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The potential role of NADPH and cytoplasmic NADP-linked dehydrogenases in protection against singlet oxygen mediated cellular toxicity
Vol. 108, No. 4, 1982 October 29, 1982
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 1709-1715
THE POTENTIAL ROLE OF NADPH AND CYTOPLASMIC NADP-LINKED DEHYDROGENASES IN PROTECTION AGAINST SINGLET OXYGEN MEDIATED CELLULAR TOXICITY Richard S. Bodaness Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Building 6, Room 235, Bethesda, Maryland 20205 Received September 20, 1982
NADPH is known to react efficiently with singlet oxygen, the predominant oxidation product being enzymatlcally active NADP +. It is suggested that this reaction acting in concert with the cytoplasmic NADPH regenerating enzymes may contribute to the protection of cytoplasmic components against singlet oxygen mediated toxicity. NADP-llnked isocitrate dehydrogenase is shown to be inactivated by slnglet oxygen photochemlcally generated with hematoporphyrin. When NADPH is present during photooxidation, the enzyme is protected from complete inactivation and regenerates the factor (NADPH) responsible for its own protection.
Singlet oxygen is a topic of intense interest in physical, chemical, and biological systems.
It has been shown to be the mediator
in many oxygen-dependent photosensitivity reactions (1,2), and may be involved in the generation of ocular cataract (3).
In addition to
photon-dependent 102 generation, it has been reported that 102 is formed from the hemoprotein-catalyzed decomposition of lipid peroxides (4).
It
has also been suggested that under certain conditions the superoxide anion radical may react with H202 to form 102 and hydroxyl radical (5). Singlet oxygen has been shown to efficiently oxidize NADPH (Eq. I) (Ref. 6) AND NADH.
The estimated reaction rate constant is
approximately 2.5 x 108 M-is -I.
The stable reduction product is H202,
and approximately 80% of the oxidized NADPH is enzymatlcally active NADP + (6). The combination of the reaction of Eq. I with NADPH regenerating enzymes such as isocitrate dehydrogenase (EC 1.1.1.42) may
Abbreviations: 10 2 = singlet molecular oxygen; HP = hematoporphyrin dihydrochloride; IC = DL-isocitrate; ICDH = NADP-llnked isocitrate dehydrogenase.
1709
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
provide a catalytic mechanism for partially protecting the cytoplasm from 102 (Eq. I and 2).
Eq. I. NADPH + 102
~ NADP + + H202
Eq. 2. NADP + + Isocitrate
ICDH Ix NADPH + CO 2 + e-ketoglutarate + H + MATERIALS AND METHODS
Chemicals were purchased from the following suppliers as indicated: deuterium oxide (99.8%), hematoporphyrin dihydrocbloride, isocitrate dehydrogenase (pig heart), DL-isocitra~e (sodium salt), L-histidine, and L-methionine (Sigma Chemical Co); NADP and NADPH (P-L Biochemicals Inc.). Photooxidation was performed in an apparatus containing a Westinghouse 15-watt 15TS-BL black light fluorescent bulb. The apparatus was designed to protect personnel from exposure to ultraviolet radiation in order to avoid possible ocular cataract formation. Light intensity was determined with a Blak-Ray long wave UV meter (Ultraviolet Products, Inc.). All photooxidations were performed in quartz cuvettes. A typical reaction mixture contained 8 ~M HP, I0 mMpotassium phosphate, pD 7.4, 5 mM MgCIg, 4 mM DL isocitrate, 0.4 mM NADPH, isocitrate dehydrogenase 46.7 pg/ml and additions as noted. The final volume was 3 ml. Control experiments revealed no inactivation of ICDH in the absence of HP and no oxidation of NADPH in the absence of HP. All photooxidatio~s were six minutes in duration at an intensity of 800 microwatts/cm ~ . RESULTS AND DISCUSSION In these experiments, 102 was generated with UV light longer than 340 nm, using HP as a photosensitizer (2,6,7).
liP
hv
)
Hpl
HP 1
) HP 3
Hp 3 + 302
/% HP + 102
Ground state HP is excited by the absorption of a photon and is transformed into a relatively shortlived HP singlet (HpI).
HP I
undergoes spin inversion to a relatively longlived HP triplet (Hp3). HP 3 is then quenched by ground state triplet molecular oxygen.
This
energy transfer reaction results in the formation of singlet molecular oxygen (102) and ground state HP. Regeneration of NADPH during 102 production.
The ability of ICDII to regenerate NADPH from NADP + under conditions of 102 production was examined using the reaction mixture as described
1710
Vol. 108, No. 4, 1982 Table I.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of the presence of an isocitrate dehydrogenase
dependent NADPH regenerating system, on the net oxidation of NADPH by 102 produced photochemically
by hematoporphyrin catalysis in deuterium
oxide. Experiment
Deletion
Final AA340 after UV irradiation
i.
none
0.22
2.
ICDH
.88
3.
Isocitrate
0.84
The experimental conditions are described under Materials and Methods. Under the conditions of photooxidation utilized, the rate of NADPH oxidation was greater than the rate of enzymatic re-reduction so that in experiment I the AA^40 continued to decrease after irradiation was halted at 6 minutes. The final stable AA340 is presented above.
under Materials and Methods. Experiment
I demonstrates
The results are presented in Table I.
that a complete ICDH-NADPH regenerating
system
provides a final AA340 of 0.22 after six minutes of UV irradiation. Under the conditions of photooxidation
utilized,
the rate of NADPH
oxidation was greater than the rate of enzymatic re-reduction experiment halted.
so that in
1 the AA340 continued to decrease after irradiation was
In contrast,
experiment
2 demonstrates
that deletion of ICDH
from the reaction mixture results in a final AA340 of 0.88, and deletion of isocitrate results in a final AA340 of 0.84. that the NADPH regenerating of 102 production.
system is functional under these conditions
The subsequent addition of the deleted item to the
curvettes of experiments
2 and 3, resulted in a decrease of AA340
(regeneration of NADPII) to a point approximately i.
These results indicate
that seen in experiment
This indicates that the inability of the regenerating
decrease AA340 below 0.22 is not due to photosensitized the enzyme, but rather represents a photosensitized approximately
25% of the NADPH to an enzymatically
system to
alteration of
conversion of inactive product.
This finding is in agreement with results previously reported NADPH as the protective
(6).
factor in the reaction mixture.
As the reaction mixture
(defined under Materials and Methods)
contained a number of components,
it was of importance to establish
1711
Vol. 108, N o . 4, 1982
B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
I
I
I
r
I
I
F 0.7
0.6 E
0.5 I-< Z
0.4
D
nO ~ 0.3 <
i
C
0.2
B
I --
I
0.1 0
I
I 4
I 3
I 2 TIME (min)
I I
I 0
Flg. i. Effect of different reaction mixture components on preservation of ICDH activity. D^O buffer was used in all experiments. Duration and intensity were as indicated under Materials and Methods. After cessation of photooxldatlon, additions were made as indicated below, a base line taken, and then the reaction started at one minute with the addition of isocltrate (for curve D isocltrate was present during+ photoirradlat~on and the reaction was initiated with 0.4 m~ NADP . A: HP and ICDII present during UV i~radiation. MgCI_, NADP , and IC then added. B: HP, ICDH, and NADP present during ~V irradlatlon. MgCI^ and IC subsequently added. C: HP, ICDH, and MgCI. present during UV i~radlation. NADP , and IC subsequently+added. D: ~P, ICDH, MgCI and IC present during UV irradiation. NADP subsequently added. E: 2 HP, ICDH, MgCI_ and NADPH present during UV irradiation. IC subsequently a~ded. F: complete reaction mixture with NADP + instead of NADPH and no UV irradiation. Baselines vertically adjusted for purposes of clarity.
clearly This
and u n a m b i g u o u s l y
is d e m o n s t r a t e d
subjected reaction
that NADPH w a s the protective
in Figure
to the H P - D 2 0 - U V mixture
m M NADP + instead
Curve A demonstrates
In these experiments,
s y s t e m in the presence
components. of NADPH,
I.
factor
The deleted
that ICDH alone w i t h
ICDH was
of the different
components
and the activity
for ICDH.
w e r e added with 0.4
of the enzyme measured.
the H P - D 2 0 - U V
system results +
in complete
inactivation
offers m i n i m a l
of the enzyme.
protection,
Curve B d e m o n s t r a t e s
as do Mg ++ and isocltrate
D, respectively.
In contrast,
by 0.4 raM N A D P H
(in the absence
curve E reveals of isocitrate). 1712
marked
as shown
that NADP in C and
protection
of ICDH
Curve F demonstrates
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table II.
Effects of slnglet oxygen reactive compounds on the + hematoporphyrin-catalyzed photooxidation of NADP -dependent isocltrate dehydrogenase. Experiment
Addition
AA340/mln x 102
i.
None
0
2.
H20 instead of D20
8.0
3.
Methlonine,
5.0
4.
Histldlne, i0 mM
6.0
5.
None, no UV
9.0
i0 mM
Photooxidation was performed for the duration and intensity noted under Materials and Methods. For the photooxidation each cuvette contained buffer, HP, ICDH, and additions or changes as indicated. At completion of UV exposure, additions were made as noted under Materials a~d Methods to achieve a complete reaction mixture except that 0.4 mM NADP was substituted for NADPH. A baseline was obtained prior to isocitrate addition, and the reduction initiated with this component.
+
the activity of ICDH in the reaction mixture NADPH) without exposure to UV.
(with NADP
substituted for
An examination of E relative to F shows
that some enzyme inactivation occurred d u r l n g t h e
six minutes of UV
exposure. Singlet oxygen as the mediator of ICDH inactivation. The results in Table II support the proposition is the mediator in the photochemical HP-D20-UV system.
Experiment
inactivation
that singlet oxygen
of ICDH by the
1 reveals that complete inactivation
occurs in the absence of any protective
components.
Singlet oxygen has
a mean lifetime of 20 ~s in D20 as compared to 2 ~s in H20 (8,9). Experiment 2 demonstrates activity.
that H20 instead of D20 protects enzymatic
Methionine and histidine are well documented scavengers of
102, with rate constants of 3 x 107, M-Is -I and 5 x 107 M-Is-1 methionine and histldine respectively
(2).
Experiments
for
3 and 4
demonstrate that both of these compounds protect the enzyme from complete inactivation. reactant
The protection afforded by NADPH, a known 102
(6), further substantiates
the propositlon that slnglet oxygen
is the mediator of the ICDH photoinactivation. 1713
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
It has been proposed that the cytoplasmic NADP-linked dehydrogenases act collectively in a concerted fashion to maintain the cytoplasmic NADPH/NADP + ratio (I0).
These are isocitrate dehydrogenase,
glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and the malic enzyme.
Potentially, under 102 generating conditions, the
task of regenerating NADPH could be shared by this enzymatic multiplicity.
Thus, while the substrate concentration of any one of the
enzymes may be inadequate to regenerate the levels of NADPH oxidized, the collective substrate levels of the different enzymes may provide an + adequate means of reducing their common cofactor - NADP . In liver, the cytoplasmic NADPH/NADP + ratio has been calculated as approximately 85, whereas the NADH/NAD + ratio is approximately 0.086 x 10-2 (Ref. I0). Therefore, although both NADPH and NADH react efficiently with 102, the predominant nucleotide of interest is clearly NADPH.
These experiments demonstrate that under 102 generating conditions, a cofactor (NADPH) may scavenge 102 to protect an enzyme from inactivation.
The protected enzyme (isocitrate dehydrogenase) can
regenerate cofactor and thus make a contribution to its own preservation.
To date, as compared to the cell membrane, little
attention has been focused upon the constituents of the cytoplasm as potential sites of singlet oxygen mediated cellular toxicity.
It is
probable, however, that 102 target sites will vary according to the specific mechanism and conditions of 102 generation.
Thus, if under
certain conditions singlet oxygen mediated oxidation of cytoplasmic components occurs then the mechanism proposed in this paper may provide some measure of protection.
Acknowledgements I thank Dr. Jin H. Kinoshita, Dr. J. Samuel Zigler, Jr., and Dr. Phillip C. Chan for constructive comments.
1714
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS REFERENCES
I. 2. 3. 4. 5. 6. 7. 8. 9. I0.
Nilsson, R., Herkel, P.B., and Kearns, D.R. (1972) Photochem. Photobiol. 16, 117-124. Suwa, K., Kimura, T., and Schaap, A.P. (1977) Biochem. Biophys. Res. Commun. 75: 785-792. Zigler, J,S., Jr., and Goosey, J.D. (1981) Photoehem. Photobiol. 33, 869-874. Haweo, F.J., O'Brlen, C.R., and O'Brien, P.J. (1977) Biochem. Biophys. Res. Commun 75, 354-361. Kellogg, E.W., III, and Frldovich, I. (1975) J. Biol. Chem. 250, 8812-8817, Bodaness, R.S., and Chan, P.C. (1977) J. Biol. Chem. 252, 8554-8560. Cannistraro, S., and Van de Vorst, A. (1977) Biochem. Biophys. Res. Commun. 74, 1177-1185. Merkel, P.B., Nilsson, R., and Kearns, D.R. (1972) J. Am. Chem. Soc. 94, 1030-1031. Merkel, P.B., and Kearns, D.R. (1972) J. Am. Chem. Soc. 94, 7244-7253. Veech, R.L., Eggleston, L.V., and Krebs, H.A. (1969) Biochem. J. 115, 609-619.
1715
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 1709-1715
THE POTENTIAL ROLE OF NADPH AND CYTOPLASMIC NADP-LINKED DEHYDROGENASES IN PROTECTION AGAINST SINGLET OXYGEN MEDIATED CELLULAR TOXICITY Richard S. Bodaness Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Building 6, Room 235, Bethesda, Maryland 20205 Received September 20, 1982
NADPH is known to react efficiently with singlet oxygen, the predominant oxidation product being enzymatlcally active NADP +. It is suggested that this reaction acting in concert with the cytoplasmic NADPH regenerating enzymes may contribute to the protection of cytoplasmic components against singlet oxygen mediated toxicity. NADP-llnked isocitrate dehydrogenase is shown to be inactivated by slnglet oxygen photochemlcally generated with hematoporphyrin. When NADPH is present during photooxidation, the enzyme is protected from complete inactivation and regenerates the factor (NADPH) responsible for its own protection.
Singlet oxygen is a topic of intense interest in physical, chemical, and biological systems.
It has been shown to be the mediator
in many oxygen-dependent photosensitivity reactions (1,2), and may be involved in the generation of ocular cataract (3).
In addition to
photon-dependent 102 generation, it has been reported that 102 is formed from the hemoprotein-catalyzed decomposition of lipid peroxides (4).
It
has also been suggested that under certain conditions the superoxide anion radical may react with H202 to form 102 and hydroxyl radical (5). Singlet oxygen has been shown to efficiently oxidize NADPH (Eq. I) (Ref. 6) AND NADH.
The estimated reaction rate constant is
approximately 2.5 x 108 M-is -I.
The stable reduction product is H202,
and approximately 80% of the oxidized NADPH is enzymatlcally active NADP + (6). The combination of the reaction of Eq. I with NADPH regenerating enzymes such as isocitrate dehydrogenase (EC 1.1.1.42) may
Abbreviations: 10 2 = singlet molecular oxygen; HP = hematoporphyrin dihydrochloride; IC = DL-isocitrate; ICDH = NADP-llnked isocitrate dehydrogenase.
1709
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
provide a catalytic mechanism for partially protecting the cytoplasm from 102 (Eq. I and 2).
Eq. I. NADPH + 102
~ NADP + + H202
Eq. 2. NADP + + Isocitrate
ICDH Ix NADPH + CO 2 + e-ketoglutarate + H + MATERIALS AND METHODS
Chemicals were purchased from the following suppliers as indicated: deuterium oxide (99.8%), hematoporphyrin dihydrocbloride, isocitrate dehydrogenase (pig heart), DL-isocitra~e (sodium salt), L-histidine, and L-methionine (Sigma Chemical Co); NADP and NADPH (P-L Biochemicals Inc.). Photooxidation was performed in an apparatus containing a Westinghouse 15-watt 15TS-BL black light fluorescent bulb. The apparatus was designed to protect personnel from exposure to ultraviolet radiation in order to avoid possible ocular cataract formation. Light intensity was determined with a Blak-Ray long wave UV meter (Ultraviolet Products, Inc.). All photooxidations were performed in quartz cuvettes. A typical reaction mixture contained 8 ~M HP, I0 mMpotassium phosphate, pD 7.4, 5 mM MgCIg, 4 mM DL isocitrate, 0.4 mM NADPH, isocitrate dehydrogenase 46.7 pg/ml and additions as noted. The final volume was 3 ml. Control experiments revealed no inactivation of ICDH in the absence of HP and no oxidation of NADPH in the absence of HP. All photooxidatio~s were six minutes in duration at an intensity of 800 microwatts/cm ~ . RESULTS AND DISCUSSION In these experiments, 102 was generated with UV light longer than 340 nm, using HP as a photosensitizer (2,6,7).
liP
hv
)
Hpl
HP 1
) HP 3
Hp 3 + 302
/% HP + 102
Ground state HP is excited by the absorption of a photon and is transformed into a relatively shortlived HP singlet (HpI).
HP I
undergoes spin inversion to a relatively longlived HP triplet (Hp3). HP 3 is then quenched by ground state triplet molecular oxygen.
This
energy transfer reaction results in the formation of singlet molecular oxygen (102) and ground state HP. Regeneration of NADPH during 102 production.
The ability of ICDII to regenerate NADPH from NADP + under conditions of 102 production was examined using the reaction mixture as described
1710
Vol. 108, No. 4, 1982 Table I.
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of the presence of an isocitrate dehydrogenase
dependent NADPH regenerating system, on the net oxidation of NADPH by 102 produced photochemically
by hematoporphyrin catalysis in deuterium
oxide. Experiment
Deletion
Final AA340 after UV irradiation
i.
none
0.22
2.
ICDH
.88
3.
Isocitrate
0.84
The experimental conditions are described under Materials and Methods. Under the conditions of photooxidation utilized, the rate of NADPH oxidation was greater than the rate of enzymatic re-reduction so that in experiment I the AA^40 continued to decrease after irradiation was halted at 6 minutes. The final stable AA340 is presented above.
under Materials and Methods. Experiment
I demonstrates
The results are presented in Table I.
that a complete ICDH-NADPH regenerating
system
provides a final AA340 of 0.22 after six minutes of UV irradiation. Under the conditions of photooxidation
utilized,
the rate of NADPH
oxidation was greater than the rate of enzymatic re-reduction experiment halted.
so that in
1 the AA340 continued to decrease after irradiation was
In contrast,
experiment
2 demonstrates
that deletion of ICDH
from the reaction mixture results in a final AA340 of 0.88, and deletion of isocitrate results in a final AA340 of 0.84. that the NADPH regenerating of 102 production.
system is functional under these conditions
The subsequent addition of the deleted item to the
curvettes of experiments
2 and 3, resulted in a decrease of AA340
(regeneration of NADPII) to a point approximately i.
These results indicate
that seen in experiment
This indicates that the inability of the regenerating
decrease AA340 below 0.22 is not due to photosensitized the enzyme, but rather represents a photosensitized approximately
25% of the NADPH to an enzymatically
system to
alteration of
conversion of inactive product.
This finding is in agreement with results previously reported NADPH as the protective
(6).
factor in the reaction mixture.
As the reaction mixture
(defined under Materials and Methods)
contained a number of components,
it was of importance to establish
1711
Vol. 108, N o . 4, 1982
B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
I
I
I
r
I
I
F 0.7
0.6 E
0.5 I-< Z
0.4
D
nO ~ 0.3 <
i
C
0.2
B
I --
I
0.1 0
I
I 4
I 3
I 2 TIME (min)
I I
I 0
Flg. i. Effect of different reaction mixture components on preservation of ICDH activity. D^O buffer was used in all experiments. Duration and intensity were as indicated under Materials and Methods. After cessation of photooxldatlon, additions were made as indicated below, a base line taken, and then the reaction started at one minute with the addition of isocltrate (for curve D isocltrate was present during+ photoirradlat~on and the reaction was initiated with 0.4 m~ NADP . A: HP and ICDII present during UV i~radiation. MgCI_, NADP , and IC then added. B: HP, ICDH, and NADP present during ~V irradlatlon. MgCI^ and IC subsequently added. C: HP, ICDH, and MgCI. present during UV i~radlation. NADP , and IC subsequently+added. D: ~P, ICDH, MgCI and IC present during UV irradiation. NADP subsequently added. E: 2 HP, ICDH, MgCI_ and NADPH present during UV irradiation. IC subsequently a~ded. F: complete reaction mixture with NADP + instead of NADPH and no UV irradiation. Baselines vertically adjusted for purposes of clarity.
clearly This
and u n a m b i g u o u s l y
is d e m o n s t r a t e d
subjected reaction
that NADPH w a s the protective
in Figure
to the H P - D 2 0 - U V mixture
m M NADP + instead
Curve A demonstrates
In these experiments,
s y s t e m in the presence
components. of NADPH,
I.
factor
The deleted
that ICDH alone w i t h
ICDH was
of the different
components
and the activity
for ICDH.
w e r e added with 0.4
of the enzyme measured.
the H P - D 2 0 - U V
system results +
in complete
inactivation
offers m i n i m a l
of the enzyme.
protection,
Curve B d e m o n s t r a t e s
as do Mg ++ and isocltrate
D, respectively.
In contrast,
by 0.4 raM N A D P H
(in the absence
curve E reveals of isocitrate). 1712
marked
as shown
that NADP in C and
protection
of ICDH
Curve F demonstrates
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
Table II.
Effects of slnglet oxygen reactive compounds on the + hematoporphyrin-catalyzed photooxidation of NADP -dependent isocltrate dehydrogenase. Experiment
Addition
AA340/mln x 102
i.
None
0
2.
H20 instead of D20
8.0
3.
Methlonine,
5.0
4.
Histldlne, i0 mM
6.0
5.
None, no UV
9.0
i0 mM
Photooxidation was performed for the duration and intensity noted under Materials and Methods. For the photooxidation each cuvette contained buffer, HP, ICDH, and additions or changes as indicated. At completion of UV exposure, additions were made as noted under Materials a~d Methods to achieve a complete reaction mixture except that 0.4 mM NADP was substituted for NADPH. A baseline was obtained prior to isocitrate addition, and the reduction initiated with this component.
+
the activity of ICDH in the reaction mixture NADPH) without exposure to UV.
(with NADP
substituted for
An examination of E relative to F shows
that some enzyme inactivation occurred d u r l n g t h e
six minutes of UV
exposure. Singlet oxygen as the mediator of ICDH inactivation. The results in Table II support the proposition is the mediator in the photochemical HP-D20-UV system.
Experiment
inactivation
that singlet oxygen
of ICDH by the
1 reveals that complete inactivation
occurs in the absence of any protective
components.
Singlet oxygen has
a mean lifetime of 20 ~s in D20 as compared to 2 ~s in H20 (8,9). Experiment 2 demonstrates activity.
that H20 instead of D20 protects enzymatic
Methionine and histidine are well documented scavengers of
102, with rate constants of 3 x 107, M-Is -I and 5 x 107 M-Is-1 methionine and histldine respectively
(2).
Experiments
for
3 and 4
demonstrate that both of these compounds protect the enzyme from complete inactivation. reactant
The protection afforded by NADPH, a known 102
(6), further substantiates
the propositlon that slnglet oxygen
is the mediator of the ICDH photoinactivation. 1713
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
It has been proposed that the cytoplasmic NADP-linked dehydrogenases act collectively in a concerted fashion to maintain the cytoplasmic NADPH/NADP + ratio (I0).
These are isocitrate dehydrogenase,
glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and the malic enzyme.
Potentially, under 102 generating conditions, the
task of regenerating NADPH could be shared by this enzymatic multiplicity.
Thus, while the substrate concentration of any one of the
enzymes may be inadequate to regenerate the levels of NADPH oxidized, the collective substrate levels of the different enzymes may provide an + adequate means of reducing their common cofactor - NADP . In liver, the cytoplasmic NADPH/NADP + ratio has been calculated as approximately 85, whereas the NADH/NAD + ratio is approximately 0.086 x 10-2 (Ref. I0). Therefore, although both NADPH and NADH react efficiently with 102, the predominant nucleotide of interest is clearly NADPH.
These experiments demonstrate that under 102 generating conditions, a cofactor (NADPH) may scavenge 102 to protect an enzyme from inactivation.
The protected enzyme (isocitrate dehydrogenase) can
regenerate cofactor and thus make a contribution to its own preservation.
To date, as compared to the cell membrane, little
attention has been focused upon the constituents of the cytoplasm as potential sites of singlet oxygen mediated cellular toxicity.
It is
probable, however, that 102 target sites will vary according to the specific mechanism and conditions of 102 generation.
Thus, if under
certain conditions singlet oxygen mediated oxidation of cytoplasmic components occurs then the mechanism proposed in this paper may provide some measure of protection.
Acknowledgements I thank Dr. Jin H. Kinoshita, Dr. J. Samuel Zigler, Jr., and Dr. Phillip C. Chan for constructive comments.
1714
Vol. 108, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS REFERENCES
I. 2. 3. 4. 5. 6. 7. 8. 9. I0.
Nilsson, R., Herkel, P.B., and Kearns, D.R. (1972) Photochem. Photobiol. 16, 117-124. Suwa, K., Kimura, T., and Schaap, A.P. (1977) Biochem. Biophys. Res. Commun. 75: 785-792. Zigler, J,S., Jr., and Goosey, J.D. (1981) Photoehem. Photobiol. 33, 869-874. Haweo, F.J., O'Brlen, C.R., and O'Brien, P.J. (1977) Biochem. Biophys. Res. Commun 75, 354-361. Kellogg, E.W., III, and Frldovich, I. (1975) J. Biol. Chem. 250, 8812-8817, Bodaness, R.S., and Chan, P.C. (1977) J. Biol. Chem. 252, 8554-8560. Cannistraro, S., and Van de Vorst, A. (1977) Biochem. Biophys. Res. Commun. 74, 1177-1185. Merkel, P.B., Nilsson, R., and Kearns, D.R. (1972) J. Am. Chem. Soc. 94, 1030-1031. Merkel, P.B., and Kearns, D.R. (1972) J. Am. Chem. Soc. 94, 7244-7253. Veech, R.L., Eggleston, L.V., and Krebs, H.A. (1969) Biochem. J. 115, 609-619.
1715