Studies on the energy metabolism of opossum Didelphis virginiana erythrocytes—III. Metabolic depletion with 2-deoxyglucose markedly accelerates methemoglobin reduction in opossum but not in human erythrocytes

Camp. Eiorhem. Physiot. Printed in Great Britain

Vol. 89A,

No. 2. pp.

0300-9629/88

119-124, 1988

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STUDIES ON THE ENERGY METABOLISM OF OPOSSUM ~r~E~P~rS ~r~G~~r~~~ ERYTHROCYTES-III. METABOLIC DEPLETION WITH 2-DEOXYGLUCOSE MARKEDLY ACCELERATES METHEMOGLOBIN REDUCTION IN OPOSSUM BUT NOT IN HUMAN ERYT~ROCYTES N. C. BETHLENFALVAY,*~ J. E. LIMA,~ E. CHADWICK~ and I. STEWART~ Departments of *Primary Care and SClinical Investigation, Fitzsimons Army Medical Center, Aurora, CO 80045-5000, USA (Received 20 May 1987)

Abstract-l. Glucose-depleted, nitrite-treated erythrocytes reduce ferriheme in vitro in an environment 100 mM to 2-deoxy-D-glucose at a rate of 2.4 p M/ml cells/hr (opossum) and 0.37 p M/ml ceIls/hr (human). 2. During the process of methemoglobin reduction the breakdown of adenine ribonucleotides is more rapid in opossum (0.9 p M/g hg/hr) than in human (0.36 FM/g hg/hr) erythrocytes. 3. Radiolabelled ribose from [U-‘4C] ATP is catabolized exclusively to [‘“Cl lactate in opossum, and to [14C] pyruvate and [V] lactate in human red cells.

INTRODUCTION

We have recently presented evidence that opossum erythrocytes suspended in an electrofyte solution in the absence of provided substrate effectively reduce nitrite-induced methemoglobin. We have suspected that this ability of opossum red ceils might be due to substrate(s) of yet undetermined nature stored in the cells in amounts sufficient to provide for the reducing energy necessary to sustain the process of methemoglobin reduction (Bethlenfalvay et al., 1983). Erythrocytes of the rabbit, rat, mouse and guinea pig have been known for a long time to be capable of reducing nitrite induced methemoglobin to some extent without added substrate in vitro. An energy source to provide for this process was named “unauswaschbares Substrat”, i.e. presumably phophorylated glycolytic intermediate(s) trapped in the cell even after multiple washings and capable of ongoing metabolism providing for the NADH coupled reduction of methemoglobin (Kiese and Weis, 1943; Kiese, 1944; Matthies, 1956). More recently it was proposed that the elusive endogenous substrate probably included ribose from the catabolism of adenine nucleotides for reducing methemoglobin by way of the NADH linked pathway (Keitt, 1972). To test this h~othesis, we have measured the changes in methemoglobin, selected glycolytic intermediates, adenine nucleotides and hypoxanthine in methemoglobin containing red cells suspended in the presence of 100 mM 2-deoxyglucose (DOG). We present comparative data on the reduction of methemoglobin in intact opossum and human erythrocytes under conditions of rapid -.. tAuthor to whom correspondence should be addressed. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. c a P m%--8

metabolic depletion. We show that, at least in the opossum, ribose cleaved from purine nucleotides suffices to provide for the reducing energy to convert methemoglobin to oxyhemoglobin in 6 hr. MATERIALS AND METHODS

Opossum blood was obtained as described previously (Bethlenfalvay et al., 1983). Human blood was drawn by venipuncture.from healthy volunteers. [U-‘4Cj Adenosine, 500 .uCi/mmol and Z-deoxv-D - 12.6 jHi glucose 46.8 Ciimmol were from New England Nuclear. 2-Deoxyglucose was purchased from Sigma Chemical Company. Other chemicals used were of reagent grade. 2-Deoxycoformycin was a gift of Warner-Lambert Company, Ann Arbor, Michigan. .I

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of adenine nucleotides with I4C adenosine Erythrocytes (I ml) were washed thrice in ice cold 150 mM KC1 and’ then suspended to 10% (v/v) in a solution containing per liter: NaCf 145mM, KC1 3mM, K,HPO, 2 mM, MgCI, 1.2mM, o-glucose IOmM and Tris 50mM pH 7.4 at 37°C. The cell suspensions (IO ml) were supplemented with 5 UCi (10 nM) BJ-‘4C1 adenosine and incubated at 37” for I hr. The absolute’concentration of adenosine in the medium was 1pM. Label&

Preparation of mezhemogiobin containing erythrocytes

Whole blood and “C-labefed suspensions were sedimented by centrifugation and the supematants discarded. The cell pellets were then washed twice with ten volumes of 150 mM KC] and the hemoglobin in the cells was converted to methemoglobin by suspending the cells in an equal volume of 1.5% potassium nitrite in distilled water. After 30 min at room temperature, the erythrocytes were sedimented by ~entrifugation and washed x 6 with 10 volumes of ice cold 150 mM KCI. Approximately 30% (v/v) suspensions of red cells were then prepared in parallel in solutions of the following composition: (a) KC1 250 mM/l, MgCl, I.2 mM/l. Tris 50mM/I; pH 7.4;’ ib) NaCl 12bmkf/l, -Na,HP& 30 mM/l, KC1 5 mM/l, MgCl, 1.2mM/l o-glucose 10 mM/l and Tris 50 mM/l pH 7.4; (c) KC1 250mM/l, MgCl, 1.2mM/l, DOG 100 mM/l, Tris 50 mM/l, pH 7.4.

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Cell counts, hemoglobin and PCV of the suspensions were determined by standard methods of 0 time and at the end of the 6 hr incubation. “‘C-labelled cells were suspended in solution “C” only. AIiquots of cell suspensions “c” were taken for the determination of baseline levels of purine nucleotides. Another portion of the suspension was sedimented and the pellet placed at - 70°C for baseline methemoglobin determination the following day.

et al

Separation of [‘“Cl and [3H] labelled metaholites of adenosine and DOG

The investigation of the metabolic trail of [U-“C] ribose (from [U-“‘C] ATP) and of [2,6 3H] DOG was done as previously described (Bethlenfaivay et al., 1988).

RESULTS

Pulse-chase experiments with 2-deoxy-D [2,6-j H] glucose ‘T labeiled nitrited red cells suspended in solution “a” were supplemented with 20 FCi 2-deoxy-u [2,6-‘H] glucose per ml (47.5 nM) and incubated at 37°C for 30min. Sufficient cold DOG was then added to achieve a final concentration of 100 mM. Reduction of methemoglobin in nitrited erythrocytes

Tubes containing cell suspensions were placed uncapped in a closed water bath at 37°C and incubated for 6 hr. The test tubes were gently shaken at hourly intervals to preclude sedimentation. At selected time intervals aiiquot volumes were withdrawn from suspension “c” and suspension “a” ([“HI DOG pulse-chase) for the determination of purine nucleotides, bases and [‘“Cl and [‘HI labelled and products of carbohydrate metabolism. Glycolytic intermediates in opossum red cells were quantitated in suspension “C” at 2 hr in the course of methemoglobin reduction. For the quantitation of methemoglob~n, aliquot suspensions “a”.-“C” were sedimented at 3 and 6 hr. The supernatants were discarded and the pellets were placed at -7o’C until further processed. Determination of red ceN glyco!ytic intermediates

G-6-P, F-6-P, F-i, 6-P. DHAP and GA-3-P were measured as described by ~inikami el al. 11965) with a Carv modei 219 recording spectrophotometer. Isoelectric ,focusing and quantitation of methemoglobin

Electrofocusing was carried out by applying 25 ~1 of 25% red cell lysates on filter paper strips to thin layer polyacrylamide gel slab containing ampholines (pH 9.5-3.3) obtained from LKB. Focusing proceeded at a constant temperature of 4’C for 45 min at a constant power of 15 W. Gels were photographed unstained immediately after completion of focusing, and then placed in a fixing solution (57.5 g trichloracetic acid and 17.25 g sulfosalycilic acid in 500 ml distilled water) for 2 hr. The fixed gels were scanned at 630 nm with a cliniscan apparatus (Helena Laboratories) which provides a hard copy of the scan on a strip chart, simultaneously providing the percentage of met, valence hybrid, and reduced hemoglobins under the corresponding peaks. Total methemoglobin in this work is the sum of methemoglobin (a,+ I(“+) pluse f of the sum of the valence hybrid hemoglobins [,I+ fi*+ +x2+ pi+ (Bunn and Drysdale, I97 I)]. HPLC rod~ochromatogruph~

The system employed in this study has been fully described in the preceding companion paper (Bethlenfalvay et al., 1988). Quantitation of purine nucleotides and bases

Red cell suspensions were processed by the alkaline extraction method (Stocchi et al., 1985) and the compounds of interest separated in a 5 pm Supelcosil LC-18 (25 cm x 4.6 mm i.d. (Supelco, Bellafonte, PA)] analytical column. The mobile phase and the chromatographic conditions were exactly as described by Stocchi et al. (1985). Peak identities were confirmed by retention times and co-elution with standards. Quantitative measurements were carried out after injection of standard solutions of known concentration to convert peak areas and heights to nanomoles.

Hematology

We chose a hypertonic buffer solution to minimize eel1 lysis. Under the conditions described, crythrocyte counts and hemoglobin remained stable. Packed red cell volumes and hematocrit rose considerably due to cell swelling. Hemolysis was barely perceptible to the naked eye. Reduct~vn vf’ met~e~loglobin

Figure IS shows that meth~tnoglobin did not reduce appreciably in human erythrocytes in the absence of a provided metabolizable substrate. Metabolic depletion in the presence of 100 mM DOG slightly accelerated the reduction of methemoglobin whereas D-&COSe, when provided to the cells, had a clearly superior effect. In contrast, methemoglobin reduction in opossum red cells incubated in the presence of 100 mM DOG proceeded to near completion in 6 hr and to the same extent as in cells provided with 10 mM u-glucose as an external energy source (Fig. 1A). The reduction of methemoglobin in opossum red cells continued unimpeded beyond 2 hr when glycolytic inte~ediates upstream from the site of NADH production were no longer detectable (data not shown). The two-hemoglobin phenotype reported to occur in Didelphis (Bethlenfalvay er LIZ., 1976) was not detected in the red cells of opossums used in this study. Purine n~cleotide cat~~o~isrn in red cells during rnet~e~loglo~~n reductivn in the presence of 100 mM DOG

Phosphate was omitted from the buffers utilized here to effect maximal nucleotide breakdown during these experiments (Whelan and Bagnara, 1979). As is shown in Fig. 2, catabotism of purine nucleotides was found to be more rapid and more extensive in opossum than in human erythrocytes. ATP was no longer detectable in opossum cells at 3 hr and more hypoxanthine was detected than in human red cell suspensions at any corresponding point in time during the reduction of methemoglobin. Opossum erythrocytes do not contain xanthine oxidase, yet sustained accumuIation of xanthine was evident. La~lling experiments using radioactive guanine suggest that G’I’P is the source for this purine base (Bethlenfalvay, in preparation). Total adenine nucleotides in opossum red cells declined at a rate of 0.9 pmoles/g hg/hr compared to 0.36pmoles catabolized/g hg/hr in human erythrocytes. Only traces of inosine (labelled and uniabe~led) were detected in red cell extracts on

chromatography suggesting that the concentration of phosphate in these experiments did not become ratelimiting for the purine nucleoside phosphorylase reaction (Fig. 4 IS]). Since neither coformycin nor deoxy-coformycin up to 300pM inhibit opossum adenosine deaminase to a significant degree (Bethlen-

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Metabolism of Didelphis RBC

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Fig. 1. Representative results of ten experiments measuring the reduction of methemoglobin in nitrited (A) opossum and (B) human erythrocytes under conditions described in the text. No substrate (-O-), D-glucose (-H-), 2-deoxyglucose (-A-). The lines are fitted to graph methemoglobin reduction within the ranges of experimental values.

falvay unpublished observations) the extent of the AMP-adenosine-inosine sidearm of adenine nucleotide catabolism (Rapoport et al., 1979; Rapoport et al., 1983) could not be defined.

DISCUSSION Earlier studies have shown that, in the absence of provided substrate, methemoglobin is reduced more rapidly in nitrited opossum than in human erythro6-

Metabolism of [U”C] ribose worn [U’4C] ATP) and of [2, 6’H] DOG in erythrocytes during the reduction of methemoglobin in the presence of 1OOmM DOG

Human and opossum erythrocytes incorporated 85% of the [Ui4C] adenosine provided during the labelling process. Almost exclusively this radioactivity chromatographed with ATP in red cells of both species prior to conversion of oxy to methemoglobin (data not shown). Figure 3 depicts a representative example of three separate experiments. At 6 hr of methemoglobin reduction, most if not all of the [‘“Cl radioactivity (from [U’“C] ribose) in opossum cell suspensions (Fig. 3A) had a retention time of 13.9 min and co-chromatographed with authentic [‘“Cl lactate in an ORH-801 column (Bethlenfalvay et al., 1988). Very little [14C] radioactivity emerged at 5.6min [sugar phosphate(s)]. In contrast, when human red cell suspensions were analyzed, less [‘“Cl radioactivity emerged at 13.9 min (lactate), more was seen at 5.6 min and a substantial amount of radioactivity was detectable with a 9.6 min retention time. This [‘“Cl radioactivity co-chromatographed with authentic [14C] pyruvate (Fig. 3B). As we have described previously in our companion paper (Bethlenfalvay et al., 1988) pulsed [2,6-3H] DOG provided to [‘“Cl labelled red cell suspensions yielded three peaks of [‘HI radioactivity. Phosphorylated [‘HI DOG and intermediates (5.7 min) unmetabolized [‘HI DOG (10.5 min) and a diffusible unidentified [3H] metabolite (12.6 min, arrow). When 30 mM KF was provided to these cell suspensions there was a measurable (IO-15%) acceleration of methemoglobin reduction. In the presence of fluoride, however, the 9.6 min and 13.9 min [“Cl and the 12.6 min [3H] radioactivities were not detectable (data not shown).

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Fig. 2. Time course of catabolism of adenine nucleotides and of hypoxanthine and xanthine formation in nitrited (A) opossum and (B) human erythrocytes in the presence of 100 mM DOG during methemoglobin reduction. Nucleotides and bases were determined by HPLC as described in the text. ATP + ADP + AMP (-•-), ATP (-a-), hypoxanthine (-O-), xanthine and hypoxanthine (-A-). The cell suspensions were incubated at 37°C. The data points are the average of six individual experiments. The vertical bars represent the ranges of experimental values.

N. C. BETHLENFALVAY

TIME

fMtNf

Fig. 3. Radiolabelled derivatives of [U-‘4C] ATP (--) and of pulsed ., 12.6-3H1 ~DOG (---) . , in extracts of (A) ._. opossum and (B) human erythrocytes at 6 hr of methemoglobin reduction. The details of the experiment are described in the text. Peak (1) phosphorylated glycolytic intermediates, peak (2) pyruvate, peak (3) f3Hl DOG, peak (4) lactate. The arrow points to an- ~u~de~tified diffusible metaboiite of DOG. Column: ORH-801. mobile Dhase 0.01 N H,SO.,. flow rate 1.6 ml/min, temp.‘35”C. [14e] adenine nucleotides and [“Cl hypoxanthine are retained on this column under the conditions of chromatography.

cytes (Bethl~nfalvay er al., 1983). Both NADH and NADPH m~themoglobin reductase activities were demonstrated in opossum red cells. While the

NADH-dependent activity was found at approximately the same level as in human red cells, the NADPH-dependent activity was detected at a somewhat higher level in opossum erythrocytes (Bethlenfalvay er al., 1982). The reduction of methemoglobin in the red cell depends primarily on a linked system of two electron carriers, cytochrome bS and NADH and the enzyme cytochrome b, reductase. The formation of NADH during the metabolism of DOG has been considered negligible (Brooks et al., 1960; Schmidt et al., 1974). Our observations are in accord with the above reports. About only 1% of the radioactivity of [l-‘“Cl DOG presented to red cells of both species was liberated as radioactive CO* per hour in the presence of 2 mM pyruvate and no radioactive material eluted in the triose region on chromatography of extracts of these cells (Bethlenfalvay et al., 1988). Yet, as is shown in Fig. I, methemoglobin reduction was accelerated by DOG in both types of erythrocytes: markedly in those of the opossum and to a much lesser extent in human cells. Extrapolation of the data from Fig. 1 reveals that in the presence of DOG or glucose, methemoglobin in opossum red cells was reduced at a rate of 2.46pM hemeiml cellsjhr. In human erythrocytes DOG sustained a reduction rate of 0.37 PM heme/ml cells/hr whereas glucose facilitated a reduction rate of 1.03 PM heme/ml cells/hr. The last value agrees well with the data of Sannes and Hultquist (1978). A comparsion of the rate and extent of reduction of methemoglobin (Fig. 1) with purine nucleotide

et al.

catabolism and of concomitant hypoxanthine production (Fig. 2) reveals one of the metabolic events that results in the disparate behavior of opossum and human erythrocytes: because ATP is more rapidly and more extensively degraded in opossum red ceUs, more ribose per unit of time becomes available for reducing methemoglobin by way of the NADHlinked pathway. The ability of human red cells to extensively convert the pentose portion of provided purine nucleosides to lactic acid has long been recognized (Lowy et al., 1958). More recently we have presented evidence that opossum erythrocytes readily utilize provided inosine and adenosine as was shown by net lactate and ATP production (Bethlenfalvay et al., 1984). As is shown in Fig. 3, most (94.3%) of the radiolabelled ribose from [U’4C] ATP is observed to chromatograph as [‘“Cl lactate at 6 hr in opossum red cell extracts. In preparations derived from human cells less total [“Cl radioactivity is present, presumably owing to lagging adenine nucleotide catabolism. Approximately 16% of this radioactivity elutes as phosphorylated sugar intermediates, and 42% each as diffusible lactate and pyruvate. The unidentified diffusible metabohte of pulsed [2.6 3II] DOG (Fig. 3, arrow) as we11as peaks 2 (pyruvate) and 4 (lactate) are not demonstrable on inhibiting enolase (Fig. 4 [6]) with 30 mM fluoride (data not shown) whereas methemoglobin reduction accelerates in the presence of this anion (Bethlenfalvay et al., 1983). The absence of [r4C] labelled pyruvate in extracts of opossum red cells at 6 hr of methemoglobin reduction is interesting. It is possible that sufficient NADH remains available in these cells for LDH to convert pyruvate to lactate (Fig. 4 [7]) or alternatively, more favorable kinetics exist between opossum red cell LDH and NADPH generated in the oxidative pentose phosphate pathway (Fig. 4 [3 and 41). This latter source of NADPH has been shown to produce reducing equivalents in glycolysis of human red cells in vitro (Rapoport et al., 1979). The proposed scheme of the energetics of methemoglobin reduction in nitrited erythrocytes in the absence of provided substrate is depicted in Fig. 4. In the presence of 100 mM DOG and in the absence of inorganic phosphate, ATP undergoes rapid catabolism (Fig. 4 [l, 8, lo]) in methemoglobin containing red cells (Ti about 1 hr). Ribose, in amounts equivalent to hypoxanthine (and xanthine in the opossum) enters the pentose phosphate pathway and emerges as a suitable metabolic intermediate to sustain the NADH-linked system of reduction of methemoglobin (Fig. 4 [S]). It is quite possible however that additional advantageous conditions exist in opossum red cells which contribute to the rapid reduction of this ferriheme. We have observed that the reduction of methemoglobin in nitrited rabbit erythrocytes in the presence of 1OOmM DOG is identical in rate to that measured in opossum red cells (Bethlenfalvay, unpublished). The Michaelis constant for cytochrome b, of the reductase in rabbit red cells is 5 p M whereas in human cells it is 30 PM (Yubishui et al., 1981). The concentration of cytochrome b5 in human red cells is about 0.5 PM (Hultquist et al., 1984). In view of the relatively high Km of the human rcductase for cytochrome b,, its velocity is far below its maximum

Metabolism of Didelphis RBC

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MGFc

Cybj

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NADPH

Fig. 4. Proposed scheme for the acceleration of methemoglobin reduction in erythrocytes in the presence of 100 mM DOG. DOG is rapidly phosphorylated by hexokinase (I) inducing rapid catabolism of ATP. The absence of inorganic phosphate from the buffer medium enhances the action of 5’ nucleotidase (10) to produce inosine. Iosine then is split by purine nucleoside phosphorylase (8) into hypoxanthine and ribose-l-phosphate. After transmutation into ribose-5-phosphate, further action by transketolases and transaldolases of the pentose phosphate cycle provides for entry of triose and hexose into the anerobic glycolytic trail and for the generation of NADH at the glyceraldehyde-3-phosphate dehydrogenase (5) step of glycolysis. Available NADH then sustains the reduction of methemoglobin catalyzed by cytochrome b, reductase (9). Pyruvate and lactate are formed as end products of ribose from ATP. Lactate is produced from pyruvate by means of lactate dehydrogenase (7) either by means of NADH not fully utilized at (9) or through NADPH generated by the metabolism of DOG at the glucose-&phosphate dehydrogenase (3) and phosphogluconate dehydrogenase (4) steps of oxydative breakdown of DOG-6-phosphate. Inhibition of enolase (6) by fluoride stops glycolytis at the 2-phosphoglycerate step of glycolysis and thus abolishes the production of pyruvate and lactate. More NADH then becomes available at step (9) to further enhance the reduction of methemoglobin. Since DOG-6-phosphate is not a substrate for glucose isomerae (2), the bulk of DOG phosphorylated (1) remains trapped in the cell and not further metabolized. The extent of AMP catabolism by deamination versus dephosphorylation (?) remains unknown.

potential. It is possible that the kinetics of opossum cytochrome b, reductase (Fig. 4 [9]) are similar to those described in rabbit red cells. Clearly more work is needed toward a fuller understanding of the energy metabolism in opossum erythrocytes. Measurements of the Km for cytochrome b, and NADH of the cytochrome b5 reductase and for NADH and NADPH of the lactate dehydrogenase (Fig. 4 [7]) may provide these insights. Acknowledgements-This research was supported by grant SO/650 from the Department of Clinical Investigation, Fitzsimons Army Medical Center. We thank Drs Ernest Beutler, H. D. -Kim and P. O’Barr for many helpful discussions. Special tribute is due to MS Chris Montoya for typing the manuscript and to MS K. Wyatt for the preparation of the figures.

Waldrup T. (1982) Cytosolic and membrane-bound methemoglobin reductases in erythrocytes of the opossum, D~de~hi~ virginiann. Camp. Biochem. Phpsiof. 73B, 591-594. Bethlenfalvay N. C., Waterman M. R., Lima J. E. and Waldrup T. (1983) Comparative aspects of methemoglobin formation and reduction in opossum (Didelphis uirginiunu) and human erythrocytes. Comp. Biochem. Physiol. 75A, 635439.

Bethlenfalvay N. C., Lima J. E. and Waldrup T. (1984) Studies on the energy me~bolism of opossum (~i~Iphis ~irgj~j~u) erythrocytes. I. Utilization of carbohydrates and purine nucleosides. J. Cell Physioi. 120, 69-74. Bethlenfalvay N. C., Lima J. E., Waldrup T., Chadwick E. and Stewart I. (1988) Studies on the energy metabolism of opossum (Didelphis oirginiunu) erythrocytes-II. Comparative aspects of 2-deoxy-D-glucose metabolism in opossum and human erythrocytes in t&o. Camp. Biochem. Physiof. 89, 113-117.

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Rapoport I., Rapoport S., Maretzki D. and Elsner R. (1979b) The breakdown of adenine nucleotides in glucosedepleted human red cells. Acta Biol. Med. Germ. 38, 1419-1429. Rapoport I., Rapoport S. M., Elsner R. and Gerber G. (1983) Breakdown of adenine nucleotides of human erythrocytes. Biomed. Biochim. Acfa 11/12, 302-305. Sannes L. J. and Hultquist D. E. (1978) Effects of hemolysate concentration, ionic strength and cytochrome b, concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes. Biochim. Biophys. Acta 544, 547-554. Schmidt F. G., Schmarz R. T. and Scholtisser C. (1974) Nucleoside-diphosphate derivatives of 2-deoxy-D-glucose in animal cells. Eur. J. Biochem. 49, 237. Stocchi V., Cucchiarini L., Magnani M., Chiarantini L., Palma P. and Crescentini G. (1985) Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal. Biochem. 146, 118-124. Whelan J. and Bagnara A. S. (1979) Factors affecting the rate of purine ribonucleotide dephosphorylation in human erythrocytes. Biochim. Biophys. Acta 563, 46&478. Yubishui T.. Tamura M. and Takeshita M. (1981) Studies on NADH-cytochrome b, reductase activities in hemolysates of human and rabbit red cells by isoelectric focusing. Biochem. Biophys. Res. Commun. 102, 860--866.