2′-deoxyadenosine metabolism in human and opossum Didelphis virginiana erythrocytes in vitro

Comp. Biochem. Physiol. Vol. 106B,No. 3, pp. 641-645, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 Pergamon Press Ltd

2'-DEOXYADENOSINE METABOLISM IN H U M A N A N D OPOSSUM D I D E L P H I S VIRGINIANA ERYTHROCYTES IN VITRO N. C. BETHLENFALVAY*~'J. , E. LIMA~and R. E. BANKS~ *Department of Primary Care and ~Clinical Investigation, Fitzimons Army Medical Center, Aurora, CO 80045, U.S.A. (Received 18 March 1993; accepted 30 April 1993 Al~traet--1. In erythrocytes of both species, deamination and phosphorylation of dADO was completely inhibited by 5/~M 2'deoxycoformycin and 10#M 5-ITU respectively. 2. Under physiologic conditions, provided with nanomolar concentrations of dADO there was complete deamination of dADO in human red cells. In opossum erythrocytes deamination and phosphorylation of the deoxynucleoside were nearly equivalent; additionally, 1-2% of the substrate was metabolized to AXP. 3. With ADA inhibited in intact red cells, apparent Km and Vm~ for dADO were 0.5 vs 0.1 mM and 0.8 vs 6.25 #mol/g hg/hr in human and opossum cells, respectively. 4. In opossum red cell lysates, GTP was superior to ATP and to dATP as a phosphate donor in the dADO kinase reaction.

INTRODUCTION

MATERIALS AND METHODS

In normal human erythrocytes, under physiologic conditions, dADO is phosphorylated only when A D A is blocked, and only at supraphysiologic (18 mM) Pi concentrations has significant deoxynucleotide formation been observed (Perrett et al., 1981). We have recently reported that red cells of the North American opossum have low A D A activity and high concentrations of dAXP (Bethlenfalvay et al., 1989). In that study, we suspected the existence of high affinity/high activity kinase(s) responsible for the accumulation of dAXP. This paper reports observations on the metabolism of dADO provided to human and opossum red cells in nanomolar concentrations under physiologic (1 mM Pi) conditions, and describes the kinetics of d A D O phosphorylation in A D A inhibited intact erythrocytes of both species.

[2,8 3H] 2'-deoxyadenosine (44 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA). [5-3H] 2'-deoxycytidine (16Ci/mmol), nucleosides, nucleotides, 2'-deoxycoformycin, pyruvate kinase (Product No. P-9136) and phosphoenolpyruvate were from Sigma Chemical Co. (St Louis, MO). Tetrahydrouridine was supplied by Calbiochem (La Jolla, CA). 5'-iodotubercidin was a gift from Professor L. Townsend, College of Pharmacy, University of Michigan (Ann Arbor, MI). Telazol was procured from Aveco Co. Inc. (Fort Dodge, IA). All other chemicals were of highest quality commercially available.

tTo whom correspondence should be addressed. Disclaimer--The opinions and assertions herein are the private views of the authors and do not necessarily reflect the views of the Department of the Army or the Department of Defense. Abbreviations--ADA, adenosine deaminase; ADO, adenosine; dADO, 2'-deoxyadenosine; dAMP, 2'-deoxyadenosine monophosphate; dADP, 2'-deoxyadenosine diphosphate; dADP, 2'-deoxyadenosine triphosphate; dAXP, total adenine deoxyribonucleotides; AXP, total adenine ribonucleotide; APRT, adenine phosphoryboryl transferase; Pi, inorganic phosphate; PEP, phosphoenolpyruvate; PK, pyruvate kinase; 2-ME, 2-mercaptoethanol; SAHH, S-adenosylhomocysteine hydrolase; 5-ITU, 5-iodotubercidin. 641

Animals Adult D. virginiana were obtained from commercial vendors in Florida and shipped by air express to Denver. The animals were individually housed and maintained at 40-60% relative humidity and 78-82°F. The animals were allowed to adjust to Denver altitude (5400 feet above sea-level) for three months before use. Collection o f blood samples The animals were first anesthetized with Telazol, 5 mg/lb body weight given intramuscularly. Sedation was rapid to permit femoral venipuncture within 5 rain. Heparinized whole blood was sedimented by centrifugation, and plasma was discarded. Red cells were washed three times with saline removing the top 3-5 mm of cells each time,

N. C, BETHLENFALVAYet aL

642

and used for the preparation of lysates and for experiments using intact erythrocytes. Human blood was obtained by antecubital venipuncture from consenting volunteers.

Preparation of lysates The packed red cells were lysed by freeze-thawing ( - 8 0 and 37°C) three times after having been suspended in 5 vol of a charcoal-dextran suspension containing 3 g charcoal, 0.3 g dextran and 5 mmol 2-mercaptoethanol/l in cold distilled water. The lysates were left for 15 min at 4°C and vortex-mixed at 2-min intervals. After centrifugation at 10,000 g for 10min, the supernatants were used as enzyme samples.

Measurements of 2'-deoxyadenosine deamination, phosphorylation and cleavage in intact red cells Opossum and human red cells (100/d) were suspended in 2ml of an electrolyte solution containing (mmol/l) 120 NaCI, 5 KCI, 1 MgCI 2, 1 Pi, 10 D-glucose, 25 Tris-HCl (pH 7.4). The cell suspensions were supplemented with [3H] dADO to yield 25, 50, 100 and 500nM of substrate (final concentration) and incubated at 37°C. Initial velocities in this system were linear for 20 min using 0.1 ml cells. For dADO kinase measurements, red cell suspensions were prepared as above and further supplemented with 2'-deoxycoformycin at 5 # M final concentration. Kinetic studies with respect to dADO in opossum red cells were conducted at seven concentrations of [3H] dADO ranging from 5 to 100/~M, and in human erythrocytes at five concentrations ranging from 20 to 100#M (constant concentrations of radioactivity). Under the above conditions, initial velocities were found to be linear for 20 min.

Phosphorylation erythrocytes

of

2'-deoxycytidine

in

intact

Packed human and opossum red cells (0.1 ml) were added to 1.9 ml of the physiologic electrolyte/glucose solution described above and incubated for 1 hr at 37°C in the presence of tetrahydrouridine (0.16 mM, final concentration) to prevent deamination of the deoxynucleoside (Hurley and Fox, 1983). Two nanomoles of [3H] 2'-deoxycytidine were then added to the suspensions and further incubated at 37°C for 1 hr.

Determination of optimum of Mg 2+ concentration and specificity of phosphate donors for dADO kinase in opossum red cell lysates The assay was performed at 37°C in stoppered tubes. Each assay contained, in a volume of I ml: 5U PK, 5 #mol, 2'-deoxycoformycin and (mmol/l) 100 PIPES (pH 7.3), 25 2-ME, 2.5 PEP, 0.5-2.5 Mg 2+ 1.0 ATP, dATP or GTP and hemolysate representing 5-10 mg hemoglobin. [3H]dADO 0.5 mmol/1 served as substrate. Initial velocities in this system were found to be linear up to 80 min and 14 mg of protein.

Preparation of extracts for radiochromatography Tubes containing cell suspensions and lysates were immersed in a boiling water bath for 3 min to precipitate protein. The resultant slurry was then filtered through 0.45/~m filter discs attached to sytinges. Of the clear filtrate, 50-100/~1 were used for chromatography.

HPLC-radiochromatography [3H]-labeled adenine ribo- and deoxyribonucleotides and unmetabolized [ 3H] dADO were quantitated as previously described (Bethlenfalvay et al., 1990).

Other determinations Hemoglobin content of cell lysates was determined with an American Optical Corp. No. 1010D Hbmeter instrument. RESULTS

Metabolism of dADO in intact erythrocytes In opossum red cells, deamination and phosphorylation of 25-500nM dADO were similar in magnitude. Table 1 shows that, in addition to dAXP, the radiolabel was detected in AXP as well. The ratio of cleaving to phosphorylating activity was 1:22-16 in the range of substrate concentrations used. Human red cells, under the conditions of these experiments, completely deaminated the substrate provided. No radiolabel was detectable in dAXP or in AXP (data not shown).

Initial velocity studies Double-reciprocal plots of initial velocity data at varying concentrations of [3H] dADO in human and opossum erythrocytes are shown in Fig. 1. They indicate an apparent Km for dADO of 0.5 mM in

1. Metabolismof [2,8-3H] 2'-deoxydaneosinein opossumerythrocytesin 20 min Dcaminase Kinase Cleavage* Relative metabolism% Deoxyadenosine (Hy) (dAXP) AXP (riM) (pmoles formed/0.1ml ceils) Unchanged Deaminas¢ Kinase Cleavage% Table

25 50 100 500

15.2 25.7 54.6 2257.4

14.2 28.7 49.7 174.6

0.86 1.8 2.8 7.0

39.6 43.8 46.5 58.9

30.3 25.7 27.3 22.5

28.4 28.7 24.8 17.8

1.7 1.8 1.4 0.8

*By action of APRT on adenine cleaved from the S-adenosylhomocysteine/2'-deoxyadenosine complex. /

643

2'-Deoxyadenosine metabolism

-10

-5

10

20

1~

20

30

40

50

=

4*

-

10

~ x l ~

30

I

~

1/S (S = ~ dADO mM)

Fig. 1. Double-reciprocal plots of the effect of 2'-deoxyadenosine concentration on 2'-dADO phosphorylating activity in (A) opossum and (B) human erythrocytes in the presence of 5 #M 2'-deoxycoformycin. human, and of 0.1 mM in opossum erythrocytes (human red cells: ATP 1.4mM, GTP 0.03mM; opossum red cells: ATP 0.4 mM, dATP 1.2 mM, GTP 0.32 mM). V~x for dADO phosphorylation is one order of magnitude greater in opossum than in human red ceils. Five micromolar 2'-deoxycoformycin completely inhibited deamination of provided substrate. At 10 #m concentration, 5-ITU, when used, abolished phosphorylation of dADO but had no effect on the amount of radiolabel accumulating in AXP (data not shown).

Phosphorylation of [3H] 2'-deoxycytidine in intact erythrocytes There was no radiochromatographic evidence of incorporation of this nucleoside into mono-, di-, or triphosphates in either human or opossum red cells (data not shown).

DISCUSSION

This study was done to (1) compare the metabolism of nanomolar dADO in human and opossum red cells under physiologic (1 m Pi) conditions, and (2) to determine the apparent kinetics of dADO phosphorylation in ADA-inhibited erythrocytes of both species. Intact red cells were chosen in this study because they may provide a better indication of the in vivo capacity of purine-metabofizing enzymes than

,~ 7 a~

017

6

5

Magnesium requirement for optimum phosphate donor activity in opossum red cell lysates Hemolysates were reacted with charcoal to remove endogenous nucleotides (Sakuma et al., 1987). Using this material, Mg 2+ concentrations required for ATP dATP and for GTP as phosphate donors were determined. Figure 2 shows that the above triphosphates have different Mg 2+ concentration requirements for optimal activity. The relative dADO phosphorylating activity of GTP was 128% of that of ATP, and dATP was only 38% as active as ATP. The distribution of the [3H] radiolabel in dADO mono-, di- and triphosphates as a function of the individual triphosphates serving as phosphate donors is depicted in Fig. 3. There is a significant relative decrease of dATP production when GTP serves a donor; most of the radiolabel accumulates with dAMP. In contrast, ATP and dATP have similar apparent activities.

w,,,,,,,~/v~_._._.. ~

0 0.0

I 0.5

I 1.0

I 1.5

I 2.0

dATP

I 2.5

M i ++ mM

Fig. 2. Effect of magnesium concentration on 2'-d¢oxyadenosine phosphorylating activity in opossum red cell lysates using 0.5 mM [3H] dADO as substrate. Nucleoside triphosphates are ! raM.

644

N.C. BETHLENFALVAYet al. Phosphate 1 O0

.4

donor ATP dATP

go

GTP 0 L~

8O

~B

7O

~1

6O

,u o

5O

0 I~

40

.~. 11

/s~.N

2o

lO

clAMP

dADP

dATP

Fig. 3. Distribution of radiolabel in 2'-deoxyadenosine mono-, di- and triphosphates formed in opossum red cell lysates using 0.5 mM [3H] 2'-deoxyadenosine as substrate. Nucleoside triphosphates are 1 mM at optimal magnesium concentrations.

cell lysates (Sahota et al., 1980; Fairbanks et al., 1987; Aragon and Sols, 1991). Our data confirm the results presented by others, i.e. that in human erythrocytes, dADO is phosphorylated only when A D A is inhibited (Snyder and Henderson, 1973; Perrett et al., 1981). In contrast, and as shown in Table 1, the extent of deamination and phosphorylation under physiologic conditions is similar in ADA-deficient red cells of the opossum. In these cells [3H] dADO is further metabolized, although to a much lesser extent, to [3H] AXP. This pathway involves the cleavage of the base [3HI adenine from the SAHH-[3H] dADO complex with subsequent phosphoribosylation of [3H] adenine via APRT to [3H] AXP (Abeles et al., 1980; Simmonds et al., 1989; Smolenski et al., 1992). The kinetic data presented in Fig. 1, using intact red cells as "synthetic units", show that opossum red cell adenosine kinase has a 5-fold greater affinity with d A D O and a 10-fold higher activity than that present in human erythrocytes. Since 20/~M 5-ITU nearly completely blocked the formation of [3H] dAXP from provided [3H]-dADO and since there was no detectable formation of nucleotides from provided [3H] deoxycytidine, our observations suggest that adenosine kinase is responsible for the phosphorylation of d A D O in red cells of both species.

In red cell lysates, opossum dADO kinase showed no specific requirement for a particular nucleoside triphosphate as a phosphate donor, although each triphosphate had a specific magnesium concentration requirement for maximum activity. Figure 2 shows that GTP was 128% as effective as ATP as a phosphate donor whereas dATP had only 38% relative efficiency. The equal or superior ability of GTP as phosphate donor for (d)ADO kinase in human (Hurley and Fox, 1983; Yamada et al., 1981) and other mammalian tissues (Lindberg et al., 1967; Murray 1968; Krygier and Momparler, 1971; Yamada et al., 1980) has been of note for some time. Of particular interest was to observe the metabolic trail of [3H] dADO in opossum red cell lysates used as the enzyme source for dADO kinase. The data shown in Fig. 3 suggest that GTP, although superior as a phosphate donor for the dADO kinase activity, is inferior to ATP and to dATP with respect to deoxyadenylate and deoxynucleoside diphosphokinase. These observations are in marked contrast to those seen in intact red cells provided with [3HI dADO. At all concentrations of the nucleoside used, dATP, dADP and dAMP were found in the same ratio (10:1:0.1) as the adenine nucleotides (data not shown) suggesting synergism of nucleoside triphosphates toward the formation of dAXP in the intact erythrocyte. The nature of the metabolic process resulting in a reciprocal relationship between red cell AXP and dAXP levels in human inherited A D A deficiency has been elucidated (Simmonds et al., 1982; Valentine et al., 1985). Such a reciprocal relationship between AXP and dAXP has also been observed in opossum erythrocytes (Bethlenfalvay et al., 1991) in which similar kinetics may prevail, although the reason(s) for the wide variations observed remain to be elucidated. A few years ago it was suspected that dAXP were mere bystanders at intracellular metabolic events (Valentine et al., 1985). Recent work has provided evidence that dATP is quite effective as an energytransferring molecule in glucose metabolism and for ion transport in both human and opossum erythrocytes (Buc et al., 1986; Nakashima et al., 1991; Park et al., 1991). The precise characterization of the true kinetic properties of opossum (d)ADO kinase must await isolation of the enzyme. research was supported by Grant No. 91/651A from the Department of Clinical Investigation, Fitzsimons Army Medical Center. We are grateful to Ms J. Grubaugh for the preparation of the illustrations, and to Ms C. Montoya for typing the manuscript. Acknowledgements--This

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