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Reduced glutathione and glutathione reductase—a comparative study of erythrocytes from various species
Comp. Biockem. PhyJiol., 1973, Vol. 46B,pp. 639 to 641. Pergamoa Peas. Prixted in Crreat Britain
SHORT COMMUNICATION REDUCED GLUTATHIONE AND GLUTATHIONE REDUCTASE--A COMPARATIVE STUDY OF ERYTHROCYTES FROM VARIOUS SPECIES* P. G. L A N K I S C H , t R. SCHROETER,~ L. L E G E § and W. V O G T Max-Planck-Institut fiir experimentelle Medizin, Abt. Biochemische Pharmakologie, D-34 G6ttingen, Germany (Received 23 .Tanuary 1973)
Abatract--1. Reduced glutathione and glutathione reductase were measured in man, guinea pig, sheep, rat, rabbit, cattle and dromedary. 2. Reduced glutathione was present in all species tested but at different concentrations. If species are placed m order of increasing concentratio~ of glutathione, the sequence is: dromedary, man, cattle, sheep, rat, guinea pig, rabbit. Similarly, species can be placed in order of increasing glutathione reductase activity: rat, cattle, sheep, dromedary, rabbit, man, guinea pig. 3. Glutathione reductase was also estimated in the erythrocyte membrane of some species which could be placed in the following order of increasing activity: rat, rabbit, guinea pig, man. 4. These species differences may be of help in further studies on the different susceptibility of erythrocytes from various species to haemolysis.
INTRODUCTION COBRA venom components show different lytic activities towards erythrocytes from various species (Condrea et al., 1964; Oldigs et al., 1971). One of them, the direct lytic factor (DLF), is supposed to lyse cells subsequent to interaction with SH-groups of membrane proteins (Vogt et al., 1970). Glutathione and glutathione reductase (GR) are known to play an important part in maintaining red cell sulphydryls thus preserving the integrity of the cells. By the action of this enzyme more than 99 per cent of glutathione is kept in the reduced form--(GSH) (Srivastava & Beutler, 1968). When this system does not function properly erythrocytes become excessively susceptible to haemolysis. * Supported by Deutsche Forsehungsgemeinschaft, Bad Godesberg. t Present address: D-34 G~ttingen, Medizinische Universit~tsklinik. Present address: D-23 Kiel, I. Medizinische Universitatsklinik. § Present address: D-78 Freiburg, Medizinische Universit~tsklinik. 639
P. G. LANKXSCH,R. SCHROETER,L. LsoE AND W. VOGT
640
This induced us to find out whether there are species differences of GSH and GR since it is known that the lyric activity of DLF varies considerably depending on the species of cells. MATERIALS AND METHODS
Reagents Reduced and oxidized glutathione was obtained from Boehringer, Mannheim; N A D P H from Sigma Chemical Company, St. Louis and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) from Aldrich Chemical Company, Milwaukee. Blood samples were taken b y venous puncture from healthy human subjects, cattle, sheep and dromedary and b y heart puncture from rabbit, guinea pig and rat. Heparin was used as an anticoagulant. G S H and G R were assayed following the procedure of Beutler (Beutler et al., 1963; Beutler, 1969). Red cell ghosts were prepared according to Condrea et al. (1965), and membrane suspensions made as described previously (Schroeter et al., 1972). RESULTS AND DISCUSSION
The results are shown in Table 1 and indeed demonstrate species differences. When the results are placed in the order from low to high values, the following sequences result: GSH: dromedary, man, cattle, sheep, rat, guinea pig, rabbit; GR activity in haemolysates: rat, cattle, sheep, dromedary, rabbit, man, guinea pig; GR, membrane-bound: rat, rabbit, guinea pig, man. TABLE 1--REDuclm GLUTATHIONE (GSH) AND GLUTATHIONE REDUCTASE (GR) on ~RYT H R O C Y T ~ FROM VARIOUS SPECIES
Species Man Guinea pig
n
GSH (rag %)
G R (haemolysate) G R (membrane) (I.U./g Hb) (I.U./10 xl ghosts)
16 14
81-55+ 8.28 141.73+14.24
15.5 + 1"9 20.9 + 4.8
12
109.5 +26.69
2"8 _+ 1.1
--*
11
122"59+13"03
2"5 + 0"81
7"0 + 0"7
6
148"5 _+16"52
11"8 + 4"0
9-7 ± 2"9
5
96"1 -+ 14'94
2-63 + 0-6
--*
2
77"5 _+ 4"5
4"1 ± 0"59
--*
28.3 + 4.4 23.8 + 5.0
(Cavia aperea porcellus) Sheep
( Ocris annnon aries) Rat
(Rattus norvegicus) Rabbit
(Oryctolagus cuniculus) Cattle
(Bos primi&enius taurus) Dromedary
(Camelus dromedarius) * Not investigated.
REDUCED GLUTATHIONE AND G L U T A T H I O N E REDUCTASE
641
According to recent investigations the G R content of equine erythrocytes is also low (Smith et al., 1972) and lies between our values for cattle and sheep. T h e results indicate that there is a correlation between G R activity and lyric activity of D L F as far as the latter has been investigated (Schroeter et al., 1972). Further studies will have to be done to clarify the involvement of the G R - G S H system in haemolysis by D L F and possibly other lytic agents. Acknowledgement--Our thanks are due to Mrs. Astrid Freund for skilful technical assistance. REFERENCES BEUTI~ E. (1969) Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. •. clin. invest. 48, 1957-1966. Bm2TZ~gRE., DLrRON O. & KELLY B. M. (1963) Improved method for determination of blood glutathione..7. Lab. Clin. Med. 61, 882-888. Cot~ E., K~TOZERSK¢J. & DE VRmS A. (1965) Binding of Ringhal's venom direct lyric factor to erythrocytes and osmotic ghosts of various animal species. Experientia (Basel) 21, 461-462. CONVR~ E., MAMMON Z., ALOOF S. & Ds VRms A. (1964) Susceptibility of erythrocytes of various animal species to the hemolytic and phospholipid splitting action of snake venom. Biochim. biophys. Acta (Amst.) 84, 365-375. OLDXOSH. D., I.~E L. & L ~ , I S C 8 P. G. (1971) Vergleichende Hamolyseversuche an Meerschweinchen- und Rattenerythrocyten in vitro rnit Phospholipase A und Direkto Lytischem-Faktor aus Cobragift (Naja naja). Naunyn-Schmiedebergs Archs Pharmak. 268, 27-32. Scmaogr~a R., LANKISCHP. G., LEO'~L. & VOOT W. (1972) Possible implication of glutathione reductase in haemolysis by the direct lyric factor of cobra venom (Naja naja). Naunyn-Schmiedebergs Archs. Pharmacol. 275, 203-211. SMITH J. E., Knn~a S. & LEE M. (1972) Glutathione reduction and other enzyme activities in equine erythrocytes. Comp. Biochem. Physiol. 43B, 413-417. SRIVAST^VAS. K. & B ~ r L ~ E. (1968) Accurate measurement of oxidized glutathionecontent of human, rabbit and rat red blood cells and tissues. Analyt. Biochem. 7,5, 70-76. VOGT W., PATZ~ P., LEOE L., OLDIOS H. D. & WILLE G. (1970) Synergism between phospholipase A and various peptides and SH-reagents in causing haemolysis. NaunynSchmiedebergs Archs Pharmak. 265, 442-454. Key Word Index--Reduced glutathione; glutathione reductase; erythrocytes; man; guinea pig; rabbit; rat; sheep; cattle; dromedary.
SHORT COMMUNICATION REDUCED GLUTATHIONE AND GLUTATHIONE REDUCTASE--A COMPARATIVE STUDY OF ERYTHROCYTES FROM VARIOUS SPECIES* P. G. L A N K I S C H , t R. SCHROETER,~ L. L E G E § and W. V O G T Max-Planck-Institut fiir experimentelle Medizin, Abt. Biochemische Pharmakologie, D-34 G6ttingen, Germany (Received 23 .Tanuary 1973)
Abatract--1. Reduced glutathione and glutathione reductase were measured in man, guinea pig, sheep, rat, rabbit, cattle and dromedary. 2. Reduced glutathione was present in all species tested but at different concentrations. If species are placed m order of increasing concentratio~ of glutathione, the sequence is: dromedary, man, cattle, sheep, rat, guinea pig, rabbit. Similarly, species can be placed in order of increasing glutathione reductase activity: rat, cattle, sheep, dromedary, rabbit, man, guinea pig. 3. Glutathione reductase was also estimated in the erythrocyte membrane of some species which could be placed in the following order of increasing activity: rat, rabbit, guinea pig, man. 4. These species differences may be of help in further studies on the different susceptibility of erythrocytes from various species to haemolysis.
INTRODUCTION COBRA venom components show different lytic activities towards erythrocytes from various species (Condrea et al., 1964; Oldigs et al., 1971). One of them, the direct lytic factor (DLF), is supposed to lyse cells subsequent to interaction with SH-groups of membrane proteins (Vogt et al., 1970). Glutathione and glutathione reductase (GR) are known to play an important part in maintaining red cell sulphydryls thus preserving the integrity of the cells. By the action of this enzyme more than 99 per cent of glutathione is kept in the reduced form--(GSH) (Srivastava & Beutler, 1968). When this system does not function properly erythrocytes become excessively susceptible to haemolysis. * Supported by Deutsche Forsehungsgemeinschaft, Bad Godesberg. t Present address: D-34 G~ttingen, Medizinische Universit~tsklinik. Present address: D-23 Kiel, I. Medizinische Universitatsklinik. § Present address: D-78 Freiburg, Medizinische Universit~tsklinik. 639
P. G. LANKXSCH,R. SCHROETER,L. LsoE AND W. VOGT
640
This induced us to find out whether there are species differences of GSH and GR since it is known that the lyric activity of DLF varies considerably depending on the species of cells. MATERIALS AND METHODS
Reagents Reduced and oxidized glutathione was obtained from Boehringer, Mannheim; N A D P H from Sigma Chemical Company, St. Louis and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) from Aldrich Chemical Company, Milwaukee. Blood samples were taken b y venous puncture from healthy human subjects, cattle, sheep and dromedary and b y heart puncture from rabbit, guinea pig and rat. Heparin was used as an anticoagulant. G S H and G R were assayed following the procedure of Beutler (Beutler et al., 1963; Beutler, 1969). Red cell ghosts were prepared according to Condrea et al. (1965), and membrane suspensions made as described previously (Schroeter et al., 1972). RESULTS AND DISCUSSION
The results are shown in Table 1 and indeed demonstrate species differences. When the results are placed in the order from low to high values, the following sequences result: GSH: dromedary, man, cattle, sheep, rat, guinea pig, rabbit; GR activity in haemolysates: rat, cattle, sheep, dromedary, rabbit, man, guinea pig; GR, membrane-bound: rat, rabbit, guinea pig, man. TABLE 1--REDuclm GLUTATHIONE (GSH) AND GLUTATHIONE REDUCTASE (GR) on ~RYT H R O C Y T ~ FROM VARIOUS SPECIES
Species Man Guinea pig
n
GSH (rag %)
G R (haemolysate) G R (membrane) (I.U./g Hb) (I.U./10 xl ghosts)
16 14
81-55+ 8.28 141.73+14.24
15.5 + 1"9 20.9 + 4.8
12
109.5 +26.69
2"8 _+ 1.1
--*
11
122"59+13"03
2"5 + 0"81
7"0 + 0"7
6
148"5 _+16"52
11"8 + 4"0
9-7 ± 2"9
5
96"1 -+ 14'94
2-63 + 0-6
--*
2
77"5 _+ 4"5
4"1 ± 0"59
--*
28.3 + 4.4 23.8 + 5.0
(Cavia aperea porcellus) Sheep
( Ocris annnon aries) Rat
(Rattus norvegicus) Rabbit
(Oryctolagus cuniculus) Cattle
(Bos primi&enius taurus) Dromedary
(Camelus dromedarius) * Not investigated.
REDUCED GLUTATHIONE AND G L U T A T H I O N E REDUCTASE
641
According to recent investigations the G R content of equine erythrocytes is also low (Smith et al., 1972) and lies between our values for cattle and sheep. T h e results indicate that there is a correlation between G R activity and lyric activity of D L F as far as the latter has been investigated (Schroeter et al., 1972). Further studies will have to be done to clarify the involvement of the G R - G S H system in haemolysis by D L F and possibly other lytic agents. Acknowledgement--Our thanks are due to Mrs. Astrid Freund for skilful technical assistance. REFERENCES BEUTI~ E. (1969) Effect of flavin compounds on glutathione reductase activity: in vivo and in vitro studies. •. clin. invest. 48, 1957-1966. Bm2TZ~gRE., DLrRON O. & KELLY B. M. (1963) Improved method for determination of blood glutathione..7. Lab. Clin. Med. 61, 882-888. Cot~ E., K~TOZERSK¢J. & DE VRmS A. (1965) Binding of Ringhal's venom direct lyric factor to erythrocytes and osmotic ghosts of various animal species. Experientia (Basel) 21, 461-462. CONVR~ E., MAMMON Z., ALOOF S. & Ds VRms A. (1964) Susceptibility of erythrocytes of various animal species to the hemolytic and phospholipid splitting action of snake venom. Biochim. biophys. Acta (Amst.) 84, 365-375. OLDXOSH. D., I.~E L. & L ~ , I S C 8 P. G. (1971) Vergleichende Hamolyseversuche an Meerschweinchen- und Rattenerythrocyten in vitro rnit Phospholipase A und Direkto Lytischem-Faktor aus Cobragift (Naja naja). Naunyn-Schmiedebergs Archs Pharmak. 268, 27-32. Scmaogr~a R., LANKISCHP. G., LEO'~L. & VOOT W. (1972) Possible implication of glutathione reductase in haemolysis by the direct lyric factor of cobra venom (Naja naja). Naunyn-Schmiedebergs Archs. Pharmacol. 275, 203-211. SMITH J. E., Knn~a S. & LEE M. (1972) Glutathione reduction and other enzyme activities in equine erythrocytes. Comp. Biochem. Physiol. 43B, 413-417. SRIVAST^VAS. K. & B ~ r L ~ E. (1968) Accurate measurement of oxidized glutathionecontent of human, rabbit and rat red blood cells and tissues. Analyt. Biochem. 7,5, 70-76. VOGT W., PATZ~ P., LEOE L., OLDIOS H. D. & WILLE G. (1970) Synergism between phospholipase A and various peptides and SH-reagents in causing haemolysis. NaunynSchmiedebergs Archs Pharmak. 265, 442-454. Key Word Index--Reduced glutathione; glutathione reductase; erythrocytes; man; guinea pig; rabbit; rat; sheep; cattle; dromedary.