Vol.
186,
No.
July
31, 1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
CERULOPLASMIN
STIMULATES NADH OXIDATION PLASMA lWEMBRANJ3
951-955
OF PIG LIVER
F.J. Alcainl, J.M. Villalba’, H. Li5w2, F.L. Crane3 and P. Navas’* ‘Departamento de Biologia Celular, Universidad de Cordoba, 14004-Cordoba, Spain 2Department of>Endocrinology, Karolinska Institute, Stockholm, Sweden 3Department of Biological Sciences,Purdue University, West Lafayette, IN 47907 Received
May 16,
1992
SUMMARY: NADH oxidationby pig liver plasmamembranes is stimulatedby ceruloplasmin (CUP) reachinga maximalvalue at 50 U/ml of CUP. NADH oxidation activatedby CUP is proportionalto the amountof protein. ConcanavalinA (Con A) which recognizesthe glucidic residuesof the CUP requiredfor bindingto the receptor inhibitsthe NADH oxidation in a dose-responsivemanner. Both adriamycin and bathophenantrolinedisulfonate @PS), previously reported as transplasma membraneelectron transport inhibitors, also inhibit the CUP-stimulatedNADH oxidationof pig liver plasmamembranes.Our resultsshow a clear interactionbetweenCUP and the NADH oxidaseof plasmamembrane,which supportsan oxidative role for CUP in its growth effect. 0 1992ncademrc Pre55,Inc.
Ceruloplasmin (CUP) is a 132 KDa glycoprotein present in the blood plasma of vertebrates (1). CUP transports the copper from the liver where is mainly synthesized (2) to recipient cells but it also has superoxide dismutase, ascorbate oxidase and ferroxidase II activities (3). Recently we reported a strong stimulation of DNA synthesis induced by CUP in CCL 29 cells (4). This stimulation was greater that observed for fetal calf serumand it was found in both normal (20%) and low (1%) oxygen tension. Transplasmamembraneelectron transport has been involved on cell growth control (5). Besides, ferricyanide and other iron-containing acceptors (6,7) and ascorbate(8), oxygen is the putative final acceptor of plasmamembraneelectron flow from the intracellular NADH (9). NADH oxidase is modulated by diferric transferrin (10) and inhibited by growth inhibitory gangliosides(11). We now show stimulation of NADH oxidase of pig liver plasmamembraneby CUP and its inhibition by both transplasmamembraneelctron flow inhibitors and Con A lectin. These findings support the oxidase role of CUP as a growth factor and its ability to interact with the NADH oxidase of liver plasmamembrane.
*To whom correspondenceshouldbe addressed.
Vol.
186,
No.
Materials
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCtI
COMMUNICATIONS
and Methods
Plasma membranes nurification. Highly-enriched plasma membrane fractions from pig liver were obtained by aqueous two-phases partitioning as described (12). Concentrated microsomal fraction were added to a mixture of 6.0% (w/w) Dextran T-500 (T’harmacia), 6.0% polyethylene glycol 3350 (Fisher), 0.25 sucrose and 15 mM TRWH2S04, pH 7.8. The content of the two-phases system were then mixed by 40 inversions of the tubes at 4”C, and the two phases were separated by centrifugation in a swinging bucket rotor of 1000 g for 5 min. The upper phase was added to clean lower phase and mixing and separation was repeated twice. The final upper phase, enriched in plasma membrane, was diluted with 1 mM sodium bicarbonate and the plasma membranes were collected by centrifugation for 20 min at 80,000 g. Membrane purity was controlled by marker enzyme analysis as in previous studies with rat liver (12). NADH oxidase activity. This activity was carried out in a medium containing 25 mM TrisHCI, 25 pM NADH, 150 mM NaCl and 5 mM KCL, pH 7.4 and 0.08 to 0.10 mg protein in a final volume of 0.5 ml. Absorbance was monitored at 340 nm, and an extinction coefftcient of 6.2 mM-l.crn-t was used for NADH. 50 U/ml of CUP contained 0.45 mg of CUP protein. ReNl1t.S Plasma membrane from pig liver contains a NADH of NADH
oxidized/min/mg
oxidase of about 4 nmoles
protein in absence of oxidants (Fig. 1). NADH
oxidation
can be stimulated by CUP in a dose-response manner reaching a maximal value about 20 nmoles/min/mg protein in the presence of 50 U/ml of CUP. Higher concentrations of CUP did not futher increase NADH oxidation (Fig. 1). CUP at the same concentration oxidation
only oxidized NADH
in the presence of CUP
at a rate of 0.1 nmoles/min was proportional
(Fig. 2). NADH
to the amount of plasma
3.0
o J,
01
0
0.0 50
Ceruloplasmin
100
0
150
02
(U/ml)
50
Protein
100
150
(pg/ml)
F&J. Oxidation of NADH by pig liver plasmamembranes in the presenceof different concentrations of CUP. Protein present in the assay was 80 pglml. Points represent the average of three experiments. m. CUP-stimulated NADH oxidasein the presenceof different amountsof protein. CUP was added at 100 U/ml. Points represent the average of three experiments. 952
200
Vol.
186,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I. Effect of different inhibitors on the rate of NADH oxidation in the presence or absence of CUP (50 U/ml). Specific activities are expressed as nmoles of NADH oxidizedlminlmg protein . Standard deviations were less than 15% (n==3). NADH oxidase Additions
- CUP
% Inhibition
+cup
% Inhibition
None
4.0
20.2
Boiled plasma membrane
0.0
100
0.1
Con A (20 Leg/ml)
1.4
65
4.3
80
Adriamycin (1 PM)
2.8
30
12.1
40
BPS (0.4 mM)
2.0
50
10.3
50
Antimycin A (1 PM)
3.9
2
19.2
5
membrane present in the assay (Fig. 2). NADPH membrane was 4.2-Ll.l
nmoles of NADPH
97
oxidase activity of pig liver plasma
oxidized/min/mg
protein and was not
activated by the presence of 50 U/ml CUP, showing a specific activity of 3.7kO.9 nmoles/minlmg
protein.
Boiled membranes NADH
showed
non oxidase activity (Table I). CUP-stimulated
oxidase was inhibited by Con A, reaching 80% inhibition with 20pg/ml (Fig.
3, Table I). NADH
oxidase itself is also inhibited by Con A but only up to 65%
(Table I). CUP-stimulated
NADH
I) and BPS (Fig. 4). Adriamycin
oxidase activity is inhibited by adriamycin
inhibited 50% of this activity (Fig 4, Table I). NADH similar way by these inhibitors transport such as antimycin
(Table
at 1 PM inhibited about 40% and 0.4 mM BPS oxidase was also affected in a
(Table I). An inhibitor
of mitochondrial
electron
A do not inhibit the plasma membrane oxidase with or
without CUP (Table I).
Discussion Addition of external oxidants to serum-free or serum limiting media stimulates the proliferation
of several transformed
oxidants used have been artificial,
cell lines (5,13).
Although
most of the
natural oxidants found in the serum as diferric
transferrin and ascorbate also stimulate cell growth (6,14,15). That they act as electron acceptors is demonstrated by their ability to oxidize NADH in whole cells or in plasma membrane preparations (5,7,16). 953
Vol.
186, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
J
, 0.0
03
Concanavalin
04
A (g/ml)
RESEARCH COMMUNICATIONS
,
0.1
0.2
0.3
Bathophenanthroline disulfonate (m&i)
w. Effect of Con A on CUP-stimulated NADH oxidation of pig liver plasma membrane. CUP was added at 50 U/ml. Points represent the average of three experiments. u. Effect of bathophenanthroline disulfonate on CUP-stimulated NADH oxidase. CUP was added at 50 U/ml. Points represent the average of three experiments.
CUP, a recognized ferrous oxidase (1) produces a high stimulation of DNA synthesis in CCL 39 cells (4). Although these cells can reduce the CUP under anaerobic conditions (Alcain, Ikw and Crane, data not published), evidence of the oxidation of cytosolic NADH induced by CUP have not been presented. Data shown in this paper demonstrate that NADH oxidation by liver plasma membrane is increasingly stimulated by CUP concentration up to 50 U/ml. This concentration representing 0.45 mg/ml is 50% higher than found in plasma (0.3 mg/ml) (17). Higher concentrations do not produce higher rates of NADH oxidation. NADH oxidase is inhibited by Con A both in the absenceor presenceof CUP. The higher inhibition observed for CUP-stimulated NADH oxidase could be causedby blocking the carbohydrate moieties of CUP by Con A required for binding to the receptor (18,19), indicating a receptor-binding mediatedaction of CUP on NADH oxidation. The antitumor agent adriamycin and the iron chelator BPS have been shownto inhibit the reduction of electron acceptors by whole cells and isolated plasma membranes (20-22). These inhibitions would relate NADH oxidase with the transplasmamembraneelectron flow to other oxidants than oxygen, and show a clear of NADH oxidase with the receptor mediated growth factor (CUP) induced cell growth (4). Transfen-in also stimulatesNADH oxidase of liver plasma membrane(10) and dependson receptor-mediatedaction (7). The findings shown here relationship
introduce additional evidence for a role of redox system on growth regulation and a clear relationship to growth factor induced proliferation. 954
0.4
Vol. 186, No. 2, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgment This work was partially supported by the Spanish DGICYT
no. PB89-0337-
co2-01. References 1. Frieden, G. (1981) In Ciba Foundation Symposium 79. Biological Roles of Copper, Experts Medica, New York, pp. 93-124. 2. Sato, M and Gitlin, J.D. (1991) J. Biol. Chem. 266, 5128-5134. 3. Goldstein, I.M, Kaplan, H.B, Hedelson, H.S. and Weissman, G. (1979) J. Biol. Chem. 254, 4040-4045. 4. Alcain, F.J., L6w, H. and Crane, F.L. (1991) Biochem. Biophys. Res. Commun. 180, 790-796. 5. Crane, F.L., Sun, I., Clark, M.G., Grebing, C. and L6w, H. (1985) Biochim. Biophys. Acta 811, 233-264. 6. Goldenberg, H., Crane, F.L. and MorrC, D.J.( 1979) J. Biol. Chem. 254, 2491-2498. 7. Sun, I.L., Navas, P., Crane, F.L., MorrB, D.J. and LKw, H. (1987) J. Biol. Chem. 262, 15915-15921. 8. Navas, P., Est&ez, A.,Bur&, M.I., Villalba, J.M. and Crane, F.L. (1988) Biochem. Biophys. Res. Commun. 154, 1029-1033. 9. Morrt?, D.J. and Brightman, A.O. (1991) J. Bioenerg. Biomembr. 23, 469-489. 10. MorrB, D.J., Crane, F.L. Eriksson, L.C., L6w, H. and Morrt$ D.M. (1991) Biochim. Biophys. Acta 1057, 140-146. 11. Morrt$ D.J. and Crane, F.L. (1990) In Oxidoreduction in the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., MorrC, D.J. and Lijw, H., Eds.), CRC Press, Boca Raton, pp. 67-84. 12. Navas, P, Nowack, D.D. and Morre, D.J. (1990) Cancer Res. 49, 2147-2158. 13. Crane, F.L., LBw, H., Sun, I.L. and Isaksson, M. (1990) In Oxidoreduction at the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., Morre, D.J. and LB,, H., Eds.), CRC Press, Boca Raton, pp. 141-170. 14. Barnes, D. and Sato, G. (1980) Cell 22, 649-654. 15. Alcaln, F.J., Bur6n, M.I., Rodriguez-Aguilera, J.C., Villalba, J.M. and Navas, P. (1990) Cancer Res. 50, 5887-5891. 16. Navas, P. and Burbn, M.I. (1990) In Oxidoreduction at the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., Morrt!, D.J. and Liiw, H.. Eds.), CRC Press, Boca Raton, pp. 225-236. 17. Tallis, G.A., Kitchiner, M.I. and Thomas, A.C. (1990) Clin. Chem. 36, 568-570. 18. Saenko, E.L. and Yaropolov, A.I. (1990) Biochem. Int. 20, 215-225 19. Saenko, E.L., Skorobogat’ko, O.V. and Yarolpolov, A.I. (1990) Biochem. Int. 22, 749757. 20. Sun, I.L., Crane, F.L., LOW, H. and Grebing, C. (1984) J. Bioenerg. Biomembr. 16, 209-22 1. 21. Grebing, C., Crane, F.L., Liiw, H. and Hall, K. (1984) J. Bioenerg. Biomembr. 16. 517533. 22. Sun, I.L., Navas, P.. Crane, F.L., Morrt!, D.J. and LOW, H. (1987) Biochem. Int. 14, 119-127.
955
186,
No.
July
31, 1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
CERULOPLASMIN
STIMULATES NADH OXIDATION PLASMA lWEMBRANJ3
951-955
OF PIG LIVER
F.J. Alcainl, J.M. Villalba’, H. Li5w2, F.L. Crane3 and P. Navas’* ‘Departamento de Biologia Celular, Universidad de Cordoba, 14004-Cordoba, Spain 2Department of>Endocrinology, Karolinska Institute, Stockholm, Sweden 3Department of Biological Sciences,Purdue University, West Lafayette, IN 47907 Received
May 16,
1992
SUMMARY: NADH oxidationby pig liver plasmamembranes is stimulatedby ceruloplasmin (CUP) reachinga maximalvalue at 50 U/ml of CUP. NADH oxidation activatedby CUP is proportionalto the amountof protein. ConcanavalinA (Con A) which recognizesthe glucidic residuesof the CUP requiredfor bindingto the receptor inhibitsthe NADH oxidation in a dose-responsivemanner. Both adriamycin and bathophenantrolinedisulfonate @PS), previously reported as transplasma membraneelectron transport inhibitors, also inhibit the CUP-stimulatedNADH oxidationof pig liver plasmamembranes.Our resultsshow a clear interactionbetweenCUP and the NADH oxidaseof plasmamembrane,which supportsan oxidative role for CUP in its growth effect. 0 1992ncademrc Pre55,Inc.
Ceruloplasmin (CUP) is a 132 KDa glycoprotein present in the blood plasma of vertebrates (1). CUP transports the copper from the liver where is mainly synthesized (2) to recipient cells but it also has superoxide dismutase, ascorbate oxidase and ferroxidase II activities (3). Recently we reported a strong stimulation of DNA synthesis induced by CUP in CCL 29 cells (4). This stimulation was greater that observed for fetal calf serumand it was found in both normal (20%) and low (1%) oxygen tension. Transplasmamembraneelectron transport has been involved on cell growth control (5). Besides, ferricyanide and other iron-containing acceptors (6,7) and ascorbate(8), oxygen is the putative final acceptor of plasmamembraneelectron flow from the intracellular NADH (9). NADH oxidase is modulated by diferric transferrin (10) and inhibited by growth inhibitory gangliosides(11). We now show stimulation of NADH oxidase of pig liver plasmamembraneby CUP and its inhibition by both transplasmamembraneelctron flow inhibitors and Con A lectin. These findings support the oxidase role of CUP as a growth factor and its ability to interact with the NADH oxidase of liver plasmamembrane.
*To whom correspondenceshouldbe addressed.
Vol.
186,
No.
Materials
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCtI
COMMUNICATIONS
and Methods
Plasma membranes nurification. Highly-enriched plasma membrane fractions from pig liver were obtained by aqueous two-phases partitioning as described (12). Concentrated microsomal fraction were added to a mixture of 6.0% (w/w) Dextran T-500 (T’harmacia), 6.0% polyethylene glycol 3350 (Fisher), 0.25 sucrose and 15 mM TRWH2S04, pH 7.8. The content of the two-phases system were then mixed by 40 inversions of the tubes at 4”C, and the two phases were separated by centrifugation in a swinging bucket rotor of 1000 g for 5 min. The upper phase was added to clean lower phase and mixing and separation was repeated twice. The final upper phase, enriched in plasma membrane, was diluted with 1 mM sodium bicarbonate and the plasma membranes were collected by centrifugation for 20 min at 80,000 g. Membrane purity was controlled by marker enzyme analysis as in previous studies with rat liver (12). NADH oxidase activity. This activity was carried out in a medium containing 25 mM TrisHCI, 25 pM NADH, 150 mM NaCl and 5 mM KCL, pH 7.4 and 0.08 to 0.10 mg protein in a final volume of 0.5 ml. Absorbance was monitored at 340 nm, and an extinction coefftcient of 6.2 mM-l.crn-t was used for NADH. 50 U/ml of CUP contained 0.45 mg of CUP protein. ReNl1t.S Plasma membrane from pig liver contains a NADH of NADH
oxidized/min/mg
oxidase of about 4 nmoles
protein in absence of oxidants (Fig. 1). NADH
oxidation
can be stimulated by CUP in a dose-response manner reaching a maximal value about 20 nmoles/min/mg protein in the presence of 50 U/ml of CUP. Higher concentrations of CUP did not futher increase NADH oxidation (Fig. 1). CUP at the same concentration oxidation
only oxidized NADH
in the presence of CUP
at a rate of 0.1 nmoles/min was proportional
(Fig. 2). NADH
to the amount of plasma
3.0
o J,
01
0
0.0 50
Ceruloplasmin
100
0
150
02
(U/ml)
50
Protein
100
150
(pg/ml)
F&J. Oxidation of NADH by pig liver plasmamembranes in the presenceof different concentrations of CUP. Protein present in the assay was 80 pglml. Points represent the average of three experiments. m. CUP-stimulated NADH oxidasein the presenceof different amountsof protein. CUP was added at 100 U/ml. Points represent the average of three experiments. 952
200
Vol.
186,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Table I. Effect of different inhibitors on the rate of NADH oxidation in the presence or absence of CUP (50 U/ml). Specific activities are expressed as nmoles of NADH oxidizedlminlmg protein . Standard deviations were less than 15% (n==3). NADH oxidase Additions
- CUP
% Inhibition
+cup
% Inhibition
None
4.0
20.2
Boiled plasma membrane
0.0
100
0.1
Con A (20 Leg/ml)
1.4
65
4.3
80
Adriamycin (1 PM)
2.8
30
12.1
40
BPS (0.4 mM)
2.0
50
10.3
50
Antimycin A (1 PM)
3.9
2
19.2
5
membrane present in the assay (Fig. 2). NADPH membrane was 4.2-Ll.l
nmoles of NADPH
97
oxidase activity of pig liver plasma
oxidized/min/mg
protein and was not
activated by the presence of 50 U/ml CUP, showing a specific activity of 3.7kO.9 nmoles/minlmg
protein.
Boiled membranes NADH
showed
non oxidase activity (Table I). CUP-stimulated
oxidase was inhibited by Con A, reaching 80% inhibition with 20pg/ml (Fig.
3, Table I). NADH
oxidase itself is also inhibited by Con A but only up to 65%
(Table I). CUP-stimulated
NADH
I) and BPS (Fig. 4). Adriamycin
oxidase activity is inhibited by adriamycin
inhibited 50% of this activity (Fig 4, Table I). NADH similar way by these inhibitors transport such as antimycin
(Table
at 1 PM inhibited about 40% and 0.4 mM BPS oxidase was also affected in a
(Table I). An inhibitor
of mitochondrial
electron
A do not inhibit the plasma membrane oxidase with or
without CUP (Table I).
Discussion Addition of external oxidants to serum-free or serum limiting media stimulates the proliferation
of several transformed
oxidants used have been artificial,
cell lines (5,13).
Although
most of the
natural oxidants found in the serum as diferric
transferrin and ascorbate also stimulate cell growth (6,14,15). That they act as electron acceptors is demonstrated by their ability to oxidize NADH in whole cells or in plasma membrane preparations (5,7,16). 953
Vol.
186, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
J
, 0.0
03
Concanavalin
04
A (g/ml)
RESEARCH COMMUNICATIONS
,
0.1
0.2
0.3
Bathophenanthroline disulfonate (m&i)
w. Effect of Con A on CUP-stimulated NADH oxidation of pig liver plasma membrane. CUP was added at 50 U/ml. Points represent the average of three experiments. u. Effect of bathophenanthroline disulfonate on CUP-stimulated NADH oxidase. CUP was added at 50 U/ml. Points represent the average of three experiments.
CUP, a recognized ferrous oxidase (1) produces a high stimulation of DNA synthesis in CCL 39 cells (4). Although these cells can reduce the CUP under anaerobic conditions (Alcain, Ikw and Crane, data not published), evidence of the oxidation of cytosolic NADH induced by CUP have not been presented. Data shown in this paper demonstrate that NADH oxidation by liver plasma membrane is increasingly stimulated by CUP concentration up to 50 U/ml. This concentration representing 0.45 mg/ml is 50% higher than found in plasma (0.3 mg/ml) (17). Higher concentrations do not produce higher rates of NADH oxidation. NADH oxidase is inhibited by Con A both in the absenceor presenceof CUP. The higher inhibition observed for CUP-stimulated NADH oxidase could be causedby blocking the carbohydrate moieties of CUP by Con A required for binding to the receptor (18,19), indicating a receptor-binding mediatedaction of CUP on NADH oxidation. The antitumor agent adriamycin and the iron chelator BPS have been shownto inhibit the reduction of electron acceptors by whole cells and isolated plasma membranes (20-22). These inhibitions would relate NADH oxidase with the transplasmamembraneelectron flow to other oxidants than oxygen, and show a clear of NADH oxidase with the receptor mediated growth factor (CUP) induced cell growth (4). Transfen-in also stimulatesNADH oxidase of liver plasma membrane(10) and dependson receptor-mediatedaction (7). The findings shown here relationship
introduce additional evidence for a role of redox system on growth regulation and a clear relationship to growth factor induced proliferation. 954
0.4
Vol. 186, No. 2, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgment This work was partially supported by the Spanish DGICYT
no. PB89-0337-
co2-01. References 1. Frieden, G. (1981) In Ciba Foundation Symposium 79. Biological Roles of Copper, Experts Medica, New York, pp. 93-124. 2. Sato, M and Gitlin, J.D. (1991) J. Biol. Chem. 266, 5128-5134. 3. Goldstein, I.M, Kaplan, H.B, Hedelson, H.S. and Weissman, G. (1979) J. Biol. Chem. 254, 4040-4045. 4. Alcain, F.J., L6w, H. and Crane, F.L. (1991) Biochem. Biophys. Res. Commun. 180, 790-796. 5. Crane, F.L., Sun, I., Clark, M.G., Grebing, C. and L6w, H. (1985) Biochim. Biophys. Acta 811, 233-264. 6. Goldenberg, H., Crane, F.L. and MorrC, D.J.( 1979) J. Biol. Chem. 254, 2491-2498. 7. Sun, I.L., Navas, P., Crane, F.L., MorrB, D.J. and LKw, H. (1987) J. Biol. Chem. 262, 15915-15921. 8. Navas, P., Est&ez, A.,Bur&, M.I., Villalba, J.M. and Crane, F.L. (1988) Biochem. Biophys. Res. Commun. 154, 1029-1033. 9. Morrt?, D.J. and Brightman, A.O. (1991) J. Bioenerg. Biomembr. 23, 469-489. 10. MorrB, D.J., Crane, F.L. Eriksson, L.C., L6w, H. and Morrt$ D.M. (1991) Biochim. Biophys. Acta 1057, 140-146. 11. Morrt$ D.J. and Crane, F.L. (1990) In Oxidoreduction in the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., MorrC, D.J. and Lijw, H., Eds.), CRC Press, Boca Raton, pp. 67-84. 12. Navas, P, Nowack, D.D. and Morre, D.J. (1990) Cancer Res. 49, 2147-2158. 13. Crane, F.L., LBw, H., Sun, I.L. and Isaksson, M. (1990) In Oxidoreduction at the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., Morre, D.J. and LB,, H., Eds.), CRC Press, Boca Raton, pp. 141-170. 14. Barnes, D. and Sato, G. (1980) Cell 22, 649-654. 15. Alcaln, F.J., Bur6n, M.I., Rodriguez-Aguilera, J.C., Villalba, J.M. and Navas, P. (1990) Cancer Res. 50, 5887-5891. 16. Navas, P. and Burbn, M.I. (1990) In Oxidoreduction at the Plasma Membrane. Relation to Growth and Transport (Crane, F.L., Morrt!, D.J. and Liiw, H.. Eds.), CRC Press, Boca Raton, pp. 225-236. 17. Tallis, G.A., Kitchiner, M.I. and Thomas, A.C. (1990) Clin. Chem. 36, 568-570. 18. Saenko, E.L. and Yaropolov, A.I. (1990) Biochem. Int. 20, 215-225 19. Saenko, E.L., Skorobogat’ko, O.V. and Yarolpolov, A.I. (1990) Biochem. Int. 22, 749757. 20. Sun, I.L., Crane, F.L., LOW, H. and Grebing, C. (1984) J. Bioenerg. Biomembr. 16, 209-22 1. 21. Grebing, C., Crane, F.L., Liiw, H. and Hall, K. (1984) J. Bioenerg. Biomembr. 16. 517533. 22. Sun, I.L., Navas, P.. Crane, F.L., Morrt!, D.J. and LOW, H. (1987) Biochem. Int. 14, 119-127.
955
