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Cytosolic and membrane-bound methemoglobin reductases in erythrocytes of the opossum, Didelphis virginiana
Comp. Biochem. Physiol. Vol, 73B, No. 3, pp. 591 to 594, 1982 Printed in Great Britain.
0305-0491/82/110591-04503.00/0 Pergamon Press Ltd
CYTOSOLIC A N D M E M B R A N E - B O U N D METHEMOGLOBIN REDUCTASES IN ERYTHROCYTES OF THE OPOSSUM, DIDELPHIS VIRGINIANA N. C. BETHLENFALVAY,*MICHAEL R. WATERMAN,tJ. E. LIMA:~and T. WALDRUP~ Departments of Primary Care* and Clinical Investigation,:[: Fitzsimons Army Medical Center, Aurora, CO 80045 and Department of Biochemistry,t University of Texas Health Science Center, Dallas, TX 75235, U,S.A.
(Received 16 February 1982) Abstract--1. NADH- and NADPH-dependent methemoglobin reductases were found to be present in opossum erythrocytes at approximately the same level as in human erythrocytes. 2. Electrofocusing studies reveal that both enzymes differ from their human counterpart with respect to pI value. 3. Increased amounts of both enzymes are observed following solubilization with either Triton X-100 or cathepsin-D. 4. Thus each activity appears to have a membrane-bound component in opossum erythrocytes as well as in the human erythrocytes.
INTRODUCTION The North American opossum, Didelphis virginiana is the first vertebrate species described to have a replacement of the distal histidine residue in one of the hemoglobin subunit types by another amino acid. In this case, the distal histidine residues of the alpha subunits at position 58 (E7) have been replaced by glutamine residues (Stenzel et al., 1979). Hemoglobin M Boston, a pathological human alpha chain mutant, has tyrosine residues at this position. Found in the heterozygous state, this human mutant hemoglobin results in methemoglobinemia of approximately 30% in affected individuals which is not ameliorated by administration of methylene blue. Thus it is concluded that as a result of the amino acid substitution, methemoglobin reductases found in human erythrocytes cannot reduce the ~-subunits of hemoglobin M Boston in vivo (Gerald & Efron, 1961). Opossum hemoglobin is more readily oxidized and reduced by a variety of reagents than is hemoglobin A (John et al., 1982), and its optical absorption properties in the visible region near 600 nm resemble those of hemoglobin M Boston (Waterman & Stenz'el, 1974). However, in this latter study it was shown that only a small amount of methemoglobin exists in freshly drawn opossum erythrocytes. Thus, in the present study, the levels of the N A D H - and N A D P H dependent methemoglobin reductases and their isoelectric points have been measured in opossum red cells and compared to the results obtained with human erythrocytes. MATERIALS A N D M E T H O D S
Blood was drawn into EDTA from anesthetized adult opossums by cardiac puncture, following which the animals were sacrificed by injection of 5% sodium pentothal. Human blood was drawn into EDTA from healthy volunteers by veinapuncture. The erythrocytes were washed several times with isotonic saline by centrifugation, and hemolysates were prepared by addition of 4 vol of distilled
water followed by centrifugation at 105,0000 for 10 rain at 4°C. Solubilization of membrane bound methemoglobin reductases was achieved by incubation of uncentrifuged hemolysates with Triton X-100 or cathepsin D as described by Choury et al. (1981a). The NADH methemoglobin reductase activity was measured according to the procedure described by Hegesh et al. (1968). The NADPH methemoglobin reductase activity was measured by the method of Huennekens et al. (1957). All determinations were carried out at 25°C using a Cary model 219 recording spectrophotometer. All chemicals used were reagent grade as obtained from Sigma Chemical Co. Electrofocusing was carried out by applying 25 ,ul of 25~o red cell lysates on filter paper strips to thin layer polyacrylamide gel slabs (245 × 110 x 1 mm) containing ampholines (pH 9.5-3.5 or pH 8.5-5.5) obtained from LKB. Electrofocusing was carried out at 10°C for 90 min at a constant wattage of 30 W. A MRA Model 158 power supply (MRA Corp., Clearwater, FL) was used with an LKB model 2117 multiphor flat bed apparatus (LKB, Rockville, Md). Specific staining for NADH and NADPH methemoglobin reductases was carried out as described by Kaplan & Beutler (1967). lsoelectric points of stained proteins were taken from a graph constructed from co-electrofocused standards of known pI (Pharmacia, Uppsula, Sweden). Gels were photographed immediately following completion of the staining procedures and electrofocusing was carried out within 24 hr following drawing of blood samples. RESULTS
Comparison of human and opossum methemoglobin reductases by electrofocusing Several differences between opossum and human methemoglobin reductases are revealed by electrofocusing on a p H 9.5-3.5 gradient (Figs 1 and 2). The N A D H specific band in human hemolysates has a pI of 6.45 while that band in opossum hemolysates has a pI of 6.5 (Fig. 1). The most cathodal formazan band of the human 105,000 g hemolysates (pI 7.8) is observed with both N A D H and N A D P H , but is more intense following staining with the latter (Figs 1 and 2). This is identified as N A D P H methemoglobin reductase
591
592
N. ('. BEIHLENFAI.VAY ('t ill.
7 (Hb)7.
(Hb)
6.
A
A,
B
Fig. 1. Isoelectric focu~,ing of NADH-dcpcndent methemoglobin reductase on a 3,5 9.5 pH gradient. Staining procedures described in the text \~crc used to visualize the reductase. (A) Membrane-free laemoNsatc from human erythrocytes. (B) Membrane-free hcmolvsate fi-om opossum
B
Fig. 3. lsoelectric focusing of NADH-dependent mcthcmoglobin reductase on a 5,5 8.5 pH gradient. (A) Membrane-free hemolysate from opossum erythroc)tes. {B! 105,000,q supernatant follo~,ing cathepsimD treatmem of washed opossum erythrocyte membranes.
el'VIhroc\ |CN
(Yubisui et al., 1979) and is not observed in opossum hemolysates. However, in opossum lysates, a band is observed at a pl of 6.1 with both N A D H and N A D P H , but stains more intensely with the latter (Figs 1 and 2). Thus the N A D H methemoglobin reductases between these two species have very similar pl values whiIe the N A D P H methemoglobin reductases between these two species have very different pl values. Attempts to electrofocus methemoglobin reductases in either crude lysates or washed red cell membranes following incubation with Triton X-100 were unsuccessful, perhaps because of the inability of the enzymes to migrate into the gel under these con-
(Hb)
[Hb)
A
B
Fig. 2. Isoelectric focusing of NADPH-dependenl methemoglobin reductase on a 3.5 9.5 pH gradient. Staining procedures described in the text were used to visualize the reductase. {A) Membrane-free hemolysate from human erythrocytes. (Bt Membrane-free hemolysate from opossum erythrocytes.
ditions. This observation has been confirmed by another laboratory (Kaplan, J. C., personal communication). However, electrofocusing of N A D H methemoglobin reductase solubilized from opossum red cell membranes with cathepsin-D revealed a sharp formazan band having a pl of 6.35 (Fig. 3), similar to that observed with human red cell membranes by Choury et a[. (1981b). In contrast, when stained with N A D P H , no distinct band was observed but rather a dark blue smear on the gel cathodal from the point of application. Comparison of human and opossum methemo~llobin reductases activities The amount of N A D H methemoglobin reductase in human and opossum erythrocytes is compared in Table 1. The activities are similar and are shown to be increased by Triton treatment in both cases. The amount of N A D P H methemoglobin reductase is somewhat greater in opossum red cells than in human red cells and this activity is markedly increased by Triton treatment. Opossum N A D H methemoglobin reductase activities were nearly identical whether human or opossum hemoglobin-ferrocyanide complex was used as substrate (data not shown). This might have been expected in view of the fact that the basic residues suggested as the binding domain for acidic groups of cytochrome b s, the lysyl residues at beta 66 (El0) and beta 95 (FG2), are not altered in opossum hemoglobin (Gacon et al., 1980; Stenzel et a/., 1979). Both methemoglobin reductase activities could be found in substantial amounts in 105,0000 supernatants following digestion of exhaustively washed opossum red cell membranes with Triton X-100 ot cathepsin-D (Table 2). DISCUSSION Both N A D H and N A D P H methemoglobin reductase activities are found in opossum red cells. The
Opossum methemoglobin reductases
593
Table 1. Erythrocyte NADH and NADPH methemoglobin reductase activity in crude hemolysates in the presence and absence of 2% Triton X-100 NADH Reduct~se
NADPB Reductase
Species
n
control
+ Triton X-100
n
control
+ Triton X-100
Opossum
5
2.31 ± ].25
3.27 ± 1.70
7
2.70 ± 0.38
7.34 ± 1.24
Human
8
2.45 ± 0.70
4.20 ± 0.87
5
1.49 ± 0.55
3.15 ± 1.24
Activitiesareexpressed aslU NADH or NADPH methemoglobin reductase perg hemoglobin.
NADH-dependent activity is found at approximately the same level as in human red cells while the NADPH-dependent activity is found at a somewhat higher level than in human red cells. On electrofocusing, the NADH-dependent enzyme is found to closely resemble the human enzyme while the NADPHdependent enzyme is greatly different with respect to pI value. It has recently been shown, that NADH methemoglobin reductase has a membrane bound component in human red cells (Choury et al., 1981b) and this enzyme appears to be entirely membranebound in the nucleated reds cells of birds and reptiles (Board et al., 1977). Triton X-100 was shown to increase the activities of both methemoglobin reductases in opossum and human red cell lysates, more so in the case of the NADPH methemoglobin reductase (Table 1). When 105,000g supernatants of washed opossum red cell membranes were assayed following incubation with Triton X-100 or cathepsin-D, a substantial increase in activity of both methemoglobin reductases was observed (Table 2). These results indicate that NADPH methemoglobin reductase also has a membrane-bound component in both species. The North American opossum is considered to be a living fossil and as such must have been able to cope with a variety of environments. As a result of the amino acid substitution at the distal side of the heme group in the alpha subunits, opossum hemoglobin is more readily oxidized than human hemoglobin (Waterman & Stenzel, 1974). Furthermore, opossum methemoglobin is more rapidly and more completely reduced by ascorbic acid in the presence or absence of inositol hexaphosphate (John & Waterman, 1981). In addition, it has been observed that opossum methe-
moglobin is more rapidly reduced in opossum erythrocytes by the methemoglobin reductase systems than is human methemoglobin in human erythrocytes (Waterman & Stenzel, 1974; John et al., 1982). The present study shows that both NADH and NADPH methemoglobin reductase activities are present in opossum erythrocytes and to approximately the same extent as found in human erythrocytes. Differences in the enzymes between the two species are illustrated by the pI values, however, it is not yet known whether the kinetic properties of the enzymes differ significantly. Perhaps the ability of ascorbate to reduce opossum hemoglobin more rapidly than human hemoglobin provides one explanation as to how the opossum maintains a readily oxidizable hemoglobin in the reduced form. However, it may also be that one or both of the opossum methemoglobin reductase activities are kinetically different such that the enzyme has a greater ability to reduce methemoglobin. Further studies on the individual enzymes will be necessary to resolve this question.
Acknowledgements--This research was supported in part by Grant No. 80-650 (NCB) from the Department of Clinical Investigation, Fitzsimons Army Medical Center and by Grant No. 1-624(MRW) from The Robert A. Welch Foundation.
REFERENCES
BOARDP. G., AGAR N. S., GRUCAM. & SHINE R. (1977) Methemoglobin and its reduction in nucleated erythrocytes from reptiles and birds. Comp. Biochem. Physiol. 57B, 265-267.
Table 2. Effect of Triton X-100 and cathepsin-D on NADH and NADPH methemoglobin reductase activity in washed opossum red cell membranes. Following incubation, 105,000.q supernatants were assayed for enzyme activity Reductase
n
+ Triton X-100
NADH
2
34.28 + 4.12
NADPH
2
14.3
± i.i
+ Catheps~n-D
107.34 -+ 1.8 76.66 ± 2.7
Activities are expressed as IU NADH or NADPH methemoglobin reductase per g protein.
594
N, C. BFTHI,ENFAI,VAYet al,
CH{}URY D. ('., LH~{}t~x A. & KAI'I AN J. ('. {1981a} Membrane-bound cytochromc bs reductase {methemoglobin reductase) in human crythrocytes. Study on normal and methemoglobincmic subjects. J. ('fin. lnt,e.st. 67, 149 155. CHOUR',' D., WAJ{'MAN H., BOISSlil J. I:'. & KAPI,AN J. C. {1981 b} Evidence for endogenous proteolytic solubilization of human red cell membrane NADH cytochromc b5 rcductase. FEBS Lett, 126, 172 174. GIIP,ALI) P. S. & EFR{}N M. R. {1961) ('heroical studies of several varieties of hemoglobin M. Proc. hath. 4{'m/, Sci. U.S.A. 47, 1758 1767. GA('ON G., [,OSqANLI.N D., LABIE D, & KAPI,AN ,I. C. (1980} Interaction between cytochrome b s and hemoglobin: involvement of 66 (El0) and 95 (FG2) lysyl residues of hemoglobin. Proc. mctn. ,4cad. Sei. [, .S.A. 77, 1917 1921. HEGESH E., CAI,MANOVICI N. & AVRON M, (1968) New method for determining fcrrihemoglobin reductase (NADH methemoglobin reductase, in erythrocytes. J. Lab. elin. Med. 72, 339-344. HUENNEKENS F, M.. CAFFERY R, W., BASI~ORD R. E. & GABRtO B. W. (1957) Erythrocytc metabolism IV. Isolation and properties of methemoglobin rcductasc. J, biol. Chem. 227, 261 272,
JOHN M. E. & WATERMAN M. R. 119811 Spectral, conformational and chemical properties of opossum methemoglobin. Eur. J. Biochem. 115, 1 6. JOHN M. E., BETHLENFAI_VAY N. C. & WA1LRMAN M. R. (1982) Oxidation-reduction properties of the hemoglobin of the opossum, Didelphis rir#iniana. Comp. Biochem. Physiol. 73B, 585-590. KAPLAN J. C. & BEUTLI-:R E. (1967) Electrophores~s of red cell NADH and N A D P H diaphorases in normal subjects and patients with congenital mcthemoglobinemia. Bioehem. Biophys. Re.~. Commun. 29, 605 610, STt!NZI~L P., BRIMHALL B., JONES R. T., BI,A('K J. A., MCLACHtAN A. & GmSON D. (1979) Opossum hemoglobin. The amino acid sequences of the x and fl chains. J. biol. Chem. 254, 2071 2076. WATZgMAN M. R. & STENZEL P. (1974) Physical and chemical properties of opossum hemoglobin. A hemoglobin containing glutamine at position 58 (E7) of the 7 subunit. Biochim. Biophys, Aeta 359, 401--410. YIJBISUI "L, MATSUKI T., TAKESHITA M. & YONEYAMA ¥. (1979) Characterization of the purified N A D P H flavin reductase of human crythrocytes. .I. Biochem. 85. 719 728.
0305-0491/82/110591-04503.00/0 Pergamon Press Ltd
CYTOSOLIC A N D M E M B R A N E - B O U N D METHEMOGLOBIN REDUCTASES IN ERYTHROCYTES OF THE OPOSSUM, DIDELPHIS VIRGINIANA N. C. BETHLENFALVAY,*MICHAEL R. WATERMAN,tJ. E. LIMA:~and T. WALDRUP~ Departments of Primary Care* and Clinical Investigation,:[: Fitzsimons Army Medical Center, Aurora, CO 80045 and Department of Biochemistry,t University of Texas Health Science Center, Dallas, TX 75235, U,S.A.
(Received 16 February 1982) Abstract--1. NADH- and NADPH-dependent methemoglobin reductases were found to be present in opossum erythrocytes at approximately the same level as in human erythrocytes. 2. Electrofocusing studies reveal that both enzymes differ from their human counterpart with respect to pI value. 3. Increased amounts of both enzymes are observed following solubilization with either Triton X-100 or cathepsin-D. 4. Thus each activity appears to have a membrane-bound component in opossum erythrocytes as well as in the human erythrocytes.
INTRODUCTION The North American opossum, Didelphis virginiana is the first vertebrate species described to have a replacement of the distal histidine residue in one of the hemoglobin subunit types by another amino acid. In this case, the distal histidine residues of the alpha subunits at position 58 (E7) have been replaced by glutamine residues (Stenzel et al., 1979). Hemoglobin M Boston, a pathological human alpha chain mutant, has tyrosine residues at this position. Found in the heterozygous state, this human mutant hemoglobin results in methemoglobinemia of approximately 30% in affected individuals which is not ameliorated by administration of methylene blue. Thus it is concluded that as a result of the amino acid substitution, methemoglobin reductases found in human erythrocytes cannot reduce the ~-subunits of hemoglobin M Boston in vivo (Gerald & Efron, 1961). Opossum hemoglobin is more readily oxidized and reduced by a variety of reagents than is hemoglobin A (John et al., 1982), and its optical absorption properties in the visible region near 600 nm resemble those of hemoglobin M Boston (Waterman & Stenz'el, 1974). However, in this latter study it was shown that only a small amount of methemoglobin exists in freshly drawn opossum erythrocytes. Thus, in the present study, the levels of the N A D H - and N A D P H dependent methemoglobin reductases and their isoelectric points have been measured in opossum red cells and compared to the results obtained with human erythrocytes. MATERIALS A N D M E T H O D S
Blood was drawn into EDTA from anesthetized adult opossums by cardiac puncture, following which the animals were sacrificed by injection of 5% sodium pentothal. Human blood was drawn into EDTA from healthy volunteers by veinapuncture. The erythrocytes were washed several times with isotonic saline by centrifugation, and hemolysates were prepared by addition of 4 vol of distilled
water followed by centrifugation at 105,0000 for 10 rain at 4°C. Solubilization of membrane bound methemoglobin reductases was achieved by incubation of uncentrifuged hemolysates with Triton X-100 or cathepsin D as described by Choury et al. (1981a). The NADH methemoglobin reductase activity was measured according to the procedure described by Hegesh et al. (1968). The NADPH methemoglobin reductase activity was measured by the method of Huennekens et al. (1957). All determinations were carried out at 25°C using a Cary model 219 recording spectrophotometer. All chemicals used were reagent grade as obtained from Sigma Chemical Co. Electrofocusing was carried out by applying 25 ,ul of 25~o red cell lysates on filter paper strips to thin layer polyacrylamide gel slabs (245 × 110 x 1 mm) containing ampholines (pH 9.5-3.5 or pH 8.5-5.5) obtained from LKB. Electrofocusing was carried out at 10°C for 90 min at a constant wattage of 30 W. A MRA Model 158 power supply (MRA Corp., Clearwater, FL) was used with an LKB model 2117 multiphor flat bed apparatus (LKB, Rockville, Md). Specific staining for NADH and NADPH methemoglobin reductases was carried out as described by Kaplan & Beutler (1967). lsoelectric points of stained proteins were taken from a graph constructed from co-electrofocused standards of known pI (Pharmacia, Uppsula, Sweden). Gels were photographed immediately following completion of the staining procedures and electrofocusing was carried out within 24 hr following drawing of blood samples. RESULTS
Comparison of human and opossum methemoglobin reductases by electrofocusing Several differences between opossum and human methemoglobin reductases are revealed by electrofocusing on a p H 9.5-3.5 gradient (Figs 1 and 2). The N A D H specific band in human hemolysates has a pI of 6.45 while that band in opossum hemolysates has a pI of 6.5 (Fig. 1). The most cathodal formazan band of the human 105,000 g hemolysates (pI 7.8) is observed with both N A D H and N A D P H , but is more intense following staining with the latter (Figs 1 and 2). This is identified as N A D P H methemoglobin reductase
591
592
N. ('. BEIHLENFAI.VAY ('t ill.
7 (Hb)7.
(Hb)
6.
A
A,
B
Fig. 1. Isoelectric focu~,ing of NADH-dcpcndent methemoglobin reductase on a 3,5 9.5 pH gradient. Staining procedures described in the text \~crc used to visualize the reductase. (A) Membrane-free laemoNsatc from human erythrocytes. (B) Membrane-free hcmolvsate fi-om opossum
B
Fig. 3. lsoelectric focusing of NADH-dependent mcthcmoglobin reductase on a 5,5 8.5 pH gradient. (A) Membrane-free hemolysate from opossum erythroc)tes. {B! 105,000,q supernatant follo~,ing cathepsimD treatmem of washed opossum erythrocyte membranes.
el'VIhroc\ |CN
(Yubisui et al., 1979) and is not observed in opossum hemolysates. However, in opossum lysates, a band is observed at a pl of 6.1 with both N A D H and N A D P H , but stains more intensely with the latter (Figs 1 and 2). Thus the N A D H methemoglobin reductases between these two species have very similar pl values whiIe the N A D P H methemoglobin reductases between these two species have very different pl values. Attempts to electrofocus methemoglobin reductases in either crude lysates or washed red cell membranes following incubation with Triton X-100 were unsuccessful, perhaps because of the inability of the enzymes to migrate into the gel under these con-
(Hb)
[Hb)
A
B
Fig. 2. Isoelectric focusing of NADPH-dependenl methemoglobin reductase on a 3.5 9.5 pH gradient. Staining procedures described in the text were used to visualize the reductase. {A) Membrane-free hemolysate from human erythrocytes. (Bt Membrane-free hemolysate from opossum erythrocytes.
ditions. This observation has been confirmed by another laboratory (Kaplan, J. C., personal communication). However, electrofocusing of N A D H methemoglobin reductase solubilized from opossum red cell membranes with cathepsin-D revealed a sharp formazan band having a pl of 6.35 (Fig. 3), similar to that observed with human red cell membranes by Choury et a[. (1981b). In contrast, when stained with N A D P H , no distinct band was observed but rather a dark blue smear on the gel cathodal from the point of application. Comparison of human and opossum methemo~llobin reductases activities The amount of N A D H methemoglobin reductase in human and opossum erythrocytes is compared in Table 1. The activities are similar and are shown to be increased by Triton treatment in both cases. The amount of N A D P H methemoglobin reductase is somewhat greater in opossum red cells than in human red cells and this activity is markedly increased by Triton treatment. Opossum N A D H methemoglobin reductase activities were nearly identical whether human or opossum hemoglobin-ferrocyanide complex was used as substrate (data not shown). This might have been expected in view of the fact that the basic residues suggested as the binding domain for acidic groups of cytochrome b s, the lysyl residues at beta 66 (El0) and beta 95 (FG2), are not altered in opossum hemoglobin (Gacon et al., 1980; Stenzel et a/., 1979). Both methemoglobin reductase activities could be found in substantial amounts in 105,0000 supernatants following digestion of exhaustively washed opossum red cell membranes with Triton X-100 ot cathepsin-D (Table 2). DISCUSSION Both N A D H and N A D P H methemoglobin reductase activities are found in opossum red cells. The
Opossum methemoglobin reductases
593
Table 1. Erythrocyte NADH and NADPH methemoglobin reductase activity in crude hemolysates in the presence and absence of 2% Triton X-100 NADH Reduct~se
NADPB Reductase
Species
n
control
+ Triton X-100
n
control
+ Triton X-100
Opossum
5
2.31 ± ].25
3.27 ± 1.70
7
2.70 ± 0.38
7.34 ± 1.24
Human
8
2.45 ± 0.70
4.20 ± 0.87
5
1.49 ± 0.55
3.15 ± 1.24
Activitiesareexpressed aslU NADH or NADPH methemoglobin reductase perg hemoglobin.
NADH-dependent activity is found at approximately the same level as in human red cells while the NADPH-dependent activity is found at a somewhat higher level than in human red cells. On electrofocusing, the NADH-dependent enzyme is found to closely resemble the human enzyme while the NADPHdependent enzyme is greatly different with respect to pI value. It has recently been shown, that NADH methemoglobin reductase has a membrane bound component in human red cells (Choury et al., 1981b) and this enzyme appears to be entirely membranebound in the nucleated reds cells of birds and reptiles (Board et al., 1977). Triton X-100 was shown to increase the activities of both methemoglobin reductases in opossum and human red cell lysates, more so in the case of the NADPH methemoglobin reductase (Table 1). When 105,000g supernatants of washed opossum red cell membranes were assayed following incubation with Triton X-100 or cathepsin-D, a substantial increase in activity of both methemoglobin reductases was observed (Table 2). These results indicate that NADPH methemoglobin reductase also has a membrane-bound component in both species. The North American opossum is considered to be a living fossil and as such must have been able to cope with a variety of environments. As a result of the amino acid substitution at the distal side of the heme group in the alpha subunits, opossum hemoglobin is more readily oxidized than human hemoglobin (Waterman & Stenzel, 1974). Furthermore, opossum methemoglobin is more rapidly and more completely reduced by ascorbic acid in the presence or absence of inositol hexaphosphate (John & Waterman, 1981). In addition, it has been observed that opossum methe-
moglobin is more rapidly reduced in opossum erythrocytes by the methemoglobin reductase systems than is human methemoglobin in human erythrocytes (Waterman & Stenzel, 1974; John et al., 1982). The present study shows that both NADH and NADPH methemoglobin reductase activities are present in opossum erythrocytes and to approximately the same extent as found in human erythrocytes. Differences in the enzymes between the two species are illustrated by the pI values, however, it is not yet known whether the kinetic properties of the enzymes differ significantly. Perhaps the ability of ascorbate to reduce opossum hemoglobin more rapidly than human hemoglobin provides one explanation as to how the opossum maintains a readily oxidizable hemoglobin in the reduced form. However, it may also be that one or both of the opossum methemoglobin reductase activities are kinetically different such that the enzyme has a greater ability to reduce methemoglobin. Further studies on the individual enzymes will be necessary to resolve this question.
Acknowledgements--This research was supported in part by Grant No. 80-650 (NCB) from the Department of Clinical Investigation, Fitzsimons Army Medical Center and by Grant No. 1-624(MRW) from The Robert A. Welch Foundation.
REFERENCES
BOARDP. G., AGAR N. S., GRUCAM. & SHINE R. (1977) Methemoglobin and its reduction in nucleated erythrocytes from reptiles and birds. Comp. Biochem. Physiol. 57B, 265-267.
Table 2. Effect of Triton X-100 and cathepsin-D on NADH and NADPH methemoglobin reductase activity in washed opossum red cell membranes. Following incubation, 105,000.q supernatants were assayed for enzyme activity Reductase
n
+ Triton X-100
NADH
2
34.28 + 4.12
NADPH
2
14.3
± i.i
+ Catheps~n-D
107.34 -+ 1.8 76.66 ± 2.7
Activities are expressed as IU NADH or NADPH methemoglobin reductase per g protein.
594
N, C. BFTHI,ENFAI,VAYet al,
CH{}URY D. ('., LH~{}t~x A. & KAI'I AN J. ('. {1981a} Membrane-bound cytochromc bs reductase {methemoglobin reductase) in human crythrocytes. Study on normal and methemoglobincmic subjects. J. ('fin. lnt,e.st. 67, 149 155. CHOUR',' D., WAJ{'MAN H., BOISSlil J. I:'. & KAPI,AN J. C. {1981 b} Evidence for endogenous proteolytic solubilization of human red cell membrane NADH cytochromc b5 rcductase. FEBS Lett, 126, 172 174. GIIP,ALI) P. S. & EFR{}N M. R. {1961) ('heroical studies of several varieties of hemoglobin M. Proc. hath. 4{'m/, Sci. U.S.A. 47, 1758 1767. GA('ON G., [,OSqANLI.N D., LABIE D, & KAPI,AN ,I. C. (1980} Interaction between cytochrome b s and hemoglobin: involvement of 66 (El0) and 95 (FG2) lysyl residues of hemoglobin. Proc. mctn. ,4cad. Sei. [, .S.A. 77, 1917 1921. HEGESH E., CAI,MANOVICI N. & AVRON M, (1968) New method for determining fcrrihemoglobin reductase (NADH methemoglobin reductase, in erythrocytes. J. Lab. elin. Med. 72, 339-344. HUENNEKENS F, M.. CAFFERY R, W., BASI~ORD R. E. & GABRtO B. W. (1957) Erythrocytc metabolism IV. Isolation and properties of methemoglobin rcductasc. J, biol. Chem. 227, 261 272,
JOHN M. E. & WATERMAN M. R. 119811 Spectral, conformational and chemical properties of opossum methemoglobin. Eur. J. Biochem. 115, 1 6. JOHN M. E., BETHLENFAI_VAY N. C. & WA1LRMAN M. R. (1982) Oxidation-reduction properties of the hemoglobin of the opossum, Didelphis rir#iniana. Comp. Biochem. Physiol. 73B, 585-590. KAPLAN J. C. & BEUTLI-:R E. (1967) Electrophores~s of red cell NADH and N A D P H diaphorases in normal subjects and patients with congenital mcthemoglobinemia. Bioehem. Biophys. Re.~. Commun. 29, 605 610, STt!NZI~L P., BRIMHALL B., JONES R. T., BI,A('K J. A., MCLACHtAN A. & GmSON D. (1979) Opossum hemoglobin. The amino acid sequences of the x and fl chains. J. biol. Chem. 254, 2071 2076. WATZgMAN M. R. & STENZEL P. (1974) Physical and chemical properties of opossum hemoglobin. A hemoglobin containing glutamine at position 58 (E7) of the 7 subunit. Biochim. Biophys, Aeta 359, 401--410. YIJBISUI "L, MATSUKI T., TAKESHITA M. & YONEYAMA ¥. (1979) Characterization of the purified N A D P H flavin reductase of human crythrocytes. .I. Biochem. 85. 719 728.