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Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes
Biochimie 84 (2002) 655–660
Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes Tatiana Bulai a, Daniela Bratosin b,*, Vlad Artenie a, Jean Montreuil c a Laboratorul de Biochimie, Facultatea de Biologie, Universitatea “Alexandru Ioan Cuza”, Bd-ul Copou no. 11, 6600 Iasi-6, Romania Institutul National de Cercetare-Dezvoltare pentru Stiinte Biologice, 296 Sp. Independentei, P.O. Box 17-16, 77748 Bucuresti, Romania c Laboratoire de chimie biologique, Université des sciences et technologies de Lille1 (USTL), Unité mixte de recherche du CNRS/USTL no. 8576, 59655 Villeneuve d’Ascq cedex, France b
Received 14 March 2002; accepted 28 May 2002
Abstract Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9-14C]Nacetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis–Menten type, Km: 1.4 ± 0.05 mM, optimal pH: 7.6 ± 0.2, optimal temperature: 70 ± 2 °C, inhibition by heavy metals: Ag+ and Hg++. These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Sialate pyruvate-lyase; Erythrocytes; Red blood cells; Mammalian lyases
1. Introduction Sialate pyruvate-lyase, also known as sialate aldolase (EC 4.1.3.3), catalyses specifically and reversibly the cleavage of free N-acetylneuraminic acid to form pyruvate and N-acetylmannosamine [1]. In 1958, this finding led to the determination by Comb and Roseman of N-acetylneuraminic acid structure [2] and synthesis [3], which results from the condensation of pyruvate with N-acetylmannosamine, and not with N-acetylglucosamine. Initially identified in Vibrio cholerae [1] and Clostridium perfringens [4], the enzyme has been detected in a number of pathogenic
Abbreviations: Neu5Ac, N-acetylneuraminic acid; CMP, cytidinemonophosphate; Hb, haemoglobin; ManNAc, N-acetylmannosamine; PBS, phosphate buffer solution, pH 7.4; RBC, red blood cell; TLC, thin-layer chromatography * Corresponding author. Tel.: +33-3-20-43-48-84; fax: +33-3-20-43-65-55. E-mail address: [email protected] (J. Montreuil).
as well as non-pathogenic bacteria. For methodological reasons, the most studied lyases are those from C. perfringens and Escherichia coli, of which their purification and crystallization have led to the determination of very similar characteristics. The bacterial enzymes are of the Michaelis–Menten type, with apparent Km values in the millimolar range (1.75–3.9 mmol). They exhibit an optimal pH varying from 7 to 7.7 and a pI between 4.5 and 5.5. The molecular mass of the denaturated enzymes as deduced from their gene structure was found to be about 33 kDa, while the value obtained from the native protein (90–110 kDa) led to the conclusion that the enzyme consists of three or four subunits (for general reviews, see [5–10]). The lyases cleave only free sialic acids, with a relative rate of 100% for N-acetylneuraminic acid, 70% for N-glycolylneuraminic acid and 33% for N-acetyl-9-O-acetylneuraminic acid, whereas they are inactive towards the other sialic acid structures (for review, see [7,10]). Microbial sialate pyruvatelyases are cytosolic enzymes and play an important role in the regulation of sialic acid metabolism in bacteria by con-
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 3 6 - 0
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trolling the intracellular concentration of sialic acid of biosynthetic or exogenous origin, thereby preventing the accumulation of toxic levels of this sugar [11]. They are typical inducible enzymes in microorganisms and are produced only in the presence of sialic acids [11,12] (for review, see [9]). The genes encoding some of these enzymes have been cloned and sequenced (for reviews, see [9,13]). In the animal kingdom, sialate pyruvate-lyase has been demonstrated in various tissues of rat, pig, guinea pig, cow and rabbit (for reviews, see [14]). The enzymes have been evidenced in rat [15] and mouse [16] intestine, frog liver and lung [14], human liver [17], murine teratocarcinoma cells [18] and promyelocytic leukaemic cells [19]. However, our knowledge of the characteristics of these enzymes is restricted to those identified in human liver and pig and bovine kidney. These lyases have properties similar to those of microbial species. They exhibit Michaelis–Menten type kinetics, with Km values from 1.7 to 3.7 and optimal pH from 7.2 to 8.0. In addition, like in bacteria, the aldolase activity has been found in the cytosol of human liver [17], pig [13] and beef [20] kidney. The substrate specificity of these enzymes is similar to that reported for the bacterial enzymes. Thus, the animal lyases cleave only free sialic acids, with a relative rate of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for 9-O,N-diacetylneuraminic acid, whereas the rate of cleavage was less than 2% for 4-O,N-diacetyl- and 7-O,Ndiacetylneuraminic acids [7]. Among the vertebrate lyases, only the pig kidney lyase isolated by affinity chromatography has been sequenced [13]. The research we developed during the last decade on the cellular and molecular mechanisms of programmed cell death of human erythrocytes [21–24] tempted us to explore the fate of sialic acid residues present in human erythrocyte membrane glycoconjugates, since it is well known that sialic acid plays a central role in the ageing of erythrocytes and their capture by macrophages (for review, see [23]). In the present paper, we describe the discovery and the main enzymatic characteristics of a sialate pyruvate-lyase present in the cytosol of the human erythrocyte.
2. Materials and methods 2.1. Materials Human blood type ORh+ collected on heparin was kindly supplied by the Centre Régional de Transfusion Sanguine de Lille. Drabkin’s reagent for the determination of haemoglobin, N-acetyl-D-mannosamine and p-dimethylaminobenzaldehyde were from Sigma Chemicals (Saint Louis, MO, USA), potassium tetraborate and N-acetylneuraminic acid from Fluka (Buchs, Switzerland) and the sodium pyruvate from Boehringer (Mannheim, RFA); Nunclon™ 96-well microtitre plates were purchased from Nunc A/S (Roskilde, Denmark). Colorimetric determination was car-
ried out using an automatic Microplate Reader, Model 550 (Biorad, Hercules, CA, USA). Solvent was eliminated from solutions using a Speed Vac Concentrator connected to a refrigerated condensation trap (Savant Instruments Inc., Farmingdale, NY, USA). TLC aluminium sheets (Silicagel 60) were from Merk Eurolab (Strasbourg, France), Biomax™ MR films from Kodak (Rochester, NY, USA) and Sep-Pak 1 cartridges from Waters Corporation (Milford, MA, USA). Cytidine 5'-monophosphate [4,5,6,7,8,914 C]sialic acid ammonium salt (25 µCi/ml) was from Amersham Pharmacia Biotech (Little Chalfont, England). 2.2. Isolation of erythrocytes Heparinized human blood was processed within 1 h of collection. Cells were sedimented by centrifugation (1000 × g; 4 °C; 5 min). After removal of plasma platelets and leukocytes by aspiration, the cells were washed three times with PBS buffer containing 0.2 mM PMSF as antiproteases. 2.3. Isolation of erythrocyte membranes and cytosol Cell ghosts of erythrocytes were prepared by lysis in 40 volumes of a hypotonic buffer (pH 7.4) obtained by the addition of nine volumes of water to the PBS buffer (pH 7.4) [25]. RBC membranes were isolated by ultracentrifugation of the lysate (10 000 × g; 4 °C; 20 min). Haemoglobin contaminating the ghosts was totally eliminated by washing the pellet three times with hypotonic PBS buffer of pH 7.4. Cytosol from the red blood cells was obtained by haemolysis of the cells by stirring for 1 min in three volumes of cold water and removing cell membranes by ultracentrifugation (10 000 × g; 4 °C; 20 min). Haemoglobin was determined using the Drabkin reagent kit. 2.4. Enzyme assay Sialate pyruvate-lyase activity was assayed by an original colorimetric micromethod for measuring the amount of N-acetylmannosamine liberated by the action of the enzyme. The protocol was as follows: to 25 µl of 32 mM N-acetylneuraminic acid in aqueous solution, 500 µl of cytosol and 75 µl of a 1 M potassium phosphate buffer of pH 7.4 were added. The blanks were carried out in the same conditions in the absence of N-acetylneuraminic acid. After incubation at 37 °C for 6 h, 3 ml of cold (– 20 °C) absolute ethanol were added while stirring. After centrifugation (1800 × g; 4 °C; 15 min), 2 ml of the supernatant were evaporated to dryness using a Speed Vac Concentrator. The “dry material” was dissolved in 100 µl of water and the liberated N-acetylmannosamine was determined. Briefly, 20 µl of the above solution were distributed in wells of a 96-well plastic microtitre plate (Nunclon™). To this, 15 µl of 0.2 M potassium tetraborate were added, and the mixture was maintained at 80 °C for 1 h. After cooling at room
T. Bulai et al. / Biochimie 84 (2002) 655–660
temperature, 180 µl of Ehrlich reagent prepared immediately before use were added. The Ehrlich reagent was made by diluting a stock solution (10 g p-dimethylaminobenzaldehyde in 87.5 ml glacial acetic acid containing 12.5 ml of concentrated hydrochloric acid) 10-fold with glacial acetic acid as described [26]. Then, after shaking for 5 min on a minishaker, the microplates were incubated at 37 °C for 30 min and the absorbances measured at 540 nm using an automatic microtitre plate reader (Microplate Reader, Model 550). For each experiment, assays were carried out in triplicate. An internal standard curve of reference was established using concentrations from 0 to 10 µg N-acetylmannosamine per 20 µl (from 0 to 2 mM). The sensitivity of the method varies in a linear way from 1 to 10 µg of N-acetylmannosamine in a final volume of 20 µl. The coloration is stable for 30 min. One unit of sialate lyase activity is defined as the amount that releases 1 µmol of N-acetylmannosamine per minute under the reaction conditions used. 2.5. Use of [14C]-labelled N-acetylneuraminic acid Labelled N-acetylneuraminic acid was prepared by hydrolysis of CMP-[4,5,6,7,8,9-14C] sialic acid (corresponding to 10 µCi) with 2 M acetic acid at 80 °C for 1 h [27]. The hydrolysate was evaporated to dryness under nitrogen stream and dissolved in 500 µl of PBS buffer of pH 7.4. The efficiency of hydrolysis was controlled by thin-layer chromatography of the hydrolysate corresponding to 20 000 dpm. Chromatography was carried out on TLC aluminium sheets (Silicagel 60) using an n-butanol/acetic acid/H2O (2:1:1.5 in volume) solvent system for 6 h at 22 °C. Autoradiography was performed with Biomax™ MR films (exposure times from 1 to 5 days). Markers of N-acetylneuraminic acid and N-acetylmannosamine were revealed with Svennerholm’s reagent [28]. Incubations with RBC cytosol were carried out as described under Section 2.4, except: (i) 2.5 µl of the solution of [14C]N-acetylneuraminic acid described above were added, corresponding to 100 000 dpm; (ii) the incubation time was extended from 6 to 12 h; (iii) the “dry material” obtained after the elimination of proteins with ethanol was dissolved in 500 µl of water and purified on a microcolumn of Sep-Pak previously washed with 5 ml of a mixture of methanol/H2O (1:1 in volume) and then with 5 ml of water. After washing the column with 3 ml of water, the obtained eluate was evaporated to dryness in vacuo. The dry residue was dissolved in 50 µl of water and was submitted to TLC as described above.
3. Results and discussion
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Fig. 1. Identification by autoradiography of thin-layer chromatogram of [14C]-N-acetylmannosamine liberated by incubation of [14C]-N-acetylneuraminic acid with blood plasma (lane 1), erythrocyte membranes (lane2) and cytosol (lane 3). Lane 4: [14C]-N-acetylneuraminic acid control and lane 5: N-acetylneuraminic acid and N-acetylmannosamine standards revealed with Svennerholm’s reagent [25].
unambiguously that the neuraminate aldolase activity is located entirely in the RBC cytosol and that the cell membranes were devoid of any activity. According to Beutler [29], the activity of the enzyme was expressed in milliunits per gram of haemoglobin or in percentage of cleaved N-acetylneuraminic acid. No activity was detected in blood plasma. The cytosolic activity was confirmed by the characterization of liberated [14C]N-acetylmannosamine by autoradiography of thin-layer chromatograms (Fig. 1). 3.2. Determination of enzyme properties 3.2.1. Effects of pH, temperature and incubation time These variables were studied using the procedures described under Section 2.4. The rate of the cleaving reaction was optimal at pH 7.6 (Fig. 2) and at a temperature of 70 ± 2 °C (Fig. 3). However, we chose to work in physiological conditions (pH 7.4 and 37 °C). The reaction rate was linear with time ranging from 0 to 6 h (Fig. 4), and the enzyme is very active, since in non-optimal conditions of temperature its activity rises 9.2 ± 0.2 mU/g Hb leading to the cleavage of 28% of the substrate in 6 h. 3.2.2. Effect of substrate concentration The effect of increasing concentrations of N-acetylneuraminic acid on the rate of the reaction clearly demonstrates that the cytosolic RBC sialate pyruvate-lyase is of the Michaelis–Menten type. Calculation of Km and Vmax according to the method of Lineweaver–Burk gave an apparent value of 1.4 ± 0.05 mM for Km and 35 ± 2 mU/g Hb for Vmax.
3.1. Evidence for a cytosolic localization of the enzyme The application of the colorimetric micromethod of N-acetylmannosamine determination we devised showed
3.2.3. Effect of RBC lysate concentration The effect of enzyme concentration on the reaction rates was determined using increasing volumes of lysate and
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Fig. 2. Effect of pH on human RBC cytosolic sialate pyruvate-lyase activity expressed in mU/g haemoglobin (in ordinates). Incubation was performed for 6 h at 37 °C in 0.25 M potassium phosphate buffer pH ranging from 5.5 to 8.5. In abcissae: pH values.
Fig. 4. Effect of incubation time on human RBC cytosolic sialate pyruvatelyase activity expressed in cleavage rate of N-acetylneuraminic acid as described in the experimental conditions in Section 2.4.
expressed in milliunits per gram Hb. As shown in Fig. 5, the increase of enzyme activity is linear. 3.2.4. Substrate specificity At enzyme-saturation substrate concentrations, N-glycolylneuraminic acid was cleaved at approximately 60% of the rate obtained with N-acetylneuraminic acid. This result is similar to that previously reported with bacterial and animal enzymes, with relative rates of 70% and 55%, respectively [7,10]. 3.2.5. Effect of metal ions The RBC lysate was preincubated (15 min; 20 °C) with metal ions at a final concentration of 1 mM. The obtained
Fig. 3. Effect of temperature on human RBC cytosolic sialate pyruvatelyase activity expressed in mU/g haemoglobin. Incubation was performed for 6 h in 0.25 M potassium phosphate buffer pH 7.4.
results show that the following cations are not inhibitors or activators of the enzyme activity (99 ± 3% of the activity): Ba2+, Ca2+, Mg2+, Mn2+, Zn2+, Fe2+, Fe3+, Cu2+. The enzyme, however, is markedly inhibited by two heavy metal ions: Ag+ and Hg2+, with inhibition values of 36 ± 5% and 43 ± 5%, respectively. These values are similar to those described for the purified enzyme from E. coli [30]. 3.2.6. Effect of pyruvate Pyruvate shows a strong inhibitory effect on aldolase activity depending on the pyruvate concentration: 0.1 mM:
Fig. 5. Kinetics of human RBC cytosolic sialate pyruvate-lyase activity in terms of increasing volumes of lysate.
T. Bulai et al. / Biochimie 84 (2002) 655–660
6%; 1 mM: 36%; 5 mM: 65%; 10 mM: 76%. The inhibitory effect was similar for N-glycolylneuraminic acid. The apparent inhibition constant, Ki, obtained from Lineweaver–Burk plots and for a final concentration of 10 mM pyruvate was 1.2 ± 0.2 mM. Similar results were obtained from microbial lyases [4,30,31].
[3]
[4]
[5] [6]
4. Concluding remarks [7]
We have, for the first time, characterized a sialate pyruvate-lyase activity in human erythrocyte cytosol that is totally absent in the cell membrane as well as in circulating blood plasma. The enzymatic parameters of this lyase are similar to those of the microbial and mammalian lyases described up to now. Obviously, the information we bring regarding this enzyme is at the moment partial, but the aim of this paper was mainly to show the localization in the human RBC cytosol of a sialate pyruvate-lyase of which we have not yet elucidated the biological role. However, we are tempted to hypothesize that this enzyme is related to the desialylation process that occurs during RBC ageing and that the role it plays is to destroy the free sialic acid residues liberated by membrane sialidases. Such a mechanism implies the presence of a sialic acid transporter in the RBC membrane, the existence of which remains to be demonstrated.
Acknowledgements This work was supported in part by grants from the Université des Sciences et Technologies de Lille, the Centre National de la Recherche Scientifique (UMR USTL/CNRS no. 8576; Director: Dr. Jean-Claude Michalski), the Etablissement Régional de Transfusion Sanguine de Lille (Director: Jean-Jacques Huart) and the MacoPharma Company (Tourcoing, France; Chairman: Hervé Dubly, and Drs. Joël Poplineau and Francis Goudaliez). We are grateful to Professors Stéphane Bouquelet, René Cacan, Philippe Delannoy, Dr. Frédéric Chirat and Monique Benaïssa for their invaluable help in the domains of enzymology and the use of radiolabelled compounds. We are indebted to Dr. Michael K. Kibe, who translated the manuscript. This work was initiated during the late Professor André Verbert’s tenure as Director of the Institute. We wish to express our heartfelt gratitude for his constant, smiling and enthusiastic support. T.B. has a doctoral fellowship from the French Government.
References [1] [2]
R. Heimer, K. Meyer, Studies on sialic acid of submaxillary mucoid, Proc. Natl. Acad. Sci. USA 42 (1956) 728–734. D.G. Comb, S. Roseman, Composition and enzymatic synthesis of N-acetylneuraminic acid (sialic acid), J. Am. Chem. Soc. 80 (1958) 497–499.
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
[21]
[22]
[23]
659
D.G. Comb, S. Roseman, The sialic acids. I. The structure and enzymatic synthesis of N-acetylneuraminic acid, J. Biol. Chem. 235 (1960) 2529–2537. S. Nees, R. Schauer, F. Mayer, Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens, Hoppe Seyler’s Z. Physiol. Chem. 357 (1976) 839–853. A. Varki, Diversity in the sialic acids, Glycobiology 2 (1992) 25–40. T. Izard, M.C. Lawrence, R.L. Malby, G.G. Lilley, P.M. Colman, The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli, Structure 2 (1994) 361–369. R. Schauer, J.P. Kamerling, Chemistry, biochemistry and biology of sialic acids, in: J. Montreuil, J.F.G. Vliegenthart, H. Schachter (Eds.), Glycoproteins, Elsevier, Amsterdam, 1997, pp. 243–402. C. Traving, R. Schauer, Structure, function and metabolism of sialic acids, Cell. Mol. Life Sci. 54 (1998) 1330–1349. R. Schauer, U. Sommer, D. Krüger, H. van Unen, C. Traving, The terminal enzymes of sialic acid metabolism: acylneuraminate pyruvate-lyases, Biosci. Rep. 19 (1999) 373–383. R. Schauer, M. Wember, Isolation and characterization of sialate lyase from pig kidney, Biol. Chem. Hoppe-Seyler 377 (1996) 293–299. E.R. Vimr, F.A. Troy, Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli, J. Bacteriol. 164 (1985) 845–853. E.R. Vimr, F.A. Troy, Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase, J. Bacteriol. 164 (1985) 854–860. U. Sommer, C. Traving, R. Schauer, The sialate pyruvate-lyase from pig kidney: purification, properties and genetic relationships, Glycoconjugate J. 16 (1999) 425–435. P. Brunetti, G.W. Jourdian, S. Roseman, The sialic acids. III. Distribution and properties of animal N-acetylneuraminic aldolase, J. Biol. Chem. 237 (1962) 2447–2453. W. Witt, H. von Nicolai, F. Zilliker, Uptake and distribution of orally applied N-acetyl-(14C)neuraminosyl-lactose and N-acetyl(14C)neuraminic acid in the organs of newborn rats, Nutr. Metab. 23 (1979) 51–61. U. Nöhle, R. Schauer, Uptake, metabolism and excretion of orally and intravenously administered, 14C- and 3H-labeled N-acetylneuraminic acid mixture in the mouse and rat, Hoppe Seyler’s Z. Physiol. Chem. 362 (1981) 1495–1506. A.P. Corfield, R. Schauer, Metabolism of sialic acids, in: R. Schauer (Ed.), Sialic Acids. Chemistry, Metabolism and Function, Cell Biology Monographs, vol 10, Springer-Verlag, Vienna, 1982, pp. 195–261. L. Warren, Sialic acid lyase in differentiating murine teratocarcinoma cells, Exp. Cell. Res. 16 (1985) 307–316. L. Warren, Sialic acid lyase in human promyelocytic leukaemic cells (HL-60) during phorbol-ester-induced differentiation, Biochim. Biophys. Acta 888 (1986) 278–281. D.A. Sirbasku, S.B. Binkley, Purification and properties of N-acetylneuraminate lyase from kidney cortex, Biochim. Biophys. Acta 206 (1970) 479–482. D. Bratosin, J. Mazurier, H. Debray, M. Lecocq, B. Boilly, C. Alonso, M. Moisei, C. Motas, J. Montreuil, Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span, Glycoconjugate J. 12 (1995) 258–267. D. Bratosin, J. Mazurier, J.P. Tissier, C. Slomianny, D. Aminoff, J. Montreuil, Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages, C. R. Acad. Sci. Paris 320 (1997) 811–818. D. Bratosin, J. Mazurier, J.P. Tissier, J. Estaquier, J.J. Huart, J.C. Ameisen, D. Aminoff, J. Montreuil, Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages, A review, Biochimie 80 (1998) 173–195.
660
T. Bulai et al. / Biochimie 84 (2002) 655–660
[24] D. Bratosin, J. Estaquier, F. Petit, D. Arnoult, B. Quatannens, J.P. Tissier, C. Slomianny, C. Sartiaux, C. Alonso, J.J. Huart, J. Montreuil, J.C. Ameisen, Programed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria, Cell Death Differ. 8 (2001) 1143–1156. [25] C.G. Gahmberg, S.I. Hakomori, External labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes, J. Biol. Chem. 248 (1973) 4311–4317. [26] J.L. Reissig, J.L. Strominger, L.F. Leloir, A modified colorimetric method for the estimation of N-acetylamino sugar, J. Biol. Chem. 217 (1955) 959–966. [27] A. Varki, S. Diaz, The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups, Anal. Biochem. 137 (1984) 236–247.
[28] E. Svennerholm, L. Svennerholm, Qualitative paper partition chromatography of sialic acids, Nature 181 (1958) 1154–1156. [29] E. Beutler, Calculation of results, in: E. Beutler (Ed.), Red Cell Metabolism: A Manual of Biochemical Methods, Grume & Stratton, New York, 1984, pp. 31–36. [30] Y. Uchida, Y. Tsukada, T. Sugimori, Purification and properties of N-acetylneuraminate-lyase from Escherichia coli, J. Biochem. 96 (1984) 507–522. [31] K. Aisaka, A. Igarashi, K. Yamaguchi, T. Uwajima, Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli, Biochem. J. 276 (1991) 541–546.
Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes Tatiana Bulai a, Daniela Bratosin b,*, Vlad Artenie a, Jean Montreuil c a Laboratorul de Biochimie, Facultatea de Biologie, Universitatea “Alexandru Ioan Cuza”, Bd-ul Copou no. 11, 6600 Iasi-6, Romania Institutul National de Cercetare-Dezvoltare pentru Stiinte Biologice, 296 Sp. Independentei, P.O. Box 17-16, 77748 Bucuresti, Romania c Laboratoire de chimie biologique, Université des sciences et technologies de Lille1 (USTL), Unité mixte de recherche du CNRS/USTL no. 8576, 59655 Villeneuve d’Ascq cedex, France b
Received 14 March 2002; accepted 28 May 2002
Abstract Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9-14C]Nacetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis–Menten type, Km: 1.4 ± 0.05 mM, optimal pH: 7.6 ± 0.2, optimal temperature: 70 ± 2 °C, inhibition by heavy metals: Ag+ and Hg++. These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Sialate pyruvate-lyase; Erythrocytes; Red blood cells; Mammalian lyases
1. Introduction Sialate pyruvate-lyase, also known as sialate aldolase (EC 4.1.3.3), catalyses specifically and reversibly the cleavage of free N-acetylneuraminic acid to form pyruvate and N-acetylmannosamine [1]. In 1958, this finding led to the determination by Comb and Roseman of N-acetylneuraminic acid structure [2] and synthesis [3], which results from the condensation of pyruvate with N-acetylmannosamine, and not with N-acetylglucosamine. Initially identified in Vibrio cholerae [1] and Clostridium perfringens [4], the enzyme has been detected in a number of pathogenic
Abbreviations: Neu5Ac, N-acetylneuraminic acid; CMP, cytidinemonophosphate; Hb, haemoglobin; ManNAc, N-acetylmannosamine; PBS, phosphate buffer solution, pH 7.4; RBC, red blood cell; TLC, thin-layer chromatography * Corresponding author. Tel.: +33-3-20-43-48-84; fax: +33-3-20-43-65-55. E-mail address: [email protected] (J. Montreuil).
as well as non-pathogenic bacteria. For methodological reasons, the most studied lyases are those from C. perfringens and Escherichia coli, of which their purification and crystallization have led to the determination of very similar characteristics. The bacterial enzymes are of the Michaelis–Menten type, with apparent Km values in the millimolar range (1.75–3.9 mmol). They exhibit an optimal pH varying from 7 to 7.7 and a pI between 4.5 and 5.5. The molecular mass of the denaturated enzymes as deduced from their gene structure was found to be about 33 kDa, while the value obtained from the native protein (90–110 kDa) led to the conclusion that the enzyme consists of three or four subunits (for general reviews, see [5–10]). The lyases cleave only free sialic acids, with a relative rate of 100% for N-acetylneuraminic acid, 70% for N-glycolylneuraminic acid and 33% for N-acetyl-9-O-acetylneuraminic acid, whereas they are inactive towards the other sialic acid structures (for review, see [7,10]). Microbial sialate pyruvatelyases are cytosolic enzymes and play an important role in the regulation of sialic acid metabolism in bacteria by con-
© 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 3 6 - 0
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trolling the intracellular concentration of sialic acid of biosynthetic or exogenous origin, thereby preventing the accumulation of toxic levels of this sugar [11]. They are typical inducible enzymes in microorganisms and are produced only in the presence of sialic acids [11,12] (for review, see [9]). The genes encoding some of these enzymes have been cloned and sequenced (for reviews, see [9,13]). In the animal kingdom, sialate pyruvate-lyase has been demonstrated in various tissues of rat, pig, guinea pig, cow and rabbit (for reviews, see [14]). The enzymes have been evidenced in rat [15] and mouse [16] intestine, frog liver and lung [14], human liver [17], murine teratocarcinoma cells [18] and promyelocytic leukaemic cells [19]. However, our knowledge of the characteristics of these enzymes is restricted to those identified in human liver and pig and bovine kidney. These lyases have properties similar to those of microbial species. They exhibit Michaelis–Menten type kinetics, with Km values from 1.7 to 3.7 and optimal pH from 7.2 to 8.0. In addition, like in bacteria, the aldolase activity has been found in the cytosol of human liver [17], pig [13] and beef [20] kidney. The substrate specificity of these enzymes is similar to that reported for the bacterial enzymes. Thus, the animal lyases cleave only free sialic acids, with a relative rate of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for 9-O,N-diacetylneuraminic acid, whereas the rate of cleavage was less than 2% for 4-O,N-diacetyl- and 7-O,Ndiacetylneuraminic acids [7]. Among the vertebrate lyases, only the pig kidney lyase isolated by affinity chromatography has been sequenced [13]. The research we developed during the last decade on the cellular and molecular mechanisms of programmed cell death of human erythrocytes [21–24] tempted us to explore the fate of sialic acid residues present in human erythrocyte membrane glycoconjugates, since it is well known that sialic acid plays a central role in the ageing of erythrocytes and their capture by macrophages (for review, see [23]). In the present paper, we describe the discovery and the main enzymatic characteristics of a sialate pyruvate-lyase present in the cytosol of the human erythrocyte.
2. Materials and methods 2.1. Materials Human blood type ORh+ collected on heparin was kindly supplied by the Centre Régional de Transfusion Sanguine de Lille. Drabkin’s reagent for the determination of haemoglobin, N-acetyl-D-mannosamine and p-dimethylaminobenzaldehyde were from Sigma Chemicals (Saint Louis, MO, USA), potassium tetraborate and N-acetylneuraminic acid from Fluka (Buchs, Switzerland) and the sodium pyruvate from Boehringer (Mannheim, RFA); Nunclon™ 96-well microtitre plates were purchased from Nunc A/S (Roskilde, Denmark). Colorimetric determination was car-
ried out using an automatic Microplate Reader, Model 550 (Biorad, Hercules, CA, USA). Solvent was eliminated from solutions using a Speed Vac Concentrator connected to a refrigerated condensation trap (Savant Instruments Inc., Farmingdale, NY, USA). TLC aluminium sheets (Silicagel 60) were from Merk Eurolab (Strasbourg, France), Biomax™ MR films from Kodak (Rochester, NY, USA) and Sep-Pak 1 cartridges from Waters Corporation (Milford, MA, USA). Cytidine 5'-monophosphate [4,5,6,7,8,914 C]sialic acid ammonium salt (25 µCi/ml) was from Amersham Pharmacia Biotech (Little Chalfont, England). 2.2. Isolation of erythrocytes Heparinized human blood was processed within 1 h of collection. Cells were sedimented by centrifugation (1000 × g; 4 °C; 5 min). After removal of plasma platelets and leukocytes by aspiration, the cells were washed three times with PBS buffer containing 0.2 mM PMSF as antiproteases. 2.3. Isolation of erythrocyte membranes and cytosol Cell ghosts of erythrocytes were prepared by lysis in 40 volumes of a hypotonic buffer (pH 7.4) obtained by the addition of nine volumes of water to the PBS buffer (pH 7.4) [25]. RBC membranes were isolated by ultracentrifugation of the lysate (10 000 × g; 4 °C; 20 min). Haemoglobin contaminating the ghosts was totally eliminated by washing the pellet three times with hypotonic PBS buffer of pH 7.4. Cytosol from the red blood cells was obtained by haemolysis of the cells by stirring for 1 min in three volumes of cold water and removing cell membranes by ultracentrifugation (10 000 × g; 4 °C; 20 min). Haemoglobin was determined using the Drabkin reagent kit. 2.4. Enzyme assay Sialate pyruvate-lyase activity was assayed by an original colorimetric micromethod for measuring the amount of N-acetylmannosamine liberated by the action of the enzyme. The protocol was as follows: to 25 µl of 32 mM N-acetylneuraminic acid in aqueous solution, 500 µl of cytosol and 75 µl of a 1 M potassium phosphate buffer of pH 7.4 were added. The blanks were carried out in the same conditions in the absence of N-acetylneuraminic acid. After incubation at 37 °C for 6 h, 3 ml of cold (– 20 °C) absolute ethanol were added while stirring. After centrifugation (1800 × g; 4 °C; 15 min), 2 ml of the supernatant were evaporated to dryness using a Speed Vac Concentrator. The “dry material” was dissolved in 100 µl of water and the liberated N-acetylmannosamine was determined. Briefly, 20 µl of the above solution were distributed in wells of a 96-well plastic microtitre plate (Nunclon™). To this, 15 µl of 0.2 M potassium tetraborate were added, and the mixture was maintained at 80 °C for 1 h. After cooling at room
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temperature, 180 µl of Ehrlich reagent prepared immediately before use were added. The Ehrlich reagent was made by diluting a stock solution (10 g p-dimethylaminobenzaldehyde in 87.5 ml glacial acetic acid containing 12.5 ml of concentrated hydrochloric acid) 10-fold with glacial acetic acid as described [26]. Then, after shaking for 5 min on a minishaker, the microplates were incubated at 37 °C for 30 min and the absorbances measured at 540 nm using an automatic microtitre plate reader (Microplate Reader, Model 550). For each experiment, assays were carried out in triplicate. An internal standard curve of reference was established using concentrations from 0 to 10 µg N-acetylmannosamine per 20 µl (from 0 to 2 mM). The sensitivity of the method varies in a linear way from 1 to 10 µg of N-acetylmannosamine in a final volume of 20 µl. The coloration is stable for 30 min. One unit of sialate lyase activity is defined as the amount that releases 1 µmol of N-acetylmannosamine per minute under the reaction conditions used. 2.5. Use of [14C]-labelled N-acetylneuraminic acid Labelled N-acetylneuraminic acid was prepared by hydrolysis of CMP-[4,5,6,7,8,9-14C] sialic acid (corresponding to 10 µCi) with 2 M acetic acid at 80 °C for 1 h [27]. The hydrolysate was evaporated to dryness under nitrogen stream and dissolved in 500 µl of PBS buffer of pH 7.4. The efficiency of hydrolysis was controlled by thin-layer chromatography of the hydrolysate corresponding to 20 000 dpm. Chromatography was carried out on TLC aluminium sheets (Silicagel 60) using an n-butanol/acetic acid/H2O (2:1:1.5 in volume) solvent system for 6 h at 22 °C. Autoradiography was performed with Biomax™ MR films (exposure times from 1 to 5 days). Markers of N-acetylneuraminic acid and N-acetylmannosamine were revealed with Svennerholm’s reagent [28]. Incubations with RBC cytosol were carried out as described under Section 2.4, except: (i) 2.5 µl of the solution of [14C]N-acetylneuraminic acid described above were added, corresponding to 100 000 dpm; (ii) the incubation time was extended from 6 to 12 h; (iii) the “dry material” obtained after the elimination of proteins with ethanol was dissolved in 500 µl of water and purified on a microcolumn of Sep-Pak previously washed with 5 ml of a mixture of methanol/H2O (1:1 in volume) and then with 5 ml of water. After washing the column with 3 ml of water, the obtained eluate was evaporated to dryness in vacuo. The dry residue was dissolved in 50 µl of water and was submitted to TLC as described above.
3. Results and discussion
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Fig. 1. Identification by autoradiography of thin-layer chromatogram of [14C]-N-acetylmannosamine liberated by incubation of [14C]-N-acetylneuraminic acid with blood plasma (lane 1), erythrocyte membranes (lane2) and cytosol (lane 3). Lane 4: [14C]-N-acetylneuraminic acid control and lane 5: N-acetylneuraminic acid and N-acetylmannosamine standards revealed with Svennerholm’s reagent [25].
unambiguously that the neuraminate aldolase activity is located entirely in the RBC cytosol and that the cell membranes were devoid of any activity. According to Beutler [29], the activity of the enzyme was expressed in milliunits per gram of haemoglobin or in percentage of cleaved N-acetylneuraminic acid. No activity was detected in blood plasma. The cytosolic activity was confirmed by the characterization of liberated [14C]N-acetylmannosamine by autoradiography of thin-layer chromatograms (Fig. 1). 3.2. Determination of enzyme properties 3.2.1. Effects of pH, temperature and incubation time These variables were studied using the procedures described under Section 2.4. The rate of the cleaving reaction was optimal at pH 7.6 (Fig. 2) and at a temperature of 70 ± 2 °C (Fig. 3). However, we chose to work in physiological conditions (pH 7.4 and 37 °C). The reaction rate was linear with time ranging from 0 to 6 h (Fig. 4), and the enzyme is very active, since in non-optimal conditions of temperature its activity rises 9.2 ± 0.2 mU/g Hb leading to the cleavage of 28% of the substrate in 6 h. 3.2.2. Effect of substrate concentration The effect of increasing concentrations of N-acetylneuraminic acid on the rate of the reaction clearly demonstrates that the cytosolic RBC sialate pyruvate-lyase is of the Michaelis–Menten type. Calculation of Km and Vmax according to the method of Lineweaver–Burk gave an apparent value of 1.4 ± 0.05 mM for Km and 35 ± 2 mU/g Hb for Vmax.
3.1. Evidence for a cytosolic localization of the enzyme The application of the colorimetric micromethod of N-acetylmannosamine determination we devised showed
3.2.3. Effect of RBC lysate concentration The effect of enzyme concentration on the reaction rates was determined using increasing volumes of lysate and
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Fig. 2. Effect of pH on human RBC cytosolic sialate pyruvate-lyase activity expressed in mU/g haemoglobin (in ordinates). Incubation was performed for 6 h at 37 °C in 0.25 M potassium phosphate buffer pH ranging from 5.5 to 8.5. In abcissae: pH values.
Fig. 4. Effect of incubation time on human RBC cytosolic sialate pyruvatelyase activity expressed in cleavage rate of N-acetylneuraminic acid as described in the experimental conditions in Section 2.4.
expressed in milliunits per gram Hb. As shown in Fig. 5, the increase of enzyme activity is linear. 3.2.4. Substrate specificity At enzyme-saturation substrate concentrations, N-glycolylneuraminic acid was cleaved at approximately 60% of the rate obtained with N-acetylneuraminic acid. This result is similar to that previously reported with bacterial and animal enzymes, with relative rates of 70% and 55%, respectively [7,10]. 3.2.5. Effect of metal ions The RBC lysate was preincubated (15 min; 20 °C) with metal ions at a final concentration of 1 mM. The obtained
Fig. 3. Effect of temperature on human RBC cytosolic sialate pyruvatelyase activity expressed in mU/g haemoglobin. Incubation was performed for 6 h in 0.25 M potassium phosphate buffer pH 7.4.
results show that the following cations are not inhibitors or activators of the enzyme activity (99 ± 3% of the activity): Ba2+, Ca2+, Mg2+, Mn2+, Zn2+, Fe2+, Fe3+, Cu2+. The enzyme, however, is markedly inhibited by two heavy metal ions: Ag+ and Hg2+, with inhibition values of 36 ± 5% and 43 ± 5%, respectively. These values are similar to those described for the purified enzyme from E. coli [30]. 3.2.6. Effect of pyruvate Pyruvate shows a strong inhibitory effect on aldolase activity depending on the pyruvate concentration: 0.1 mM:
Fig. 5. Kinetics of human RBC cytosolic sialate pyruvate-lyase activity in terms of increasing volumes of lysate.
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6%; 1 mM: 36%; 5 mM: 65%; 10 mM: 76%. The inhibitory effect was similar for N-glycolylneuraminic acid. The apparent inhibition constant, Ki, obtained from Lineweaver–Burk plots and for a final concentration of 10 mM pyruvate was 1.2 ± 0.2 mM. Similar results were obtained from microbial lyases [4,30,31].
[3]
[4]
[5] [6]
4. Concluding remarks [7]
We have, for the first time, characterized a sialate pyruvate-lyase activity in human erythrocyte cytosol that is totally absent in the cell membrane as well as in circulating blood plasma. The enzymatic parameters of this lyase are similar to those of the microbial and mammalian lyases described up to now. Obviously, the information we bring regarding this enzyme is at the moment partial, but the aim of this paper was mainly to show the localization in the human RBC cytosol of a sialate pyruvate-lyase of which we have not yet elucidated the biological role. However, we are tempted to hypothesize that this enzyme is related to the desialylation process that occurs during RBC ageing and that the role it plays is to destroy the free sialic acid residues liberated by membrane sialidases. Such a mechanism implies the presence of a sialic acid transporter in the RBC membrane, the existence of which remains to be demonstrated.
Acknowledgements This work was supported in part by grants from the Université des Sciences et Technologies de Lille, the Centre National de la Recherche Scientifique (UMR USTL/CNRS no. 8576; Director: Dr. Jean-Claude Michalski), the Etablissement Régional de Transfusion Sanguine de Lille (Director: Jean-Jacques Huart) and the MacoPharma Company (Tourcoing, France; Chairman: Hervé Dubly, and Drs. Joël Poplineau and Francis Goudaliez). We are grateful to Professors Stéphane Bouquelet, René Cacan, Philippe Delannoy, Dr. Frédéric Chirat and Monique Benaïssa for their invaluable help in the domains of enzymology and the use of radiolabelled compounds. We are indebted to Dr. Michael K. Kibe, who translated the manuscript. This work was initiated during the late Professor André Verbert’s tenure as Director of the Institute. We wish to express our heartfelt gratitude for his constant, smiling and enthusiastic support. T.B. has a doctoral fellowship from the French Government.
References [1] [2]
R. Heimer, K. Meyer, Studies on sialic acid of submaxillary mucoid, Proc. Natl. Acad. Sci. USA 42 (1956) 728–734. D.G. Comb, S. Roseman, Composition and enzymatic synthesis of N-acetylneuraminic acid (sialic acid), J. Am. Chem. Soc. 80 (1958) 497–499.
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18] [19]
[20]
[21]
[22]
[23]
659
D.G. Comb, S. Roseman, The sialic acids. I. The structure and enzymatic synthesis of N-acetylneuraminic acid, J. Biol. Chem. 235 (1960) 2529–2537. S. Nees, R. Schauer, F. Mayer, Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens, Hoppe Seyler’s Z. Physiol. Chem. 357 (1976) 839–853. A. Varki, Diversity in the sialic acids, Glycobiology 2 (1992) 25–40. T. Izard, M.C. Lawrence, R.L. Malby, G.G. Lilley, P.M. Colman, The three-dimensional structure of N-acetylneuraminate lyase from Escherichia coli, Structure 2 (1994) 361–369. R. Schauer, J.P. Kamerling, Chemistry, biochemistry and biology of sialic acids, in: J. Montreuil, J.F.G. Vliegenthart, H. Schachter (Eds.), Glycoproteins, Elsevier, Amsterdam, 1997, pp. 243–402. C. Traving, R. Schauer, Structure, function and metabolism of sialic acids, Cell. Mol. Life Sci. 54 (1998) 1330–1349. R. Schauer, U. Sommer, D. Krüger, H. van Unen, C. Traving, The terminal enzymes of sialic acid metabolism: acylneuraminate pyruvate-lyases, Biosci. Rep. 19 (1999) 373–383. R. Schauer, M. Wember, Isolation and characterization of sialate lyase from pig kidney, Biol. Chem. Hoppe-Seyler 377 (1996) 293–299. E.R. Vimr, F.A. Troy, Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli, J. Bacteriol. 164 (1985) 845–853. E.R. Vimr, F.A. Troy, Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase, J. Bacteriol. 164 (1985) 854–860. U. Sommer, C. Traving, R. Schauer, The sialate pyruvate-lyase from pig kidney: purification, properties and genetic relationships, Glycoconjugate J. 16 (1999) 425–435. P. Brunetti, G.W. Jourdian, S. Roseman, The sialic acids. III. Distribution and properties of animal N-acetylneuraminic aldolase, J. Biol. Chem. 237 (1962) 2447–2453. W. Witt, H. von Nicolai, F. Zilliker, Uptake and distribution of orally applied N-acetyl-(14C)neuraminosyl-lactose and N-acetyl(14C)neuraminic acid in the organs of newborn rats, Nutr. Metab. 23 (1979) 51–61. U. Nöhle, R. Schauer, Uptake, metabolism and excretion of orally and intravenously administered, 14C- and 3H-labeled N-acetylneuraminic acid mixture in the mouse and rat, Hoppe Seyler’s Z. Physiol. Chem. 362 (1981) 1495–1506. A.P. Corfield, R. Schauer, Metabolism of sialic acids, in: R. Schauer (Ed.), Sialic Acids. Chemistry, Metabolism and Function, Cell Biology Monographs, vol 10, Springer-Verlag, Vienna, 1982, pp. 195–261. L. Warren, Sialic acid lyase in differentiating murine teratocarcinoma cells, Exp. Cell. Res. 16 (1985) 307–316. L. Warren, Sialic acid lyase in human promyelocytic leukaemic cells (HL-60) during phorbol-ester-induced differentiation, Biochim. Biophys. Acta 888 (1986) 278–281. D.A. Sirbasku, S.B. Binkley, Purification and properties of N-acetylneuraminate lyase from kidney cortex, Biochim. Biophys. Acta 206 (1970) 479–482. D. Bratosin, J. Mazurier, H. Debray, M. Lecocq, B. Boilly, C. Alonso, M. Moisei, C. Motas, J. Montreuil, Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span, Glycoconjugate J. 12 (1995) 258–267. D. Bratosin, J. Mazurier, J.P. Tissier, C. Slomianny, D. Aminoff, J. Montreuil, Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages, C. R. Acad. Sci. Paris 320 (1997) 811–818. D. Bratosin, J. Mazurier, J.P. Tissier, J. Estaquier, J.J. Huart, J.C. Ameisen, D. Aminoff, J. Montreuil, Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages, A review, Biochimie 80 (1998) 173–195.
660
T. Bulai et al. / Biochimie 84 (2002) 655–660
[24] D. Bratosin, J. Estaquier, F. Petit, D. Arnoult, B. Quatannens, J.P. Tissier, C. Slomianny, C. Sartiaux, C. Alonso, J.J. Huart, J. Montreuil, J.C. Ameisen, Programed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria, Cell Death Differ. 8 (2001) 1143–1156. [25] C.G. Gahmberg, S.I. Hakomori, External labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes, J. Biol. Chem. 248 (1973) 4311–4317. [26] J.L. Reissig, J.L. Strominger, L.F. Leloir, A modified colorimetric method for the estimation of N-acetylamino sugar, J. Biol. Chem. 217 (1955) 959–966. [27] A. Varki, S. Diaz, The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups, Anal. Biochem. 137 (1984) 236–247.
[28] E. Svennerholm, L. Svennerholm, Qualitative paper partition chromatography of sialic acids, Nature 181 (1958) 1154–1156. [29] E. Beutler, Calculation of results, in: E. Beutler (Ed.), Red Cell Metabolism: A Manual of Biochemical Methods, Grume & Stratton, New York, 1984, pp. 31–36. [30] Y. Uchida, Y. Tsukada, T. Sugimori, Purification and properties of N-acetylneuraminate-lyase from Escherichia coli, J. Biochem. 96 (1984) 507–522. [31] K. Aisaka, A. Igarashi, K. Yamaguchi, T. Uwajima, Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli, Biochem. J. 276 (1991) 541–546.