexes between CAP37 and CP from various mammalian species suggests evolutionary conservatism of the structural motifs in CP interacting with CAP37. Effect of CP on enzymatic activity of serprocidins. We studied an effect of CP on activity of NE and PR3 towards NBA (Fig. 2a) and on activity of CG displayed with BTE (Fig. 2b). In all cases CP did not affect kcat but increased Km, i.e. it behaved as a reversible competitive inhibitor (Table 1). For each proteinase Ki was ~ 1 µM, which is some three times less than CP concentration in healthy donors’ blood. However, since plasma content of classical serpins and their affinity towards proteinases studied are higher, CP in the human body is not likely to be the phys iological inhibitor of the latter, especially on account of its extra high susceptibility to proteolytic degradation. BIOCHEMISTRY (Moscow) Vol. 75 No. 11 2010

Studying the dependence of inhibition by CP of serpro cidin activity at minimum of substrate concentration on ionic strength showed that at NaCl content above 300 mM the inhibitory effect of CP is virtually absent (Fig. 3). This is another confirmation of our suggestion about the ionic character of interaction between CP and serprocidins. Effect of serprocidins on activity of CP. Preliminary study of the effect of serprocidins on oxidase activity of CP displayed towards various substrates (Fe2+, synthetic and biogenic amines) demonstrated that serprocidins affect exclusively the oxidation of pPD. They suppressed pPD oxidation as reversible competitive inhibitors, increasing Km without changing Vmax (Fig. 4). Maximum effect was displayed by CAP37 (Ki ~ 20 nM) (Table 2). Considering that CPcatalyzed pPD oxidation runs at pH 5.0, the inhibiting effect of serprocidins could not occur due to proteolytic degradation of CP. First, in our control experiments we observed no proteolysis of CP in the course of its incubation with serprocidins equal to the duration of oxidase reaction measurements (data not shown). This supported the notion that serprocidins are inactive at pH 5.0. Second, which is the most important, CAP37 that has the most pronounced effect on activity of CP is not a proteinase. Dissociation constant of CAP37 complex with immo bilized CP. On account that Ki is equal to the dissociation constant (Kd) of an enzyme–inhibitor complex and reflects the affinity of interaction directly affecting the enzyme’s catalytic properties, to evaluate correctly the affinity of CAP37 towards CP we attempted to determine Kd of a complex formed by CAP37 with immobilized CP. Measuring concentration of nonbound CAP37 after incubation with CPSepharose and with the control por tion of Sepharose allowed evaluating the specific binding of CAP37 with immobilized CP. Scatchard plots based on

1.0 1 – V(+CP)/V(–CP)

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NE + CP PR3 + CP CG + CP

0.8 0.6 0.4 0.2 0.0 50

150

250

350

NaCl, mM Fig. 3. Dependence on NaCl concentration of inhibitory effect of CP (3 µM) on activity of NE, PR3, and CG (30 nM) towards 0.1 mM NBA or 0.5 mM BTE in 50 mM sodiumphosphate buffer, pH 7.4.

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Table 1. Effect of CP on kinetic properties of serprocidins Proteinase/substrate

Km, µM

Km(+CP), µM

kcat, sec–1

kcat(+CP), sec–1

Ki, µM

NE/NBA

178 ± 16

295 ± 17

11.6 ± 0.2

11.3 ± 0.2

1.14 ± 0.08

PR3/NBA

436 ± 24

691 ± 21

15.8 ± 0.2

15.6 ± 0.2

1.28 ± 0.09

CG/BTE

1250 ± 80

2410 ± 80

2.1 ± 0.1

2.1 ± 0.1

0.81 ± 0.09

Table 2. Effect of serprocidins on kinetics of CPcatalyzed pPD oxidation Km, mM

Vmax, A530/min

Ki, nM

CP

0.53 ± 0.01

0.33 ± 0.01



CP + NE

0.94 ± 0.02

0.33 ± 0.01

131 ± 8

CP + PR3

0.83 ± 0.01

0.34 ± 0.01

182 ± 9

CP + CG

1.34 ± 0.02

0.32 ± 0.01

67 ± 5

CP + CAP37

3.27 ± 0.02

0.34 ± 0.01

20 ± 3

Serprocidins

results of adding CAP37 to CPSepharose aliquots with ~0.5 and 1 µM of immobilized CP gave the same values of Kd (Fig. 5). The approximating straight lines crossed the abscissa at 53 and 111 nM. Therefore, the relation of binding center concentrations is somewhat close to the relation of CPSepharose aliquots used in our experi ments (0.5/1 ~ 53/111). Summarizing the data obtained, one can draw some conclusions concerning the selectivity of interaction between CP and CAP37. It should be noted that CAP37

12

CP + PR3 CP

СР + CАP37 СР + CG CP + NE

6

2

4 [B]/[F]

[pPD]/V, mM·min

16

is the only representative of its family that interacts with CP in blood plasma. This means that serpins of the blood did not compete with CP for binding to CAP37 as it hap pens when CP interacts with proteolytically active serpro cidins. There are grounds to suggest that CAP37 involves the same site in its molecule for binding both to CP and to heparin. Interactions of CP with LF, protamine, and MPO follow the same pattern [7, 8]. Under certain conditions CP seems to be able to affect antimicrobial and heparinbinding properties of

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0 0.5

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[pPD], mM Fig. 4. Graphs plotted in Hanes–Woolf coordinates showing the inhibition by serprocidins (100 nM) of enzymatic activity of CP (50 nM) towards pPD (0.251.25 mM) in 0.1 M sodiumacetate buffer, pH 5.0.

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[B], nM

Fig. 5. Scatchard plots reflecting binding of CAP37 with CP Sepharose (0.5 and 1 µM CP). 1, 2) Kd = 13.7 ± 0.4 and 12.8 ± 0.4 nM, respectively.

BIOCHEMISTRY (Moscow) Vol. 75 No. 11 2010

CERULOPLASMIN AND SERPROCIDINS CAP37. It is noteworthy that Kd value obtained for inter action between CP and CAP37 coincides with Kd for highaffinity LFbinding site [36], with all this going on CAP37 competes for binding to CP not with LF, but with MPO. To all appearances the CP molecule has a common site for binding both CAP37 and MPO, since these two proteins affect the affinity of CP towards pPD. Displacement of MPO from its complex with CP is in line with the fact that Ka ~ 0.2 µM for activating effect of MPO [13] is 10 times higher than Ki ~ 20 nM for inhibito ry effect of CAP37.

14.

15. 16.

17. 18.

The authors are grateful to Prof. V. N. Kokryakov (Institute of Experimental Medicine, St. Petersburg) for generous supply by buffy coat. This study was supported by the Russian Foundation for Basic Research grants 090401059 and 100400820.

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