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Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes
Vol. 160,
No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 28, 1989
Pages
954-960
OCCURRRNCE Of THE ERYTHROID CELL SPECIFIC ARACHIDONATE 1 S-LIPOXYGENASB IN HUMAN RETICULOCYTRS’
Peter Kroschwald, Alexandra Kroschwald. Hartmut Kuhn#, Peter Ludwig, Bernd J. Thiele, Marina Hohne,Tankred Scheweand Samuel M. Rapoport Institute of Biochemistry, Schoolof Medicine (Char&e), Humboldt University, HessischeStr. 3-4, DDR-1040Berlin, GDR Received
March
29,
1989
Summary: Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous II- 14C]-arachidonicacid to 15hydroxy5,8,11,13(2,2,&E)-eicosatetraenoic acid ( 15-HETE1. Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase.Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is aiso present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrenceof an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradationof mitochondria. 0 1989 Academic Press, Inc.
The erythroid cell-specific lipoxygenase has been shown to be involved in the degradationof mitochondria in rabbit reticulocytes ( 1,2). A remarkable property of this enzyme is its ability to oxygenate phospholipids and biological membranes without preceding action of a phospholipase (3,451. Only little information is available as to an analogous lipoxygenase in red cells of other species. A lipoxygenase pathway was demonstrated in reticulocytes of rats, mice and rhesus monkeys (61. An arachidonate 12-lipoxygenase was claimed to occur in erythrocytes of various animal species(7). This finding was not confirmed in our * Parts of this work was presented at the 14th Internat. Congress of Biochem., Prague,Czechoslovakia,July IO- 15 ( 19881Abstr.No. TH 116. # To whom correspondenceshould be addressed. Abbreviations: 15HETE - 15S-hydroxy - 5,8,11,13(2,Z,Z,E1-eicosatetraenoic acid, 13HODE - 13S-hydroxy-9,ll (Z,El-octadecadienoicacid, BTYA - 5,8,11,14eicosatetraynoic acid, SP-HPLC - straight phase high pressure liquid chromatography, SDS- sodium dodecyl sulfate, ETP - electron transfer particles (from bovine heart) 0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
954
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(8). Recently the amino acid sequence of a human erythroid cell specific lipoxygenase has been deduced from its cloned cDNA (9). However, no experimental data are available as to the enzymatic properties of this lipoxygenase. Here we present evidence for the occurrence of an enxymatically active iipoxygenase in immature human red blood cellsobtained from patients suffering from various hemolytic disorders.
laboratory.
Materials
and Methods
Chemicals: The chemicals used were from the following sources: [l- 14C]arachidonic acid (2.07 MBq/mmol) from Amersham (UK); heparin from Richter (FRG);precoated silica gel thin layer plates from Silufol (CSSR);nitrocellulose paper from Schleicher and Schull (FRG).ETYA was a kind gift from Prof. R.P.Evstigneeva (Institute of Fine Chemical Technology, Moscow, USSR). Preparations: Reticulocyte-rich blood was collected with 500 IU heparin and washed twice with isotonic saline. The buffy coat was removed. The cells were lyzed with two volumes of water and the stroma was removed by centrifugation (20,000 g). Ammonium sulfate precipitation of the stroma free supernatant was carried out as described in (8). The lipoxygenase from rabbit reticulocytes was purified according to (10). A polyclonal guinea pig antiserum against the pure rabbit reticulocyte lipoxygenase was prepared as described in (11). Using this antiserum no cross-reacting material was detected in any tissue of rabbits except reticulocytes but not in normal human blood. The lipoxygenase cDNA probe used for the Northern Blot was prepared according to ( 12). Authentic standards of 13SHODEand 1SS-HETEwere prepared with soybean lipoxygenase ( 13). Bovine heart electron transfer particles (ETP) were prepared as described in (14). Total polysomal RNA was prepared according to ( 151. Analytics: Radio thin-layer chromatography and SP-HPLC were carried out as described in (8). Electrophoresis was performed according to (16). Afterwards the proteins were transferred from the gel onto a nitrocellulose paper by semi-dry electroblotting ( 17). Immuno dot blotting was carried out as in ( 18) and Northern blotting was performed as described in (19). RNA samples were run in 2.2 M formaldehyde/ 1.2% agarose gel after denaturation in formamide, transferred to nitrocellulose and probed with a nick-translated lipoxygenase plasmid pGF6 ( 12). Results Human reticulocytes metabolize I1 -14CI-arachidonic acid to hydroxy eicosatetraenoic acid as shown by co-migration with an authentic standard of 15HETEin radio thin-layer chromatography (Fig. 1, trace A). Preincubation of the cells with the lipoxygenase inhibitor ETYA strongly decreased the formation of this product (Fig. 1, trace B). SP-HPLCwhich separates the different positional isomers of the hydroxylated derivatives of arachidonic acid indicated that the main product formed was 15-HETE (data not shown). This product has been shown before to be the main oxygenation product of arachidonic acid metabolism by intact rabbit reticulocytes and by the pure rabbit reticulocyte lipoxygenase (20). Linoleic acid was converted by human reticulocytes mainly to 13HODE (data not shown). 955
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
min
acid by human reticulocytes. 2 ml of a reticulocyte rich blood cell suspension( 10.4 % reticulocytes; haematocrit of 0.1I from a newborn with a Rh-incompatibility syndrom were incubated with 7.4 kBq I1-14Cl-arachidonic acid at 370 C for 10 min. The lipids were extracted with ethylacetate and analyxed by radio thin-layer chromatography. Trace A reticulocyte-rich blood cell suspension; trace B - reticulocyte-rich blood cell suspensionafter 5 min. preincubation with 100 CMERA.
Fig. 1. Conversion of Il-14Chrachidonic
Fig. 2. Inactivation of the NADHoxidase by human reticulocyte lipoxygenase.The lipoxyeenasepreparation (ammonium sulfate precipitate at 0.55 saturation) from a reticulocyte-rich blood cell suspension of a newborn was preincubated with electron transfer particles (0.32 mg/ml protein) for 1S min. The NADH oxidase activity of the preincubation sample was assayedoxygraphically after addition of 80 pM NADH. Assay volume 2 ml. AI control (no lipoxygenase), BI 50 pg/ml lipoxygenaseprotein, Cl 100 pg/ml lipoxygenaseprotein.
The reticulocyte lipoxygenase possesses the unique property to oxygenate phospholipids (4) and mitochondrial membranes (3). This reaction is accompanied by an irreversible inactivation of the NADH- and succinateoxidase activities and a drastic change of the passive electric membrane properties (21). From Fig. 2 it can
be seenthat the NADH oxidase activity of ETPwas inactivated in a dose dependent manner by an ammonium sulfate precipitate (0.55 saturation) of a reticulocyterich blood cell suspension obtained from a newborn with a Rh-incompatibility
syndrom. The lipoxygenase inhibitor salicylhydroxamic acid (1 mM) completely prevented this inactivation. The ammonium sulfate precipitates from this and a variety of other anaemic blood specimens were tested for the presence of the lipoxygenase by immunological dot blot experiments using an antiserum against the pure rabbit reticulocyte lipoxygenase. In aD cases strongly positive responses were observed
(Fig. 3, ICI. In contrast, a fraction of human leukocytes, prepared in an identical way, did not exhibit any immunoreactivity (Fig. 3, IB). The cross-reactivity with 956
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-.
FRONT
.I
,,
f=I
‘--- ORIGIN
B
Fig. 3. Immunological crossreactivity of the human reticulocyte lipoxygenasewith an antiserum againstthe rabbit reticulocyte lipoxygenases.J1 dot blot 30 pg of an ammonium sulfate precipitation (0.55 saturation) of a reticulocyte rich blood cell suspension (53 X reticulocytes) of a patient suffering from pyruvate kinase deficiency was used as source of the human lipoxygenase. A) control (no lipoxygenase) B) human leukocyte preparation (ammonium sulfate precipitate at 0.55 saturation) C) human reticulocyte liporygenase JII Western blot: Ammonium sulfate precipitate (0.55 saturation) of a reticulocyte rich blood cell suspension (10.4 X reticulocytes)of a newborn with a Rh-incompatibility syndrom as sourceof the human lipoxygenase.A - human lipoxygenase,B - rabbit lipoxygenase
the polyclonal antiserum against the rabbit reticulocyte enzyme was used to determine the molecular weight of the human reticulocyte lipoxygenase. Fig. 4 shows the Western blot of a human reticulocyte lipoxygenase preparation (ammonium sulfate precipitation at 0.55 saturation) from a newborn with a Rhincompatibility syndrom. It can be seen that the human reticulocyte lipoxygenase (lane 1) has the same apparent molecular weight (about 70 kDa1 as the lipoxygenase from rabbit reticulocytes (lane 2). The molecular weight determined by SDSelectrophoresisappears to be underestimated since the complete primary structure recently established yields an Mr of 75 kDa for both the rabbit and the human enzyme ( 12,9). 957
Vol. 160, No. 2, 1989
BIOCHEMICAL
LOX
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mRNA-
1
2
3
4
5
Northern blotting of human reticulocyte RNAsprobed with a rabbit reticulocyte cDNA.1) rabbit reticulocyte RNA(0.5 pg), 2-5) Humanreticulcyte total polysomalRNA (20 pg): 2) G-6-PDdeficiency,3) spherocytosis,4) premature newborn, 5) PK-deficiency(abbreviations seeTable1).
Pig.4
In Table 1 the results of the experiments with human reticulocytes obtained from patients suffering from various haemolytic disorders are summarized. It can be seen that the lipoxygenase was detected in reticulocytes of all patients tested with either the dot blot technique or by the formation of 15-HETEfrom exogenous arachidonic acid. In red cells of five healthy blood donors and in human leukocytes we did not detect the lipoxygenase with either of the methods. It should be mentioned that in two cases (one PK-deficiency, one B-thalassaemia) the lipoxygenase protein was detectedwith the dot-blot technique but no formation of 15-HETEfrom exogenousarachidonic acid was observed. It may be suggestedthat Table 1. The occurrenceof the lipoxygenasein reticulocyte-rich blood cell suspensionsobtained from patients suffering from various haemolytic disorders Number of specimens
disorder
reticulocyte immuno 15-HETE content dot blot formation
5 erythrocytes (healthy donors) 1 leukocytes(healthy donors)
0.2-0.5 0.2
-
-
2 R-thalassaemia 3 PK-deficiency 1 G-6-PDdeficiency
36; 20 50; 53; 55 41
+ + +
+l) ns.
3 normal newborns 2 Rh-incompatibility
2.7; 2.9; 3.4 10; 7
-2) +
+
+l)
PK- pyruvate kinase,G-6-PD- glucose-6-phosphatedehydrogenase t-1 tested negative; (+I tested positive; n.t. not tested; t)one out of these sampleswere tested positive in the dot blot, but no 15-HETEformation was observed; g)the samplewith 3.4 %reticulocyte content turned out questionable positive in the immuno dot blot test. 958
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the lipoxygenase was enzymatically inactive in these cells. This inactive lipoxygenase might be the consequenceof the suicidal reaction of the enzyme (22) in later maturational stagesof the reticulocytes. With the polysomal fraction of the haemolysates of four additional anaemic patients Northern blottings with a rabbit reticulocyte cDNA probe was performed. As shown in Fig. 4 in all cases positive mRNA signals were detected. Control experiments with RNA from human leukocytes did not reveal any hybridization. Discussion
Human reticulocytes obtained from patients with various haemolytic disorders contain a lipoxygenase which is very similar to the enzyme present in rabbit reticulocytes after experimental bleeding anaemia. Several fines of experimental evidence support this conclusion:i) identical pattern of the products formed from exogenous polyenoic fatty acids, ii) immunological cross-reactivity with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase, iii) identical apparent molecular weight, iv) reactivity towards mitochondrial membranes leading to inactivation of respiratory enzymes and VI positive hybridization of human reticulocyte mRNA with the cloned reticulocyte lipoxygenase cDNA probe under stringent conditions. The structural similarity as evidencedfrom both immunological cross-reactivity and from the hybridization experiments on one hand and the agreement of the enzymatic properties on the other hand strongly suggest that the reticulocyte lipoxygenase of rabbits and man are enzymes of the same type. This conclusion is supported by the comparison of the complete primary structure of the human erythroid cell lipoxygenase (9) with that from rabbit reticulocytes (23) indicating a high degree of similarity. The possibility to detect the lipoxygenase by means of various methods in anaemic patients but not in normal subjects suggestsa clinical significance of this finding which will be studied in future work. The presence of the erythroid cell specific lipoxygenase in reticulocytes of various animal species(8) and man suggests that this enzyme generally occurs in immature red blood cells. For rabbit reticulocytes the lipoxygenase has been shown to be involved in the maturational breakdown of mitochondria (1,2). The occurrence of the lipoxygenase in reticulocytes of other animal species suggests that this enzyme is also involved in the maturational degradation of mitochondria in animals other than rabbits. The principles for the maturational breakdown of mitochondria appearto be similar in reticulocytes of all animal species. References
1. Rapoport,S.M.(1986) The Reticulocyte,CRCPress Inc., BocaRaton.FA 1986. 2. Rapoport,S.M.and Schewe,T (1986) Biochim. Biophys. Acta && 471-495. 959
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Lankin, V.Z.,Kuhn, H.,Hiebsch,C.,Schewe,T., Rapoport,SM., Tikhase,A.K. and N.T.Gordeeva(1985) Biomed. B&him. Acta fi 655-664. 4. Murray, J.J.and Brash A.R. ( 1988) Arch. B&hem. Biophys.2(& 5 14-523. 5. Fiskum, G.,Bryant, R.W.,Low, C.,Pease,A. and Bailey, J.M. (1985) In Prostaglandins,Leukotrienes, and Lipoxins. Biochemistry, Mechanismof Action and Clinical Applications (J.M.Bailey, Ed.),pp, 87-95 Plenum Press, New York. 6. Kroschwald, P., Kroschwald, A., Wiesner, R.,Schewe,T. and Kuhn, H. (1987) Biomed. B&him. Acta fi 1237- 1247. 7. Kobayashi,T. and Levine, L. (1983) J. Biol. Chem.2ss. 9116-9121. 8. Ludwig, P.,Hohne,M., Kuhn, H., Schewe,T.and Rapoport,S.M. (1988) Biomed. B&him. Acta 42 593-608. 9. Sigal, E.,Craig,C.S.,Highland, E.,Gruneberger,D.,Costello,L.L.,Dixon,R.A.F.and Nadel,J.A. (1988) B&hem. Biophys. Res.Commun. 157,457-464. 10.Schewe,T.,Wiesner, R. and Rapoport,S.M.(198 1) Methods Enxymol. Lf, 430441. 11. Wiesner, R.,Tanner&C.,Hausdorf, G.,Schewe,T. and Rapoport,SM. (1977)Acta Biol. Med. Germ. 36,393-403. 12.Thiele, B.J.,Fleming , J.,Kasturi, K.,O’Prey,J., Black, E., Chester,J., Rapoport, S.M. and Harris, P.R.( 1987) GenejZ, 111- 119. 13.Crawford, C.G.,van Alpen, G.H.W.,Cook,H.W. and Lands,W.E.M.(1978) Life Sci. 211,1255-1262. 14. Crane,F.L.,Glenn,J.L. and Green,D.E.(1956) Biochim. Biophys. Acta 22 475487. 15. Thiele, B.J.,Andree, H., Hohne,M. and Rapoport,S.M.( 1982) Eur. J. B&hem. m133-141. 16. Laemmli, U.K.(1970) Nature 222 680-685. 17. Kyhse-Anderson,J. ( 1984) J. Biophys. Biochem.Methods Ip, 203209. 18. Gershoni,J.M. and Palade,G.E.(1983) Anal. B&hem. l.3l, l-15. 19. Maniatis, T., Fritsch, E.Fand Sambrook,J, ( 1982) Molecular Cloning,A Laboratory Manual, ColdSpring Harbor Laboratory, ColdSpring Harbor, New York. 20. Bryant, R.W.Bailey, J.M.,Schewe,T. and Rapoport,SM. (1982) J. Biol. Chem. 2v,6050-6055. 21. H. Kuhn, F. Pliquett, W. Wunderlich, T. Scheweand W. Krause (1983) Biochim. Biophys. Acta Z283-290 22. B. Hartel, H. Kuhn and S.M.Rapoport(1984) Proceedingsof the 16th FEBS Congress,Moscow 1984, pp. 299-304, VNU SciencePress BV. 23. Fleming, J..Thiele, B.J.,Chester,J.,O’Prey,J.. Anton, I., Aitken, A., Rapoport, S.M.and Harrison, P.R.(1988) Gene,in press.
960
No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 28, 1989
Pages
954-960
OCCURRRNCE Of THE ERYTHROID CELL SPECIFIC ARACHIDONATE 1 S-LIPOXYGENASB IN HUMAN RETICULOCYTRS’
Peter Kroschwald, Alexandra Kroschwald. Hartmut Kuhn#, Peter Ludwig, Bernd J. Thiele, Marina Hohne,Tankred Scheweand Samuel M. Rapoport Institute of Biochemistry, Schoolof Medicine (Char&e), Humboldt University, HessischeStr. 3-4, DDR-1040Berlin, GDR Received
March
29,
1989
Summary: Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous II- 14C]-arachidonicacid to 15hydroxy5,8,11,13(2,2,&E)-eicosatetraenoic acid ( 15-HETE1. Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase.Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is aiso present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrenceof an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradationof mitochondria. 0 1989 Academic Press, Inc.
The erythroid cell-specific lipoxygenase has been shown to be involved in the degradationof mitochondria in rabbit reticulocytes ( 1,2). A remarkable property of this enzyme is its ability to oxygenate phospholipids and biological membranes without preceding action of a phospholipase (3,451. Only little information is available as to an analogous lipoxygenase in red cells of other species. A lipoxygenase pathway was demonstrated in reticulocytes of rats, mice and rhesus monkeys (61. An arachidonate 12-lipoxygenase was claimed to occur in erythrocytes of various animal species(7). This finding was not confirmed in our * Parts of this work was presented at the 14th Internat. Congress of Biochem., Prague,Czechoslovakia,July IO- 15 ( 19881Abstr.No. TH 116. # To whom correspondenceshould be addressed. Abbreviations: 15HETE - 15S-hydroxy - 5,8,11,13(2,Z,Z,E1-eicosatetraenoic acid, 13HODE - 13S-hydroxy-9,ll (Z,El-octadecadienoicacid, BTYA - 5,8,11,14eicosatetraynoic acid, SP-HPLC - straight phase high pressure liquid chromatography, SDS- sodium dodecyl sulfate, ETP - electron transfer particles (from bovine heart) 0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
954
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(8). Recently the amino acid sequence of a human erythroid cell specific lipoxygenase has been deduced from its cloned cDNA (9). However, no experimental data are available as to the enzymatic properties of this lipoxygenase. Here we present evidence for the occurrence of an enxymatically active iipoxygenase in immature human red blood cellsobtained from patients suffering from various hemolytic disorders.
laboratory.
Materials
and Methods
Chemicals: The chemicals used were from the following sources: [l- 14C]arachidonic acid (2.07 MBq/mmol) from Amersham (UK); heparin from Richter (FRG);precoated silica gel thin layer plates from Silufol (CSSR);nitrocellulose paper from Schleicher and Schull (FRG).ETYA was a kind gift from Prof. R.P.Evstigneeva (Institute of Fine Chemical Technology, Moscow, USSR). Preparations: Reticulocyte-rich blood was collected with 500 IU heparin and washed twice with isotonic saline. The buffy coat was removed. The cells were lyzed with two volumes of water and the stroma was removed by centrifugation (20,000 g). Ammonium sulfate precipitation of the stroma free supernatant was carried out as described in (8). The lipoxygenase from rabbit reticulocytes was purified according to (10). A polyclonal guinea pig antiserum against the pure rabbit reticulocyte lipoxygenase was prepared as described in (11). Using this antiserum no cross-reacting material was detected in any tissue of rabbits except reticulocytes but not in normal human blood. The lipoxygenase cDNA probe used for the Northern Blot was prepared according to ( 12). Authentic standards of 13SHODEand 1SS-HETEwere prepared with soybean lipoxygenase ( 13). Bovine heart electron transfer particles (ETP) were prepared as described in (14). Total polysomal RNA was prepared according to ( 151. Analytics: Radio thin-layer chromatography and SP-HPLC were carried out as described in (8). Electrophoresis was performed according to (16). Afterwards the proteins were transferred from the gel onto a nitrocellulose paper by semi-dry electroblotting ( 17). Immuno dot blotting was carried out as in ( 18) and Northern blotting was performed as described in (19). RNA samples were run in 2.2 M formaldehyde/ 1.2% agarose gel after denaturation in formamide, transferred to nitrocellulose and probed with a nick-translated lipoxygenase plasmid pGF6 ( 12). Results Human reticulocytes metabolize I1 -14CI-arachidonic acid to hydroxy eicosatetraenoic acid as shown by co-migration with an authentic standard of 15HETEin radio thin-layer chromatography (Fig. 1, trace A). Preincubation of the cells with the lipoxygenase inhibitor ETYA strongly decreased the formation of this product (Fig. 1, trace B). SP-HPLCwhich separates the different positional isomers of the hydroxylated derivatives of arachidonic acid indicated that the main product formed was 15-HETE (data not shown). This product has been shown before to be the main oxygenation product of arachidonic acid metabolism by intact rabbit reticulocytes and by the pure rabbit reticulocyte lipoxygenase (20). Linoleic acid was converted by human reticulocytes mainly to 13HODE (data not shown). 955
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
min
acid by human reticulocytes. 2 ml of a reticulocyte rich blood cell suspension( 10.4 % reticulocytes; haematocrit of 0.1I from a newborn with a Rh-incompatibility syndrom were incubated with 7.4 kBq I1-14Cl-arachidonic acid at 370 C for 10 min. The lipids were extracted with ethylacetate and analyxed by radio thin-layer chromatography. Trace A reticulocyte-rich blood cell suspension; trace B - reticulocyte-rich blood cell suspensionafter 5 min. preincubation with 100 CMERA.
Fig. 1. Conversion of Il-14Chrachidonic
Fig. 2. Inactivation of the NADHoxidase by human reticulocyte lipoxygenase.The lipoxyeenasepreparation (ammonium sulfate precipitate at 0.55 saturation) from a reticulocyte-rich blood cell suspension of a newborn was preincubated with electron transfer particles (0.32 mg/ml protein) for 1S min. The NADH oxidase activity of the preincubation sample was assayedoxygraphically after addition of 80 pM NADH. Assay volume 2 ml. AI control (no lipoxygenase), BI 50 pg/ml lipoxygenaseprotein, Cl 100 pg/ml lipoxygenaseprotein.
The reticulocyte lipoxygenase possesses the unique property to oxygenate phospholipids (4) and mitochondrial membranes (3). This reaction is accompanied by an irreversible inactivation of the NADH- and succinateoxidase activities and a drastic change of the passive electric membrane properties (21). From Fig. 2 it can
be seenthat the NADH oxidase activity of ETPwas inactivated in a dose dependent manner by an ammonium sulfate precipitate (0.55 saturation) of a reticulocyterich blood cell suspension obtained from a newborn with a Rh-incompatibility
syndrom. The lipoxygenase inhibitor salicylhydroxamic acid (1 mM) completely prevented this inactivation. The ammonium sulfate precipitates from this and a variety of other anaemic blood specimens were tested for the presence of the lipoxygenase by immunological dot blot experiments using an antiserum against the pure rabbit reticulocyte lipoxygenase. In aD cases strongly positive responses were observed
(Fig. 3, ICI. In contrast, a fraction of human leukocytes, prepared in an identical way, did not exhibit any immunoreactivity (Fig. 3, IB). The cross-reactivity with 956
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-.
FRONT
.I
,,
f=I
‘--- ORIGIN
B
Fig. 3. Immunological crossreactivity of the human reticulocyte lipoxygenasewith an antiserum againstthe rabbit reticulocyte lipoxygenases.J1 dot blot 30 pg of an ammonium sulfate precipitation (0.55 saturation) of a reticulocyte rich blood cell suspension (53 X reticulocytes) of a patient suffering from pyruvate kinase deficiency was used as source of the human lipoxygenase. A) control (no lipoxygenase) B) human leukocyte preparation (ammonium sulfate precipitate at 0.55 saturation) C) human reticulocyte liporygenase JII Western blot: Ammonium sulfate precipitate (0.55 saturation) of a reticulocyte rich blood cell suspension (10.4 X reticulocytes)of a newborn with a Rh-incompatibility syndrom as sourceof the human lipoxygenase.A - human lipoxygenase,B - rabbit lipoxygenase
the polyclonal antiserum against the rabbit reticulocyte enzyme was used to determine the molecular weight of the human reticulocyte lipoxygenase. Fig. 4 shows the Western blot of a human reticulocyte lipoxygenase preparation (ammonium sulfate precipitation at 0.55 saturation) from a newborn with a Rhincompatibility syndrom. It can be seen that the human reticulocyte lipoxygenase (lane 1) has the same apparent molecular weight (about 70 kDa1 as the lipoxygenase from rabbit reticulocytes (lane 2). The molecular weight determined by SDSelectrophoresisappears to be underestimated since the complete primary structure recently established yields an Mr of 75 kDa for both the rabbit and the human enzyme ( 12,9). 957
Vol. 160, No. 2, 1989
BIOCHEMICAL
LOX
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mRNA-
1
2
3
4
5
Northern blotting of human reticulocyte RNAsprobed with a rabbit reticulocyte cDNA.1) rabbit reticulocyte RNA(0.5 pg), 2-5) Humanreticulcyte total polysomalRNA (20 pg): 2) G-6-PDdeficiency,3) spherocytosis,4) premature newborn, 5) PK-deficiency(abbreviations seeTable1).
Pig.4
In Table 1 the results of the experiments with human reticulocytes obtained from patients suffering from various haemolytic disorders are summarized. It can be seen that the lipoxygenase was detected in reticulocytes of all patients tested with either the dot blot technique or by the formation of 15-HETEfrom exogenous arachidonic acid. In red cells of five healthy blood donors and in human leukocytes we did not detect the lipoxygenase with either of the methods. It should be mentioned that in two cases (one PK-deficiency, one B-thalassaemia) the lipoxygenase protein was detectedwith the dot-blot technique but no formation of 15-HETEfrom exogenousarachidonic acid was observed. It may be suggestedthat Table 1. The occurrenceof the lipoxygenasein reticulocyte-rich blood cell suspensionsobtained from patients suffering from various haemolytic disorders Number of specimens
disorder
reticulocyte immuno 15-HETE content dot blot formation
5 erythrocytes (healthy donors) 1 leukocytes(healthy donors)
0.2-0.5 0.2
-
-
2 R-thalassaemia 3 PK-deficiency 1 G-6-PDdeficiency
36; 20 50; 53; 55 41
+ + +
+l) ns.
3 normal newborns 2 Rh-incompatibility
2.7; 2.9; 3.4 10; 7
-2) +
+
+l)
PK- pyruvate kinase,G-6-PD- glucose-6-phosphatedehydrogenase t-1 tested negative; (+I tested positive; n.t. not tested; t)one out of these sampleswere tested positive in the dot blot, but no 15-HETEformation was observed; g)the samplewith 3.4 %reticulocyte content turned out questionable positive in the immuno dot blot test. 958
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the lipoxygenase was enzymatically inactive in these cells. This inactive lipoxygenase might be the consequenceof the suicidal reaction of the enzyme (22) in later maturational stagesof the reticulocytes. With the polysomal fraction of the haemolysates of four additional anaemic patients Northern blottings with a rabbit reticulocyte cDNA probe was performed. As shown in Fig. 4 in all cases positive mRNA signals were detected. Control experiments with RNA from human leukocytes did not reveal any hybridization. Discussion
Human reticulocytes obtained from patients with various haemolytic disorders contain a lipoxygenase which is very similar to the enzyme present in rabbit reticulocytes after experimental bleeding anaemia. Several fines of experimental evidence support this conclusion:i) identical pattern of the products formed from exogenous polyenoic fatty acids, ii) immunological cross-reactivity with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase, iii) identical apparent molecular weight, iv) reactivity towards mitochondrial membranes leading to inactivation of respiratory enzymes and VI positive hybridization of human reticulocyte mRNA with the cloned reticulocyte lipoxygenase cDNA probe under stringent conditions. The structural similarity as evidencedfrom both immunological cross-reactivity and from the hybridization experiments on one hand and the agreement of the enzymatic properties on the other hand strongly suggest that the reticulocyte lipoxygenase of rabbits and man are enzymes of the same type. This conclusion is supported by the comparison of the complete primary structure of the human erythroid cell lipoxygenase (9) with that from rabbit reticulocytes (23) indicating a high degree of similarity. The possibility to detect the lipoxygenase by means of various methods in anaemic patients but not in normal subjects suggestsa clinical significance of this finding which will be studied in future work. The presence of the erythroid cell specific lipoxygenase in reticulocytes of various animal species(8) and man suggests that this enzyme generally occurs in immature red blood cells. For rabbit reticulocytes the lipoxygenase has been shown to be involved in the maturational breakdown of mitochondria (1,2). The occurrence of the lipoxygenase in reticulocytes of other animal species suggests that this enzyme is also involved in the maturational degradation of mitochondria in animals other than rabbits. The principles for the maturational breakdown of mitochondria appearto be similar in reticulocytes of all animal species. References
1. Rapoport,S.M.(1986) The Reticulocyte,CRCPress Inc., BocaRaton.FA 1986. 2. Rapoport,S.M.and Schewe,T (1986) Biochim. Biophys. Acta && 471-495. 959
Vol. 160, No. 2, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Lankin, V.Z.,Kuhn, H.,Hiebsch,C.,Schewe,T., Rapoport,SM., Tikhase,A.K. and N.T.Gordeeva(1985) Biomed. B&him. Acta fi 655-664. 4. Murray, J.J.and Brash A.R. ( 1988) Arch. B&hem. Biophys.2(& 5 14-523. 5. Fiskum, G.,Bryant, R.W.,Low, C.,Pease,A. and Bailey, J.M. (1985) In Prostaglandins,Leukotrienes, and Lipoxins. Biochemistry, Mechanismof Action and Clinical Applications (J.M.Bailey, Ed.),pp, 87-95 Plenum Press, New York. 6. Kroschwald, P., Kroschwald, A., Wiesner, R.,Schewe,T. and Kuhn, H. (1987) Biomed. B&him. Acta fi 1237- 1247. 7. Kobayashi,T. and Levine, L. (1983) J. Biol. Chem.2ss. 9116-9121. 8. Ludwig, P.,Hohne,M., Kuhn, H., Schewe,T.and Rapoport,S.M. (1988) Biomed. B&him. Acta 42 593-608. 9. Sigal, E.,Craig,C.S.,Highland, E.,Gruneberger,D.,Costello,L.L.,Dixon,R.A.F.and Nadel,J.A. (1988) B&hem. Biophys. Res.Commun. 157,457-464. 10.Schewe,T.,Wiesner, R. and Rapoport,S.M.(198 1) Methods Enxymol. Lf, 430441. 11. Wiesner, R.,Tanner&C.,Hausdorf, G.,Schewe,T. and Rapoport,SM. (1977)Acta Biol. Med. Germ. 36,393-403. 12.Thiele, B.J.,Fleming , J.,Kasturi, K.,O’Prey,J., Black, E., Chester,J., Rapoport, S.M. and Harris, P.R.( 1987) GenejZ, 111- 119. 13.Crawford, C.G.,van Alpen, G.H.W.,Cook,H.W. and Lands,W.E.M.(1978) Life Sci. 211,1255-1262. 14. Crane,F.L.,Glenn,J.L. and Green,D.E.(1956) Biochim. Biophys. Acta 22 475487. 15. Thiele, B.J.,Andree, H., Hohne,M. and Rapoport,S.M.( 1982) Eur. J. B&hem. m133-141. 16. Laemmli, U.K.(1970) Nature 222 680-685. 17. Kyhse-Anderson,J. ( 1984) J. Biophys. Biochem.Methods Ip, 203209. 18. Gershoni,J.M. and Palade,G.E.(1983) Anal. B&hem. l.3l, l-15. 19. Maniatis, T., Fritsch, E.Fand Sambrook,J, ( 1982) Molecular Cloning,A Laboratory Manual, ColdSpring Harbor Laboratory, ColdSpring Harbor, New York. 20. Bryant, R.W.Bailey, J.M.,Schewe,T. and Rapoport,SM. (1982) J. Biol. Chem. 2v,6050-6055. 21. H. Kuhn, F. Pliquett, W. Wunderlich, T. Scheweand W. Krause (1983) Biochim. Biophys. Acta Z283-290 22. B. Hartel, H. Kuhn and S.M.Rapoport(1984) Proceedingsof the 16th FEBS Congress,Moscow 1984, pp. 299-304, VNU SciencePress BV. 23. Fleming, J..Thiele, B.J.,Chester,J.,O’Prey,J.. Anton, I., Aitken, A., Rapoport, S.M.and Harrison, P.R.(1988) Gene,in press.
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