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Stimulation of glucose oxidation and transport in isolated rat adipocytes by riboflavin and visible light
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 208, No. 2, May, pp.380-387,1981
Stimulation
of Glucose Oxidation and Transport in Isolated Adipocytes by Riboflavin and Visible Light JOEL
M. GOODMAN’
AND PAUL
Rat
HOCHSTEIN2
Institute for Toxicology, School of Pharmacy, and Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California 90038 Received August 27, 1980 Riboflavin, which is known to cause photooxidative damage in biological systems, is now shown to stimulate glucose transport and oxidation in isolated rat adipocytes in the presence of visible light. At low riboflavin concentrations, well within normal blood levels, there is a small but reproducible stimulation of Cl-labeled glucose oxidation to labeled CO1 (30% stimulation at 1O-s M), which does not require light. However, at higher concentrations (10e5 M and above), light greatly potentiates this effect on C1-glucose oxidation as well as stimulates &-glucose oxidation (two- to three-fold over controls). These apparent effects on the hexose monophosphate shunt and glycolytic-tricarboxylic acid pathways are blocked by 10 pM cytochalasin B, a glucose transport inhibitor. Riboflavin in light is further shown to stimulate uptake of 3Gmethylglucose, a nonmetabolizable glucose analog. These light-dependent effects are not affected by catalase or superoxide dismutase, but they are inhibited by dimethylfuran, a singlet oxygen scavenger. This latter agent has no effect on glucose metabolism in untreated or insulin-treated cells. The results suggest a physiologically important potential effect of riboflavin and visible light.
The possible effects of visible and nearultraviolet light on metabolic processes (other than photosynthesis) are generally recognized since cells contain photosensitizing substances with the capacity to initiate oxidative events. Among these substances are the flavins. The range of biological materials affected by flavin photooxidation is very broad (1) and includes several amino acids, purines, pyrimidines, ascorbic acid, DNA, RNA, and enzymes such as urease, tyrosinase, and trypsin. Visible and near-uv light are toxic to procaryotic and eucaryotic cells (2) and there is evidence in the cases of lens tissue (3), human diploid fibroblasts in culture (4), and isolated mitochondria (5) that the phototoxicity is dependent on endogenous flavins. The photoxidative effects of riboflavin 1 Present address: Department of Biological Chemistry, University of California, Los Angeles, Calif. 90024.
2 To whom all correspondence should be addressed. 0003-9861/81/060380-08$02.00/O Copyright All rights
0 1981 by Academic Press, of reproduction in any form
380 Inc. reserved.
have also been expoited for therapeutic purposes. Newburger et al. (6) reported the in vitro photodegradation of uric acid by riboflavin and suggested the use of the vitamin with phototherapy in hyperuricemic patients. Flavins and other dyes in combination with phototherapy have been found effective for lesions caused by herpesvirus (7, 8). The vitamin has also been found effective in potentiating the effects of phototherapy for moderate hyperbilirubinemia in infants (9) and it has been shown that it causes degradation of bilirubin in vitro (10). The mechanism(s) of most riboflavin-catalyzed photoxidations remains obscure, although many species of activated oxygen may be active intermediates (11, 12) including superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical. Such oxidants might also have been specific effects on certain physiological processes. For example, hydrogen peroxide, H202, may stimulate the hexose monophosphate shunt
STIMULATION
OF GLUCOSE
OXIDATION
(HMS)” by causing enzymatic oxidation of glutathione (13) and hence of NADPH (14, 15). Czech has demonstrated (16) the activation of the facilitated diffusion of glucose (hereafter referred to as glucose transport) by the oxidants H202, K,( 1-hydroxy-B methyl-4-am.inonaphthalene), and methylene blue. He postulated that these agents produce an active disulfide-containing form of the transporter. Since riboflavin, a compound present in all tissues, is able to generate oxygen radicals and H,Oz we have examined its effects on glucose oxidation and transport in isolated adipocytes. MATERIALS
AND METHODS
All flavins, fraction V bovine serum albumin (BSA), cytochalaain B, catalase, superoxide dismutase, and xanthine oxida#se were supplied by Sigma Chemical Company. The riboflavin was twice recrystallized in 2 N acetic acid before use. Dimethylfuran (DMF) and dinonylphthalate were purchased from Aldrich. Crystallized bovin insulin was obtained from Mann, and type I collagenase from Worthington Corporation. All radiolabeled compounds and Econofluor were purchased from New England Nuclear. The other chemicals utilized were at least of reagent grade. Isolated adipocytes were prepared from the parametrial tissue of 299 to 259-g female rats (Simonsen Farms, Gilroy, Calif.) by the Mukherjee and Lynn (15) modification of the Rodbell technique (161, except that BSA was dialyzed against 10 IIIM sodium phosphate buffer, pH 7.4, for 36 h. Cells, typically containing about 5 pg DNA eq/ml, were incubated in 5-ml plastic beakercups in a shaking water bath at 37”C, either completely covered with aluminum foil (dark incubations) or with the top of a clear plastic Petri dish to prevent evaporation and eliminate exposure to uv light. Cells were exposed to light from two 75-W General Electric floodlamps with tungsten filaments and frosted-glass surfaces which served to diffuse the light. The lamps were spaced 28 cm apart and 30 cm above the beaker-cups. The light intensity incident on the suspensions was 6.4 W/m*, as measured by a Gossen Lunasix light meter. Flavin solutions were made and added to cells in very dim light. To prepare cell homogenates, concentrated cell suspensions prepared as above except with undialyzed BSA (typically 20 pg DNA eq/ml) were homogenized at room temperature with a Potter-Elvehjem tissue grinder with Teflon pestle. After centriguation (10 3 Abbreviations used: HMS, hexose monophosphate shunt; TCA, tricarboxylic acid; BSA, bovine serum albumin; SOD, superoxide dismutase; DMF, dimethylfuran.
BY RIBOFLAVIN
AND LIGHT
381
min at loOOg), the infranatant (referred to as the homogenate) was diluted with 3 parts phosphate buffer (17) and incubated in a fashion similar to that used for intact cells. Glucose oxidation to CO, was measured by trapping the gas in Hyamine hydroxide (18), and uptake of 30 methylglucose was determined by a slight modification of the method of Livingston and Lockwood (191, in which values in the presence of 50 pM cytochalasin B were subtracted from those in its absence to correct for nonspecific diffusion and trapping in the extracellular space. Activities of catalase, superoxide dismutase, and hexokinase were determined by the methods of Aebi (201, McCord and Fridovich (211, and Beutler (22), respectively. DNA content of adipocytes was determined after extraction of the bulk lipids with 20 vol of chlorofornnmethanol2:l by the Giles and Myers (23) modification of the Burton (24) assay. RESULTS
Incubation of isolated rat adipocytes with riboflavin stimulates the oxidation of [14Cl]glucose to 14C0,, as illustrated in the left panel of Fig. 1. It can be seen that riboflavin stimulates oxidation even in the absence of light. This effect was always observed at concentrations of 1O-6 M and above, well within the normal human blood levels (25). In the two representative experiments shown in Fig. 1, the stimulation at 10e6 M was an average of 33% above controls without riboflavin. (In five total experiments under slightly varying conditions, stimulation at 10e6 M was 30 5 8.0% SE.) The presence of visible light, however, markedly potentiated the riboflavin stimulation of oxidation of 1O-5 M and above reaching maximal stimulation of 90% over the dark riboflavin control at 3 x 10p5. Effects of similar magnitude occurred with both FAD and FMN (not shown). These data suggest that riboflavin in the dark causes a significant stimulation of the hexose monophosphate pathway of glucose oxidation, which is potentiated above lop6 M in the presence of visible light. The stimulation of CO, production from glucose was not limited to the hexose monophosphate shunt as illustrated in the right panel of Fig. 1. Oxidation of [14CB]glucose, an index of carbon flux through glycolysis
382
GOODMAN
AND HOCHSTEIN
FIG. 1. Effect of riboflavin on C,- and &-glucose oxidation in isolated rat adipocytes. Adipocytes were isolated and incubated in beakercups for 1 h with appropriate riboflavin concentrations in the absence or presence of light. Six aliquots from each beakercup were then placed in tubes containing either [W,]- or [W,]glucose (glucose concentration was 0.2 mM) and again incubated for 30 min in the dark. Each point represents the mean value of triplicate samples. Circles and squares represent experiments on two different days. Open symbols, light incubation; closed symbols, dark incubation. Further details are described under Materials and Methods.
and the tricarboxylic acid cycle, was also affected. Unlike its effect on the HMS, riboflavin in the dark did not significantly stimulate this pathway at low levels. However in the light it had a dramatic effect. Oxidation was stimulated 110% in the presence of lop5 M riboflavin and 390% at low4 M compared to the dark controls. Since the effects of photooxidation in biological systems are typically destructive in nature! it appeared possible that the effects with hght and riboflavin seen in Fig. 1 might be caused by increased intracellular availability of glucose produced by increased nonspecific diffusion of the hexose through damaged membranes. To test this possibility, we measured glucose oxidation in the presence of cytochalasin B, which specifically blocks D-glucose transport but not nonspecific diffusion (26). If the effects in Fig. 1 are a consequence of photooxidation which increases nonspecific diffusion, cytochalasin B should not affect glucose oxidation. Figure 2 again demonstrates the dark stimulation of the HMS by riboflavin (at 3 x lop5 M) and the profound effects of the vitamin in light on the HMS and the glycolytic-TCA pathway. Moreover, it can be readily seen that these effects are abolished by 10 PM cytochalasin B. These results clearly demonstrate that the effects of
riboflavin and light are dependent, in part, on glucose transport, rather than nonspecific diffusion of glucose through damaged membranes. The apparent stimulation by riboflavin and light of glucose oxidation may be a result of increased glucose “push” or “pull” through the membrane or a combination of both. That is, either/both glucose transport per se is stimulated, or/and intracellular enzymes of glucose oxidation are activated. To first address this question, we determined the effect of riboflavin and light on uptake of 3-O-methylglucose, a nonmetabolizable analog. Neither light alone nor riboflavin at 3 X lop5 M in the dark significantly stimulated 3-0-methylglucose uptake (data not shown). However, as can be seen in Fig. 3, 3 x lop5 M riboflavin in light produced a 140% stimulation of transport compared to transport without the vitamin. For comparison, the effect of insulin is also shown. These results demonstrate that glucose transport is activated by riboflavin in light independently of any activation of intracellular utilization of glucose. The second possibility, that activation of intracellular enzymes of glucose oxidation might contribute to the stimulation by riboflavin and light, was tested by observing whether oxidation is stimulated under con-
STIMULATION
so tP
OF GLUCOSE
OXIDATION
0.07
5
1.5
=. E
1.2
q
14 1opM
Cytochalorin
B
C,-Glucose
BY RIBOFLAVIN
AND LIGHT
383
C,-glucose oxidation, as expected. In combination, the effects of the two agents in light are additive on C,-glucose oxidation, suggesting independent pathways of stimulation. The effects on C6-glucose oxidation are more than twice that of additive. This latter puzzling finding on &-glucose oxidation may be due to an indirect effect of riboflavin on insulin action or vice versa and was not explored further. The ability of adipoeyte homogenates to oxidize &-glucose to CO2 was exploited to further investigate the role of riboflavin in stimulating the hexose monophosphate shunt activity independent of transport. Figure 4 illustrates C,-glucose oxidation to CO, in ATP-fortified homogenates with increasing NADP+ concentrations. There is
FIG. 2. Effect of cytochalasin B on glucose oxidation stimulated by riboflavin. Conditions as in Fig. 1 except cytochalasin B (hatched bars) or ethanol (open bars) at 1% total volume was added to appropriate beakercups after the l-h incubation with or without riboflavin at 3 x lo+ M. Cells were allowed to incubate for 15 min and then were pipetted into tubes containing the [Wlglucose.
ditions where glucose is accessible for utilization. This condition was met by two different approaches. In the first, the effect of riboflavin and light on oxidation was assayed in the presence of insulin, so that transport was maximally activated by the hormone. Under these conditions, stimulation of glucose oxidation above that by insulin alone would strongly suggest direct effects on the pathways of glucose utilization. Cells were incubated with 0.5 mu/ml insulin and/or 3 x 10e5 M riboflavin. Although the data are not shown, the transport of 3Gmethylglucose in the presence of both agents did not exceed that of insulin alone. On the other hand, the effects of the two agents in combination on glucose oxidation, demonstrated in Table I, are striking. This table illustrates the effects of riboflavin seen in previous figures, and that of insulin, which stimulates both C1- and
R,boflovm
/
Time,
sec.
FIG. 3. Effect of riboflavin in light on transport of 34-methylglucose. Isolated adipocytes were incubated in light for 1 h with insulin (0.5 mu/ml) or riboflavin (3 x 10m5M) in the appropriate beakercup. Cells were transferred into tubes and the uptake of 3-Omethylglucose was measured at 20°C over the indicated times. The difference of the means of triplicate determinations in the absence and presence of cytochalasin B was calculated for each time point. This figure illustrates the composite result of four experiments in which the effects of either insulin or riboflavin were measured. The bars represent the range of values in the experiments.
384
GOODMAN
AND HOCHSTEIN
evidently insufficient endogenous pyridine nucleotide in the homogenate to support signif-lcant glucose oxidation. At low (presumably physiological) levels of NADP+, it can be seen that riboflavin in the light greatly stimulates this oxidation, increasing it 140% at 0.25 mM NADP+. At higher nucleotide concentrations the effect is less pronounced and at 2 mM NADP+ riboflavin inhibits oxidation (not shown). A simple explanation of these results is that riboflavin in light stimulates C,-glucose oxidation in homogenates by reoxidizing the NADPH produced by the HMS dehydrogenases, thereby regenerating cofactor. Thus the effect is most pronounced at low NADP+ concentrations. At higher NADP+ levels the apparent inhibition by riboflavin may represent damage to the shunt pathway that was masked by low NADP+ levels. For example, endogenous hexokinase activity in the homogenate was depressed 48% after incubation with riboflavin compared to its absence (data not shown). It can be seen that in the presence of 1 mM oxidized glutathione (GSSG), which is expected to reoxidize NADPH through glutathione reducTABLE EFFECT
I
OF INSULIN AND RIBOFLAVIN OXIDATION IN ADIPOCYTE~
ON GLUCOSE
Glucose oxidation to CO, (nmol/pg DNA) [‘*CJGlucose as substrate
[14Ce]Glucose as substrate
Addition
Dark
Light
Dark
Light
None Insulin Riboflavin Insulin + riboflavin
0.259 1.198 0.504 1.436
0.272 1.286 1.070 2.450
0.013 0.255 0.021 0.226
0.016 0.250 0.117 0.719
a Isolated adipocytes were incubated in light or dark for 1 h with insulin (0.5 mu/ml) and/or riboflavin (3 X 10m5 M). Aliquots were removed for triplicate determination of glucose oxidation during a 30-min pulse. Values are the means of triplicate values (range 2 10%) of an experiment, which was repeated twice with similar results. Glucose concentration was 0.2 mM. Values are based on the specific activity of labeled glucose added. The intensity of visible light was 6.4 W/mZ.
L
0
025
0.5 NADP*(mM)
I
0.75
1.0
FIG. 4. Effect of riboflavin in light on C,-glucose oxidation in adipocyte homogenates. Homogenates were incubated in uncovered beakercups for 1 h with or without 1O-5 M riboflavin (Rf) in light. Aliquots were pipetted into tubes containing ATP(2.5 mM, final concentration), NADP+ (indicated concentrations), GSSG (0 or 1 mM, final concentration), and [‘4C,]glucose (0.2 mM, final concentration) and incubated at 37°C for 20 min.
no longer stimulates tase, riboflavin oxidation but depresses it about 30%. These data suggest that the stimulation by riboflavin with light on glucose oxidation in rat adipocytes may be a consequence of both activation of glucose transport increasing the availability of glucose intracellularly and stimulation of pathways of glucose oxidation. The possible role of oxygen radicals produced by riboflavin photoxidation in the stimulation of glucose oxidation and transport shown above was investigated by including catalase, superoxide dismutase (SOD), dimethylfuran (DMF), and mannitol, which may scavenge or destroy hydrogen peroxide, superoxide, singlet oxygen, and hydroxyl radical, respectively. Figure 5 illustrates the effect of these agents on C,-glucose oxidation stimulated by riboflavin. It can be readily observed that cata-
STIMULATION
OF GLUCOSE
14
OXIDATION
BY RIBOFLAVIN
AND LIGHT
385
C,-Glucose
0
P I Rib< a Cd
FIG. 5. Effect of catalase, superoxide dismutase, dimethylfuran, and mannitol on the stimulation of &glucose oxidation by riboflavin. Conditions as in Fig. 1, except the appropriate agent was present in the cell suspensions with riboflavin (3 X 10m5M) and only &glucose oxidation was measured. The final activities or concentrations of catalase, SOD, DMF, and mannitol were 430 u/ml, 27 u/ml, 1.5 mM, and 5 mM, respectively. D, dark incubation; L, light incubation.
lase, SOD, and mannitol do not inhibit the riboflavin effect. The lack of effect of the enzymes catalase and SOD was not a result of their destruction by riboflavin since incubation of riboflavin and the enzymes alone produced no effect on SOD activity and only a 20% drop of that of catalase (not shown). In contrast, DMF, a singlet oxygen scavenger, abolished the stimulation of oxidation by riboflavin in light while it did not affect the control dark incubations. DMF had a similar effect on the stimulation of C,-glucose oxidation and the transport of 3Gmethylglucose by riboflavin, but not by insulin, whi1.e SOD and catalase did not have inhibitory effects. These data suggests that singlet oxygen may be an activator of glucose transport and oxidation in intact rat adipocytes, although further studies are necessary to confirm this hypothesis. DISCUSSION
The physiological role of riboflavin has long been known to be as a participant in intracellular electron transport. Riboflavin may also catalyze photooxidative damage. In this report we demonstrate a new aspect of the action of this vitamin in the stimulation of glucose oxidation and transport.
A stimulatory effect, even in the dark, on the hexose monophosphate shunt of intact rat adipocytes was observed at low concentrations of riboflavin (lop6 M). It should be noted that this dark effect was only seen if dialyzed BSA was used. Presumably the crude BSA contained some riboflavin-like contaminants. The dark effect on shunt activity is mimicked to similar degrees by both FAD and FMN. These latter phosphorylated compounds would not be expected to cross the plasma membrane. Thus, it is possible that the plasma membrane is the site of action of the dark effect of the flavins. For example, reduction of the vitamin (extracellularly?) by intracellular NADPH would generate the oxidized cofactor of the HMS. Although a plasma membrane NADPH-flavin oxidoreductase has not been reported, it would not be the first case of a transmembrane oxidationreduction, e.g., NAD(P)H can reduce 0, in phagocytes (27). Mukherjee and Lynn (17) have reported an adipocyte plasma membrane NADPH: 0, oxidoreductase that is activated by insulin which might be related topographically to the activity reported here. Light has a striking effect on glucose oxidation and transport in the presence of riboflavin. It should be mentioned that the ri-
386
GOODMAN
AND
boflavin concentrations at which these effects are seen are not totally unphysiological. For example, the riboflavin concentration in lens epithelium, a tissue that certainly is exposed to light much in excess of that used in these experiments, is reported to be 1.2 x 10e5 M (28). Further in viwo studies will be required to determine the physiological importance of this new aspect in the action of the vitamin. The unexpected lack of inhibition by catalase and the effect of dimethylfuran suggest that singlet oxygen may be the primary agent mediating the activation of glucose transport and the stimulation of glucose oxidation, although corroborating evidence is necessary to confirm this conclusion. In this regard, it is noted that hematoporphyn, another photosensitizer, can oxidize NADPH to NADP via singlet oxygen (29), which may be involved with the stimulation of C&glucose oxidation in whole cells and homogenates which we observe. The mechanism by which singlet oxygen may activate transport is unknown, but this concept is not inconsistent with Czech’s model of disulfide activation (16). While the product detected from the interaction of cysteine with singlet oxygen (generated by dyes) is usually cysteic acid, cystine can be produced at neutral pH (30). The mechanism by which oxidants activate glucose transport is far from clear. Insulin has been shown to prevent the autophosphorylation of membrane proteins (31, 32) which may be important in activation of transport. Vanadate, recently shown to have insulin-mimetic effects (33), is a potent inhibitor of phosphotransferase enzymes (34, 35), and perhaps these enzymes are the targets of oxidants which lead to transport activation. However, it should not be assumed that the initial event of the oxidative activation of transport by oxidants occurs at the plasma membrane. Recent evidence from two laboratories (36, 37) suggests that insulin “activates” glucose transport in adipocytes by causing the rapid translocation of transporters from internal membranes to the cell surface; the cellular locale of initiation of this activity is not known. Further study of the insulin-mimetic effects of oxidants such as riboflavin
HOCHSTEIN
may be of value in unraveling nism of this process.
the mecha-
ACKNOWLEDGMENTS We are grateful to Ms. Jill Reeves and Ms. Lloyd Wong for excellent technical assistance. This work was supported in part by Grant AG 00471 from the National Institute of Aging. REFERENCES 1. TAYLOR, M. B., AND RADDA, G. K. (1971) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 18B, pp. 496506, Academic Press, New York. 2. EPEL, B. L. (1973) Photophysiology 8, 209-229. 3. VARMA, S. D., KUMAR, S., AND RICHARDS, R. D. (1979) Proc. Nut. Acad. Ski. USA 76, 35043596. 4. PEREIRA, 0. M., SMITH, J. R., AND PACKER, L. (1976) Photo&m. Photobiol. 24, 237-242. 5. AGGARWAL, B. B., QUINTANILHA, A. T., CAMMACH, R., AND PACKER, L. (1978) Biochim. Biophys. Acta 502, 367-382. 6. NEWBURGER, J., COMBS, A. B., AND Hsu, T. F. (1977) J. Pharrn. Sci. 66, 1561-1564. 7. FELBER, T. D., SMITH, E. B.. KNOX. J. M.. WALLIS, L., AND MELNICK, ‘J. L. (;973) J: Amer. Med. Assoc. 223, 289-292. 8. KAUFMAN, R. H., GARDNER, H. L., BROWN, D., WALLIS, C., RAWLS, W. E., AND MELNICK, J. L. (1973) Amer. J. Obstet. Gynecol. 117, 11441146. 9. PASCALE, J. A., MIMS, L. C., GREENBERG, M. H., GOODEN, D. S., AND CHRONISTER, E. (1976) Pediat. Res. 10, 854-856. 10. LIN, J.-K., SU, I.-J., AND Hsu, S.-M. (1977) J. Formosun Med. Assoc. 76, 293-300. 11. PENZER, G. R. (1970) Biochem. J. 116, 733-743. 12. FOOTE, C. S. (1968) Science 162, 963-970. 13. COHEN, G., AND HOCHSTEIN, P. (1961) Science 134, 1574-75. 14. REED, P. W. (1969) J. Biol. Chm. 244, 24592464. 15. MUKHERJEE, S. P., LANE, R. H., AND LYNN, W. S. (1978) Biochem. Pharmucol. 27, 2589-2594. 16. CZECH, M. P. (1976) J. Biol. Chem. 251, 1X41170. 17. MUKHERJEE, S. P., AND LYNN, W. S. (1977) Arch. Biochem. Biophys. 184, 69-77. 18. RODBELL, M. (1964) J. Biol. Chem. 239,375-380. 19. LIVINGSTON, J. N., AND LOCKWOOD, D. H. (1975) J. Biol. Chem. 250, 8353-8360. 20. AEBI, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.1, 2nd Engl. ed., pp. 673-684, Academic Press, New York. 21. MCCORD, J. E., AND FRIDOVICH, I. (1969) J. Biol. Chem. 244, 6049-6055.
STIMULATION
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OXIDATION
22. BEUTLER, E. (1975) in Red Cell Metabolism: A Manual Iof Biochemical Methods, 2nd ed., pp. 38-40, Grune & Stratton, New York. 23. GILES, K. W., AND MYERS, A. (1965) Nature (London:) 206, 93. 24. BURTON, K.. (1956) Biochem. J. 62, 315-323. 25. BAKER, H., FRANK, O., FEINGOLD, S., GEL LENE, R. A., LEEVY, C. M., AND HUTNER, S. H. (1966)Amer. J. Clin. Nub. 19, 17-26. 26. CZECH, M. P., LYNN, D. G., AND LYNN, W. S. (1973) J. Biol. Chem. 248, 3636-3641. 27. BABIOR, B. M. (1978) N. Engl. J. Med. 298, 659668, 721--725. 28. KINSEY, V. E., AND JACKSON, B. (1951) AMA Arch. Ophthalmol. 46, 536-541. 29. BODANESS, R. S., AND CHAN, P. C. (1977) J. Biol. Chum. 252, 8651-8560.
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30. JORI, G., GALIAZZO, G., AND SCOFFONE, E. (1969) Int. J. Protein Res. 1, 289-298. 31. SEALS, J. R., MCDONALD, J. M., AND JARETT, L. (1979) J. Biol. Chem. 254, 6991-6996. 32. SEALS, J. R., MCDONALD, J. M., AND JARE~, L. (1979) J. Biol. Chem. 254, 6997-7001. 33. DUBYAK, G. R., AND KLEINZELLER, A. (1980) J. Biol. Chem. 255, 5306-5312. 34. GOODNO, C. C. (1979) Proc. Nat. Acad. Sci. USA 76, 2620-2624. 35. LINDQUIST, R. N., LYNN, J. L., AND LEINHARD, G. E. (1973) J. Amer. Chem. Sot. 95, 87628768. 36. SUZUKI, K., AND KONO, T. (1980) Proc. Nat. Acad. Sci. USA 77, 2542-2545. 37. CUSHMAN, S. W., AND WARDZALA, L. J. (1980) J. Biol. Chem. 255, 4758-4762.
Stimulation
of Glucose Oxidation and Transport in Isolated Adipocytes by Riboflavin and Visible Light JOEL
M. GOODMAN’
AND PAUL
Rat
HOCHSTEIN2
Institute for Toxicology, School of Pharmacy, and Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California 90038 Received August 27, 1980 Riboflavin, which is known to cause photooxidative damage in biological systems, is now shown to stimulate glucose transport and oxidation in isolated rat adipocytes in the presence of visible light. At low riboflavin concentrations, well within normal blood levels, there is a small but reproducible stimulation of Cl-labeled glucose oxidation to labeled CO1 (30% stimulation at 1O-s M), which does not require light. However, at higher concentrations (10e5 M and above), light greatly potentiates this effect on C1-glucose oxidation as well as stimulates &-glucose oxidation (two- to three-fold over controls). These apparent effects on the hexose monophosphate shunt and glycolytic-tricarboxylic acid pathways are blocked by 10 pM cytochalasin B, a glucose transport inhibitor. Riboflavin in light is further shown to stimulate uptake of 3Gmethylglucose, a nonmetabolizable glucose analog. These light-dependent effects are not affected by catalase or superoxide dismutase, but they are inhibited by dimethylfuran, a singlet oxygen scavenger. This latter agent has no effect on glucose metabolism in untreated or insulin-treated cells. The results suggest a physiologically important potential effect of riboflavin and visible light.
The possible effects of visible and nearultraviolet light on metabolic processes (other than photosynthesis) are generally recognized since cells contain photosensitizing substances with the capacity to initiate oxidative events. Among these substances are the flavins. The range of biological materials affected by flavin photooxidation is very broad (1) and includes several amino acids, purines, pyrimidines, ascorbic acid, DNA, RNA, and enzymes such as urease, tyrosinase, and trypsin. Visible and near-uv light are toxic to procaryotic and eucaryotic cells (2) and there is evidence in the cases of lens tissue (3), human diploid fibroblasts in culture (4), and isolated mitochondria (5) that the phototoxicity is dependent on endogenous flavins. The photoxidative effects of riboflavin 1 Present address: Department of Biological Chemistry, University of California, Los Angeles, Calif. 90024.
2 To whom all correspondence should be addressed. 0003-9861/81/060380-08$02.00/O Copyright All rights
0 1981 by Academic Press, of reproduction in any form
380 Inc. reserved.
have also been expoited for therapeutic purposes. Newburger et al. (6) reported the in vitro photodegradation of uric acid by riboflavin and suggested the use of the vitamin with phototherapy in hyperuricemic patients. Flavins and other dyes in combination with phototherapy have been found effective for lesions caused by herpesvirus (7, 8). The vitamin has also been found effective in potentiating the effects of phototherapy for moderate hyperbilirubinemia in infants (9) and it has been shown that it causes degradation of bilirubin in vitro (10). The mechanism(s) of most riboflavin-catalyzed photoxidations remains obscure, although many species of activated oxygen may be active intermediates (11, 12) including superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical. Such oxidants might also have been specific effects on certain physiological processes. For example, hydrogen peroxide, H202, may stimulate the hexose monophosphate shunt
STIMULATION
OF GLUCOSE
OXIDATION
(HMS)” by causing enzymatic oxidation of glutathione (13) and hence of NADPH (14, 15). Czech has demonstrated (16) the activation of the facilitated diffusion of glucose (hereafter referred to as glucose transport) by the oxidants H202, K,( 1-hydroxy-B methyl-4-am.inonaphthalene), and methylene blue. He postulated that these agents produce an active disulfide-containing form of the transporter. Since riboflavin, a compound present in all tissues, is able to generate oxygen radicals and H,Oz we have examined its effects on glucose oxidation and transport in isolated adipocytes. MATERIALS
AND METHODS
All flavins, fraction V bovine serum albumin (BSA), cytochalaain B, catalase, superoxide dismutase, and xanthine oxida#se were supplied by Sigma Chemical Company. The riboflavin was twice recrystallized in 2 N acetic acid before use. Dimethylfuran (DMF) and dinonylphthalate were purchased from Aldrich. Crystallized bovin insulin was obtained from Mann, and type I collagenase from Worthington Corporation. All radiolabeled compounds and Econofluor were purchased from New England Nuclear. The other chemicals utilized were at least of reagent grade. Isolated adipocytes were prepared from the parametrial tissue of 299 to 259-g female rats (Simonsen Farms, Gilroy, Calif.) by the Mukherjee and Lynn (15) modification of the Rodbell technique (161, except that BSA was dialyzed against 10 IIIM sodium phosphate buffer, pH 7.4, for 36 h. Cells, typically containing about 5 pg DNA eq/ml, were incubated in 5-ml plastic beakercups in a shaking water bath at 37”C, either completely covered with aluminum foil (dark incubations) or with the top of a clear plastic Petri dish to prevent evaporation and eliminate exposure to uv light. Cells were exposed to light from two 75-W General Electric floodlamps with tungsten filaments and frosted-glass surfaces which served to diffuse the light. The lamps were spaced 28 cm apart and 30 cm above the beaker-cups. The light intensity incident on the suspensions was 6.4 W/m*, as measured by a Gossen Lunasix light meter. Flavin solutions were made and added to cells in very dim light. To prepare cell homogenates, concentrated cell suspensions prepared as above except with undialyzed BSA (typically 20 pg DNA eq/ml) were homogenized at room temperature with a Potter-Elvehjem tissue grinder with Teflon pestle. After centriguation (10 3 Abbreviations used: HMS, hexose monophosphate shunt; TCA, tricarboxylic acid; BSA, bovine serum albumin; SOD, superoxide dismutase; DMF, dimethylfuran.
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min at loOOg), the infranatant (referred to as the homogenate) was diluted with 3 parts phosphate buffer (17) and incubated in a fashion similar to that used for intact cells. Glucose oxidation to CO, was measured by trapping the gas in Hyamine hydroxide (18), and uptake of 30 methylglucose was determined by a slight modification of the method of Livingston and Lockwood (191, in which values in the presence of 50 pM cytochalasin B were subtracted from those in its absence to correct for nonspecific diffusion and trapping in the extracellular space. Activities of catalase, superoxide dismutase, and hexokinase were determined by the methods of Aebi (201, McCord and Fridovich (211, and Beutler (22), respectively. DNA content of adipocytes was determined after extraction of the bulk lipids with 20 vol of chlorofornnmethanol2:l by the Giles and Myers (23) modification of the Burton (24) assay. RESULTS
Incubation of isolated rat adipocytes with riboflavin stimulates the oxidation of [14Cl]glucose to 14C0,, as illustrated in the left panel of Fig. 1. It can be seen that riboflavin stimulates oxidation even in the absence of light. This effect was always observed at concentrations of 1O-6 M and above, well within the normal human blood levels (25). In the two representative experiments shown in Fig. 1, the stimulation at 10e6 M was an average of 33% above controls without riboflavin. (In five total experiments under slightly varying conditions, stimulation at 10e6 M was 30 5 8.0% SE.) The presence of visible light, however, markedly potentiated the riboflavin stimulation of oxidation of 1O-5 M and above reaching maximal stimulation of 90% over the dark riboflavin control at 3 x 10p5. Effects of similar magnitude occurred with both FAD and FMN (not shown). These data suggest that riboflavin in the dark causes a significant stimulation of the hexose monophosphate pathway of glucose oxidation, which is potentiated above lop6 M in the presence of visible light. The stimulation of CO, production from glucose was not limited to the hexose monophosphate shunt as illustrated in the right panel of Fig. 1. Oxidation of [14CB]glucose, an index of carbon flux through glycolysis
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FIG. 1. Effect of riboflavin on C,- and &-glucose oxidation in isolated rat adipocytes. Adipocytes were isolated and incubated in beakercups for 1 h with appropriate riboflavin concentrations in the absence or presence of light. Six aliquots from each beakercup were then placed in tubes containing either [W,]- or [W,]glucose (glucose concentration was 0.2 mM) and again incubated for 30 min in the dark. Each point represents the mean value of triplicate samples. Circles and squares represent experiments on two different days. Open symbols, light incubation; closed symbols, dark incubation. Further details are described under Materials and Methods.
and the tricarboxylic acid cycle, was also affected. Unlike its effect on the HMS, riboflavin in the dark did not significantly stimulate this pathway at low levels. However in the light it had a dramatic effect. Oxidation was stimulated 110% in the presence of lop5 M riboflavin and 390% at low4 M compared to the dark controls. Since the effects of photooxidation in biological systems are typically destructive in nature! it appeared possible that the effects with hght and riboflavin seen in Fig. 1 might be caused by increased intracellular availability of glucose produced by increased nonspecific diffusion of the hexose through damaged membranes. To test this possibility, we measured glucose oxidation in the presence of cytochalasin B, which specifically blocks D-glucose transport but not nonspecific diffusion (26). If the effects in Fig. 1 are a consequence of photooxidation which increases nonspecific diffusion, cytochalasin B should not affect glucose oxidation. Figure 2 again demonstrates the dark stimulation of the HMS by riboflavin (at 3 x lop5 M) and the profound effects of the vitamin in light on the HMS and the glycolytic-TCA pathway. Moreover, it can be readily seen that these effects are abolished by 10 PM cytochalasin B. These results clearly demonstrate that the effects of
riboflavin and light are dependent, in part, on glucose transport, rather than nonspecific diffusion of glucose through damaged membranes. The apparent stimulation by riboflavin and light of glucose oxidation may be a result of increased glucose “push” or “pull” through the membrane or a combination of both. That is, either/both glucose transport per se is stimulated, or/and intracellular enzymes of glucose oxidation are activated. To first address this question, we determined the effect of riboflavin and light on uptake of 3-O-methylglucose, a nonmetabolizable analog. Neither light alone nor riboflavin at 3 X lop5 M in the dark significantly stimulated 3-0-methylglucose uptake (data not shown). However, as can be seen in Fig. 3, 3 x lop5 M riboflavin in light produced a 140% stimulation of transport compared to transport without the vitamin. For comparison, the effect of insulin is also shown. These results demonstrate that glucose transport is activated by riboflavin in light independently of any activation of intracellular utilization of glucose. The second possibility, that activation of intracellular enzymes of glucose oxidation might contribute to the stimulation by riboflavin and light, was tested by observing whether oxidation is stimulated under con-
STIMULATION
so tP
OF GLUCOSE
OXIDATION
0.07
5
1.5
=. E
1.2
q
14 1opM
Cytochalorin
B
C,-Glucose
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C,-glucose oxidation, as expected. In combination, the effects of the two agents in light are additive on C,-glucose oxidation, suggesting independent pathways of stimulation. The effects on C6-glucose oxidation are more than twice that of additive. This latter puzzling finding on &-glucose oxidation may be due to an indirect effect of riboflavin on insulin action or vice versa and was not explored further. The ability of adipoeyte homogenates to oxidize &-glucose to CO2 was exploited to further investigate the role of riboflavin in stimulating the hexose monophosphate shunt activity independent of transport. Figure 4 illustrates C,-glucose oxidation to CO, in ATP-fortified homogenates with increasing NADP+ concentrations. There is
FIG. 2. Effect of cytochalasin B on glucose oxidation stimulated by riboflavin. Conditions as in Fig. 1 except cytochalasin B (hatched bars) or ethanol (open bars) at 1% total volume was added to appropriate beakercups after the l-h incubation with or without riboflavin at 3 x lo+ M. Cells were allowed to incubate for 15 min and then were pipetted into tubes containing the [Wlglucose.
ditions where glucose is accessible for utilization. This condition was met by two different approaches. In the first, the effect of riboflavin and light on oxidation was assayed in the presence of insulin, so that transport was maximally activated by the hormone. Under these conditions, stimulation of glucose oxidation above that by insulin alone would strongly suggest direct effects on the pathways of glucose utilization. Cells were incubated with 0.5 mu/ml insulin and/or 3 x 10e5 M riboflavin. Although the data are not shown, the transport of 3Gmethylglucose in the presence of both agents did not exceed that of insulin alone. On the other hand, the effects of the two agents in combination on glucose oxidation, demonstrated in Table I, are striking. This table illustrates the effects of riboflavin seen in previous figures, and that of insulin, which stimulates both C1- and
R,boflovm
/
Time,
sec.
FIG. 3. Effect of riboflavin in light on transport of 34-methylglucose. Isolated adipocytes were incubated in light for 1 h with insulin (0.5 mu/ml) or riboflavin (3 x 10m5M) in the appropriate beakercup. Cells were transferred into tubes and the uptake of 3-Omethylglucose was measured at 20°C over the indicated times. The difference of the means of triplicate determinations in the absence and presence of cytochalasin B was calculated for each time point. This figure illustrates the composite result of four experiments in which the effects of either insulin or riboflavin were measured. The bars represent the range of values in the experiments.
384
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evidently insufficient endogenous pyridine nucleotide in the homogenate to support signif-lcant glucose oxidation. At low (presumably physiological) levels of NADP+, it can be seen that riboflavin in the light greatly stimulates this oxidation, increasing it 140% at 0.25 mM NADP+. At higher nucleotide concentrations the effect is less pronounced and at 2 mM NADP+ riboflavin inhibits oxidation (not shown). A simple explanation of these results is that riboflavin in light stimulates C,-glucose oxidation in homogenates by reoxidizing the NADPH produced by the HMS dehydrogenases, thereby regenerating cofactor. Thus the effect is most pronounced at low NADP+ concentrations. At higher NADP+ levels the apparent inhibition by riboflavin may represent damage to the shunt pathway that was masked by low NADP+ levels. For example, endogenous hexokinase activity in the homogenate was depressed 48% after incubation with riboflavin compared to its absence (data not shown). It can be seen that in the presence of 1 mM oxidized glutathione (GSSG), which is expected to reoxidize NADPH through glutathione reducTABLE EFFECT
I
OF INSULIN AND RIBOFLAVIN OXIDATION IN ADIPOCYTE~
ON GLUCOSE
Glucose oxidation to CO, (nmol/pg DNA) [‘*CJGlucose as substrate
[14Ce]Glucose as substrate
Addition
Dark
Light
Dark
Light
None Insulin Riboflavin Insulin + riboflavin
0.259 1.198 0.504 1.436
0.272 1.286 1.070 2.450
0.013 0.255 0.021 0.226
0.016 0.250 0.117 0.719
a Isolated adipocytes were incubated in light or dark for 1 h with insulin (0.5 mu/ml) and/or riboflavin (3 X 10m5 M). Aliquots were removed for triplicate determination of glucose oxidation during a 30-min pulse. Values are the means of triplicate values (range 2 10%) of an experiment, which was repeated twice with similar results. Glucose concentration was 0.2 mM. Values are based on the specific activity of labeled glucose added. The intensity of visible light was 6.4 W/mZ.
L
0
025
0.5 NADP*(mM)
I
0.75
1.0
FIG. 4. Effect of riboflavin in light on C,-glucose oxidation in adipocyte homogenates. Homogenates were incubated in uncovered beakercups for 1 h with or without 1O-5 M riboflavin (Rf) in light. Aliquots were pipetted into tubes containing ATP(2.5 mM, final concentration), NADP+ (indicated concentrations), GSSG (0 or 1 mM, final concentration), and [‘4C,]glucose (0.2 mM, final concentration) and incubated at 37°C for 20 min.
no longer stimulates tase, riboflavin oxidation but depresses it about 30%. These data suggest that the stimulation by riboflavin with light on glucose oxidation in rat adipocytes may be a consequence of both activation of glucose transport increasing the availability of glucose intracellularly and stimulation of pathways of glucose oxidation. The possible role of oxygen radicals produced by riboflavin photoxidation in the stimulation of glucose oxidation and transport shown above was investigated by including catalase, superoxide dismutase (SOD), dimethylfuran (DMF), and mannitol, which may scavenge or destroy hydrogen peroxide, superoxide, singlet oxygen, and hydroxyl radical, respectively. Figure 5 illustrates the effect of these agents on C,-glucose oxidation stimulated by riboflavin. It can be readily observed that cata-
STIMULATION
OF GLUCOSE
14
OXIDATION
BY RIBOFLAVIN
AND LIGHT
385
C,-Glucose
0
P I Rib< a Cd
FIG. 5. Effect of catalase, superoxide dismutase, dimethylfuran, and mannitol on the stimulation of &glucose oxidation by riboflavin. Conditions as in Fig. 1, except the appropriate agent was present in the cell suspensions with riboflavin (3 X 10m5M) and only &glucose oxidation was measured. The final activities or concentrations of catalase, SOD, DMF, and mannitol were 430 u/ml, 27 u/ml, 1.5 mM, and 5 mM, respectively. D, dark incubation; L, light incubation.
lase, SOD, and mannitol do not inhibit the riboflavin effect. The lack of effect of the enzymes catalase and SOD was not a result of their destruction by riboflavin since incubation of riboflavin and the enzymes alone produced no effect on SOD activity and only a 20% drop of that of catalase (not shown). In contrast, DMF, a singlet oxygen scavenger, abolished the stimulation of oxidation by riboflavin in light while it did not affect the control dark incubations. DMF had a similar effect on the stimulation of C,-glucose oxidation and the transport of 3Gmethylglucose by riboflavin, but not by insulin, whi1.e SOD and catalase did not have inhibitory effects. These data suggests that singlet oxygen may be an activator of glucose transport and oxidation in intact rat adipocytes, although further studies are necessary to confirm this hypothesis. DISCUSSION
The physiological role of riboflavin has long been known to be as a participant in intracellular electron transport. Riboflavin may also catalyze photooxidative damage. In this report we demonstrate a new aspect of the action of this vitamin in the stimulation of glucose oxidation and transport.
A stimulatory effect, even in the dark, on the hexose monophosphate shunt of intact rat adipocytes was observed at low concentrations of riboflavin (lop6 M). It should be noted that this dark effect was only seen if dialyzed BSA was used. Presumably the crude BSA contained some riboflavin-like contaminants. The dark effect on shunt activity is mimicked to similar degrees by both FAD and FMN. These latter phosphorylated compounds would not be expected to cross the plasma membrane. Thus, it is possible that the plasma membrane is the site of action of the dark effect of the flavins. For example, reduction of the vitamin (extracellularly?) by intracellular NADPH would generate the oxidized cofactor of the HMS. Although a plasma membrane NADPH-flavin oxidoreductase has not been reported, it would not be the first case of a transmembrane oxidationreduction, e.g., NAD(P)H can reduce 0, in phagocytes (27). Mukherjee and Lynn (17) have reported an adipocyte plasma membrane NADPH: 0, oxidoreductase that is activated by insulin which might be related topographically to the activity reported here. Light has a striking effect on glucose oxidation and transport in the presence of riboflavin. It should be mentioned that the ri-
386
GOODMAN
AND
boflavin concentrations at which these effects are seen are not totally unphysiological. For example, the riboflavin concentration in lens epithelium, a tissue that certainly is exposed to light much in excess of that used in these experiments, is reported to be 1.2 x 10e5 M (28). Further in viwo studies will be required to determine the physiological importance of this new aspect in the action of the vitamin. The unexpected lack of inhibition by catalase and the effect of dimethylfuran suggest that singlet oxygen may be the primary agent mediating the activation of glucose transport and the stimulation of glucose oxidation, although corroborating evidence is necessary to confirm this conclusion. In this regard, it is noted that hematoporphyn, another photosensitizer, can oxidize NADPH to NADP via singlet oxygen (29), which may be involved with the stimulation of C&glucose oxidation in whole cells and homogenates which we observe. The mechanism by which singlet oxygen may activate transport is unknown, but this concept is not inconsistent with Czech’s model of disulfide activation (16). While the product detected from the interaction of cysteine with singlet oxygen (generated by dyes) is usually cysteic acid, cystine can be produced at neutral pH (30). The mechanism by which oxidants activate glucose transport is far from clear. Insulin has been shown to prevent the autophosphorylation of membrane proteins (31, 32) which may be important in activation of transport. Vanadate, recently shown to have insulin-mimetic effects (33), is a potent inhibitor of phosphotransferase enzymes (34, 35), and perhaps these enzymes are the targets of oxidants which lead to transport activation. However, it should not be assumed that the initial event of the oxidative activation of transport by oxidants occurs at the plasma membrane. Recent evidence from two laboratories (36, 37) suggests that insulin “activates” glucose transport in adipocytes by causing the rapid translocation of transporters from internal membranes to the cell surface; the cellular locale of initiation of this activity is not known. Further study of the insulin-mimetic effects of oxidants such as riboflavin
HOCHSTEIN
may be of value in unraveling nism of this process.
the mecha-
ACKNOWLEDGMENTS We are grateful to Ms. Jill Reeves and Ms. Lloyd Wong for excellent technical assistance. This work was supported in part by Grant AG 00471 from the National Institute of Aging. REFERENCES 1. TAYLOR, M. B., AND RADDA, G. K. (1971) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 18B, pp. 496506, Academic Press, New York. 2. EPEL, B. L. (1973) Photophysiology 8, 209-229. 3. VARMA, S. D., KUMAR, S., AND RICHARDS, R. D. (1979) Proc. Nut. Acad. Ski. USA 76, 35043596. 4. PEREIRA, 0. M., SMITH, J. R., AND PACKER, L. (1976) Photo&m. Photobiol. 24, 237-242. 5. AGGARWAL, B. B., QUINTANILHA, A. T., CAMMACH, R., AND PACKER, L. (1978) Biochim. Biophys. Acta 502, 367-382. 6. NEWBURGER, J., COMBS, A. B., AND Hsu, T. F. (1977) J. Pharrn. Sci. 66, 1561-1564. 7. FELBER, T. D., SMITH, E. B.. KNOX. J. M.. WALLIS, L., AND MELNICK, ‘J. L. (;973) J: Amer. Med. Assoc. 223, 289-292. 8. KAUFMAN, R. H., GARDNER, H. L., BROWN, D., WALLIS, C., RAWLS, W. E., AND MELNICK, J. L. (1973) Amer. J. Obstet. Gynecol. 117, 11441146. 9. PASCALE, J. A., MIMS, L. C., GREENBERG, M. H., GOODEN, D. S., AND CHRONISTER, E. (1976) Pediat. Res. 10, 854-856. 10. LIN, J.-K., SU, I.-J., AND Hsu, S.-M. (1977) J. Formosun Med. Assoc. 76, 293-300. 11. PENZER, G. R. (1970) Biochem. J. 116, 733-743. 12. FOOTE, C. S. (1968) Science 162, 963-970. 13. COHEN, G., AND HOCHSTEIN, P. (1961) Science 134, 1574-75. 14. REED, P. W. (1969) J. Biol. Chm. 244, 24592464. 15. MUKHERJEE, S. P., LANE, R. H., AND LYNN, W. S. (1978) Biochem. Pharmucol. 27, 2589-2594. 16. CZECH, M. P. (1976) J. Biol. Chem. 251, 1X41170. 17. MUKHERJEE, S. P., AND LYNN, W. S. (1977) Arch. Biochem. Biophys. 184, 69-77. 18. RODBELL, M. (1964) J. Biol. Chem. 239,375-380. 19. LIVINGSTON, J. N., AND LOCKWOOD, D. H. (1975) J. Biol. Chem. 250, 8353-8360. 20. AEBI, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.1, 2nd Engl. ed., pp. 673-684, Academic Press, New York. 21. MCCORD, J. E., AND FRIDOVICH, I. (1969) J. Biol. Chem. 244, 6049-6055.
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22. BEUTLER, E. (1975) in Red Cell Metabolism: A Manual Iof Biochemical Methods, 2nd ed., pp. 38-40, Grune & Stratton, New York. 23. GILES, K. W., AND MYERS, A. (1965) Nature (London:) 206, 93. 24. BURTON, K.. (1956) Biochem. J. 62, 315-323. 25. BAKER, H., FRANK, O., FEINGOLD, S., GEL LENE, R. A., LEEVY, C. M., AND HUTNER, S. H. (1966)Amer. J. Clin. Nub. 19, 17-26. 26. CZECH, M. P., LYNN, D. G., AND LYNN, W. S. (1973) J. Biol. Chem. 248, 3636-3641. 27. BABIOR, B. M. (1978) N. Engl. J. Med. 298, 659668, 721--725. 28. KINSEY, V. E., AND JACKSON, B. (1951) AMA Arch. Ophthalmol. 46, 536-541. 29. BODANESS, R. S., AND CHAN, P. C. (1977) J. Biol. Chum. 252, 8651-8560.
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30. JORI, G., GALIAZZO, G., AND SCOFFONE, E. (1969) Int. J. Protein Res. 1, 289-298. 31. SEALS, J. R., MCDONALD, J. M., AND JARETT, L. (1979) J. Biol. Chem. 254, 6991-6996. 32. SEALS, J. R., MCDONALD, J. M., AND JARE~, L. (1979) J. Biol. Chem. 254, 6997-7001. 33. DUBYAK, G. R., AND KLEINZELLER, A. (1980) J. Biol. Chem. 255, 5306-5312. 34. GOODNO, C. C. (1979) Proc. Nat. Acad. Sci. USA 76, 2620-2624. 35. LINDQUIST, R. N., LYNN, J. L., AND LEINHARD, G. E. (1973) J. Amer. Chem. Sot. 95, 87628768. 36. SUZUKI, K., AND KONO, T. (1980) Proc. Nat. Acad. Sci. USA 77, 2542-2545. 37. CUSHMAN, S. W., AND WARDZALA, L. J. (1980) J. Biol. Chem. 255, 4758-4762.