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Studies on the energy metabolism of opossum Didelphis Virginiana erythrocytes-IV. Red cells have low adenosine deaminase activity and high levels of deoxyadenosine nucleotides
Life Sciences, Vol. 44, pp. 963-970 Printed in the U.S.A.
Pergamon Press
STUDIES ON THE ENERGY M E T A B O L I S M OF O P O S S U M D I D E L P H I S V I R G I N I A N A ERYTHROCYTES-IV. RED CELLS HAVE LOW A D E N O S I N E D E A M I N A S E A C T I V I T Y AND HIGH LEVELS OF D E O X Y A D E N O S I N E N U C L E O T I D E S N.C.
Bethlenfalvay,
Department
E. C h a d w i c k *
and J.E.
Lima*
of Primary Care and Clinical I n v e s t i g a t i o n F i t z s i m o n s Army Medical Center Aurora, C o l o r a d o 80045 (Received in final form February 3, 1989) Summary
A l k a l i n e extracts of adult o p o s s u m red cells were used to d e t e r m i n e t r i p h o s p h a t e s of adenosine, d e o x y a d e n o s i n e and g u a n o s i n e by anion e x c h a n g e HPLC. Mean (nm/g Hg) ATP content of e r y t h r o c y t e s was 3713 and that of dATP 1913 (n = 12). S o n i c a t e s of red cells d e a m i n a t e d a d e n o s i n e (ADO) at a rate of 1.55 nm/mg Hg/h and d e o x y a d e n o s i n e (dADO) at 1.82 nm/mg Hg/h. dATP s y n t h e s i s from provided dADO was one order of m a g n i t u d e greater in o p o s s u m than in human e r y t h r o c y t e s at both low and high d A D O and Pi concentrations. During our current studies of the salvage and d e - n o v o s y n t h e s i s of purine n u c l e o t i d e s by o p o s s u m e r y t h r o c y t e s (in preparation) we have o b s e r v e d a s t r i k i n g l y altered HPLC pattern of red cell extracts, c h a r a c t e r i z e d by well d e f i n e d d o u b l e t s at p o s i t i o n s where a d e n o s i n e d i p h o s p h a t e (ADP) and a d e n o s i n e t r i p h o s p h a t e (ATP) usually elute. Such e r y t h r o c y t e purine n u c l e o t i d e pattern has, to date, not been d e s c r i b e d in either e u t h e r i a n (I) or meta t h e r i a n (2) species. Here we present e v i d e n c e of the p r e s e n c e of d e o x y a d e n o s i n e t r i p h o s p h a t e (dATP) in h a l f - m i l l i m o l a r quantities in o p o s s u m e r y t h r o c y t e s . We further show that red cells, but not white cells of this species have low a d e n o s i n e d e a m i n a s e (ADA) a c t i v i t y and p o s t u l a t e the e x i s t e n c e of high a f f i n i t y / h i g h a c t i v i t y kinase(s) which, in concert, provide for s p e c t a c u l a r a c c u m u l a t i o n of dATP in red cells. Materials
and M e t h o d s
O p o s s u m blood (15ml) was obtained as p r e v i o u s l y d e s c r i b e d (3). Nucleotides, d e o x y n u c l e o t i d e s , ribo and d e o x y r i b o n u c l e o sides, bases and deoxy c o f o r m y c i n were obtained from Sigma C h e m i c a l Co. (St. Louis, Mo). D e o x y a d e n o s i n e [8-~H], (18 Ci/mmole) was p u r c h a s e d from ICN R a d i o c h e m i c a l s (Irvine, Ca). ADO and dADO t r i p h o s p h a t e s : E r y t h r o c y t e s sedimented from whole blood were washed thrice in saline, removing the buffy coat each time. Cells (20-30%) were suspended in an electrolyte solution (basic buffer) c o n t a i n i n g (mmoles/liter) : 150 NaCI, 5 KCI, 1.5 MgCI2, 10 D - g l u c o s e and 100 TRIS-HCI buffer (pH 7.4). A l i q u o t s of these s u s p e n s i o n s were used for the 0024-3205/89 $3.00 + .00
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Opossum Red Cell dATP and ADA
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d e t e r m i n a t i o n of cell count, h e m o g l o b i n , and b a s e l i n e n u c l e o t i d e t r i p h o s p h a t e d e t e r m i n a t i o n s w i t h i n 45 min of c a r d i o c e n t h e s i s . ADA a c t i v i t y : E n z y m i c a c t i v i t y was i n v e s t i g a t e d by isoe l e c t r i c focusing (IEF) in red cell (RBC) and p e r i p h e r a l blood m o n o n u c l e a r cell (MNC) s o n i c a t e s and by r e v e r s e phase HPLC in RBC s o n i c a t e s p r o v i d e d to a known a m o u n t of substrate. After s e d i m e n t a t i o n in F i c o l l - D e x t r a n , RBC and MNC were washe~ thrice and then r e s u s p e n d e d in saline (RBC = 50%, MNC = 6-7x10" c e l l s / ml). The s u s p e n s i o n s were s o n i c a t e d and c e n t r i f u g e d for lh at Ixl0 b RPM and IEF was p e r f o r m e d as p r e v i o u s l y d e s c r i b e d (4)~ 20ui of s o n i c a t e s c o r r e s p o n d i n g to 1-1.5 mg Hg and i-1.2x10 MNC w e r e focused and the gel stained for ADA a c t i v i t y as d e s c r i b e d (5). For HPLC, the r e a c t i o n m i x t u r e (I ml) c o n t a i n e d ADO or d A D O (100 nmoles), EDTA (i umole) TRIS-HCI buffer (100 u m o l e s pH 7.4) and RBC s o n i c a t e c o r r e s p o n d i n g to 10-15 mg h e m o g l o b i n . After i n c u b a t i o n for 30 min at 37 ° C, the r e a c t i o n was term i n a t e d by i m m e r s i o n of the vessel into b o i l i n g water for 4 min, and u n r e a c t e d ADO or d A D O in the c l e a r e d s u p e r n a t a n t d e t e r m i n e d as d e s c r i b e d below. P r o d u c t i o n of d A T P from dADO: Both short (10 min) and long (120 min) i n c u b a t i o n s were p e r f o r m e d . For short incubations, p a c k e d RBC (200 ul) were s u s p e n d e d in 1 ml of basic buffer supp l e m e n t e d to c o n t a i n 1 u m o l e Pi and 0.55 n m o l e s [8-JH] d A D O (10 uCi). For the 2h i n c u b a t i o n , 200 ul of packed RBC were added to 800 ul of basic buffer s o l u t i o n s u p p l e m e n t e d to c o n t a i n 20 umoles of Pi and 180 nmoles of [8-~H] d A D O (167 u C i / u m o l e ) . Identical cell s u s p e n s i o n s w i t h o u t r a d i o l a b e l l e d dADO were used for the d e t e r m i n a t i o n of h e m a t o l o g i c p a r a m e t e r s at h o u r l y intervals. C e l l s were p e l l e t e d after the a p p r o p r i a t e period of inc u b a t i o n , w a s h e d thrice and r e s u s p e n d e d to original volume (i ml) in the b a s i c buffer s o l u t i o n and i m m e d i a t e l y e x t r a c t e d . Extracts not c h r o m a t o g r a p h e d i m m e d i a t e l y were kept at -70 ° C until analyzed. HPLC/Radiochromato@raphy: A l k a l i n e e x t r a c t s (6) were analyzed. The c h r o m a t o g r a p h i c s y s t e m (Waters) c o n s i s t e d of an 840 c h r o m a t o g r a p h y station, three model 510 pumps, a digital prof e s s i o n a l model 350 solvent p r o g r a m m e r , a model 710-B s a m p l e p r o c e s s o r , a TCM t e m p e r a t u r e control m o d u l e and a No. 490 programmable multi-wavelength detector. R a d i o a c t i v i t y was m e a s u r e d using a Flo-one beta model I-C flow d e t e c t o r e q u i p p e d with a 2500 ul flow-cell ( R a d i o m a t i c I n s t r u m e n t s , Addison, Ii, USA). The d e t e c t o r unit was c o n n e c t e d to the HPLC system in series e n a b l i n g the column e f f l u e n t to pass from the UV d e t e c t o r dir e c t l y to the r a d i o a c t i v i t y d e t e c t o r . M i n i - s c i n t (Radiomatic Instruments) liquid s c i n t i l l a t o r fluid was used for all a n a l y s e s in a ratio r e c o m m e n d e d by the m a n u f a c t u r e r . A p p a r e n t flow cell c o u n t i n g e f f i c i e n c i e s were c a l c u l a t e d as d e s c r i b e d e l s e w h e r e (7). S e p a r a t i o n of n u c l e o t i d e di and t r i p h o s p h a t e s was d o n e in a B e c k m a n U l t r a s i l AX (4.6x250mm) anion e x c h a n g e c o l u m n with 0.2M p h o s p h a t e buffer, pH 6.4, in an i s o c r a t i c m o d e at 19 ° C as d e s c r i b e d (8). Ribo and d e o x y r i b o n u c l e o s i d e s were s e p a r a t e d in a r e v e r s e - p h a s e W a t e r s u B o n d a p a k phenyl (3.6x300mm) c o l u m n with a m o b i l e phase c o n s i s t i n g of an a m m o n i u m a c e t a t e - m e t h a n o l a c e t o n i t r i l e t e t r a h y d r o f u r a n g r a d i e n t at 23 ° C as d e s c r i b e d (9). Peak i d e n t i t i e s were c o n f i r m e d by r e t e n t i o n time of and c o e l u tion with s t a n d a r d s , by e n z y m a t i c p e a k - s h i f t t e c h n i q u e s and by
Vol. 44, No. 14, 1989
Opossum Red Cell dATP and ADA
965
resistance of d e o x y n u c l e o t i d e s to o x i d a t i v e d e s t r u c t i o n by p e r i o d a t e (10). Total area integration of r a d i o a c t i v i t y (CPM) was p o s s i b l e using the Flo-9~e system m i c r o c o m p u t e r which can simultaneously quantitate [ ~ C ] and [3H] r a d i o a c t i v i t y . Results Nucleotide triphosphate (NTP) c o n t e n t of RBC: Anione x c h a n g e HPLC a n a l y s i s revealed a 2:1 ratio of ATP : dATP. Of 12 adult o p o s s u m s control values of NTP were (mean ± SEM nmoles/g Hg): 3713 ~ 46 ATP, 1913 + 71 dATP, and 1116 ~ 20 GTP. No dATP was seen in extracts of MNC (data not shown). ADA a c t i v i t y in RBC and MNC: At s u b s t r a t e concentration of 100 uM, ADA a c t i v i t y in sonicates of RBC d e t e r m i n e d . b y reversephase HPLC were (nM ADO or dADO d e a m i n a t e d / m g Hg/h ~ S E M ) : 1.54 ~ 0.09 ADO, 1.82 ~ 0.24 dADO (n=5). 10uM d - c o f o r m y c i n fully a b o l i s h e d ADA activity. These values are s l i g h t l y higher than those reported in horse (0.2nm), but an order of m a g n i t u d e below the a c t i v i t y (60nm) d e t e c t e d in human RBC (ii). Fig. 1 shows IEF patterns of s o n i c a t e s of human (H) o p o s s u m (O) RBC and MNC stained for ADA a c t i v i t y for 15 min.
Fig.
1
I s o e l e c t r i c focusing of human (H), o p o s s u m (O), red (RBC), and m o n o n u c l e a r (MNC) cell s o n i c a t e s followed by staining for ADA activity. A c t i v i t i e s in (H) RBC and MNC are similar, staining is less intense in (O) MNC and can barely be seen in (O) RBC. Hg = hemoglobin. pl of ADA is a p p r o x i m a t e l y 4.5.
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Opossum Red Cell dATP and ADA
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A representative radiochroma t o g r a m (n = 3) of human (A) and o p o s s u m (B) RBC incubated with 1 umgle Pi and 0.55 nmoles [8-~H] dADO (10 uCi) per ml suspension for 10 min. N u c l e o s i d e and d e o x y n u c l e o side t r i p h o s p h a t e s from an extract (50ui) c o r r e s p o n d i n g to 0.5 mg Hg were separated isocratically. Flow cell (2500 ul) counting e f f i c i e n c y is 4.5 - 4.8% under the cond i t i o n s used.
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ADA a c t i v i t y is c l e a r l y visible in s o n i c a t e s of both (H) and (O) MNC and of (H) RBC. O p o s s u m RBC sonicate on IEF reveals b a r e l y v i s i b l e ADA band.
a
P r o d u c t i o n of dATP from dADO in RBC: Fig. 2 depicts the inc o r p o r a t i o n of 0.55 nmoles of [8-3 H] dADO (10uCi)/ml cell susp e n s i o n into (A) human and (B) o p o s s u m RBC in 10 min. No detectable r a d i o a c t i v i t y is seen i n c o r p o r a t e d in the human material. This o b s e r v a t i o n c o n f i r m s data reported by others (12,13) that in human RBC s i g n i f i c a n t dATP formation in-vitro occurs only at h i g h s u b s t r a t e c o n c e n t r a t i o n and at u n p h y s i o l o g i c a l Pi levels. As shown in Fig. 3, at high (20 mM) Pi and (180 uM, 167 uCi/umole) [8-~H] dADO c o n c e n t r a t i o n , o p o s s u m RBC i n c o r p o r a t e d dADO into dATP one order of m a g n i t u d e greater than did human RBC (784 vs. 73 nmoles/g Hg/h [n=3], compare Fig. 3D and 3C). While ATP in human RBC (Fig. 3A and C) remains e s s e n t i a l l y u n c h a n g e d d u r i n g the 2 h incubation, ATP in o p o s s u m RBC d e c l i n e d to 23%, w h e r e a s dATP rose to 177% of control value (Fig. 3B and D). The above results o b t a i n e d using RBC of this m a r s u p i a l species having inherent low ADA a c t i v i t y are similar to those reported by others using in-vitro ADA inhibited human RBC (14). In that study a similar d e c l i n e of ATP a c c o m p a n i e d by the synthesis of d A T P was seen at high (20 mM) Pi and (0.2 - 2.0 mM) dADO concentrations. O p o s s u m RBC whose ADA was inhibited with deoxycoformycin (10 uM) were able to synthesize dATP (dADO 180 uM, Pi 20 mM) 1209 nmoles/g Hg/h compared to the synthesis of 310 nmoles/g Hg/h in ADA inhibited human RBC (values are the means of 3 d e t e r m i n a t i o n s ) .
Vol. 44, No. 14, 1989
Opossum Red Cell dATP and ADA
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A representative (n=3) r a d i o c h r o m a t o g r a m of human (A,C) and o p o s s u m (B,D) RBC incubated for 2 h with 20 umoles Pi and 180 nmoles [8-3H] dADO (167 uCi/uM) per ml suspension. ADO, dADO and GUO di and t r i p h o s p h a t e s from an extract (75 ul) c o r r e s p o n d i n g to 0.85 mg Hg were s e p a r a t e d isocratically. Flow cell (2500 ul) c o u n t i n g e f f i c i e n c y is 4.5 - 4.8% under the c o n d i t i o n s used. Hematolo@~: H e m a t o l o g i c p a r a m e t e r s obtained in whole blood, plasma e l e c t r o l y t e c o m p o s i t i o n and o s m o l a r i t y agreed c l o s e l y with values reported e l s e w h e r e (15-16)~ MNC in cell suspensions were c o n s i s t e n t l y below 8.7 x 10=/ul and p l a t e l e t s were not observed. C o n s i d e r a b l e lysis of o p o s s u m RBC occurs when incubated for 3 or more hours in isotonic media (17). To m a i n t a i n c o n s t a n c y of the number of m e t a b o l i z i n g RBC in suspensions, the use of a h y p e r t o n i c buffer was n e c e s s a r y (17). As Fig. 4 indicates, o p o s s u m RBC, HGB and MCH remain stable in such a buffer (460 mosM) w h e r e a s mean c o r p u s c u l a r volume (MCV) and h e m a t o c r i t (HCT) increase to 111% of control value. A corr e s p o n d i n g d e c l i n e to 89% of control value is seen to occur in mean c o r p u s c u l a r h e m o g l o b i n c o n c e n t r a t i o n (MCHC) after a 2 h incubation. Cells incubated for 2 h in a buffer (34g mosM) c o m p a r a b l e to plasma o s m o l a r i t y behaved as those s u s p e n d e d in a h y p e r o s m o l a r buffer, but with loss of cells. For the sake of c o m p a r a b i l i t y of the c o n d i t i o n s used in this study, and that d e s c r i b i n g the salvage and of the d e - n o v o synthesis of p u r i n e n u c l e o t i d e s requiring a 4 h incubation of red cells (in preparation), a h y p e r t o n i c buffer was used to m a i n t a i n the number of m e t a b o l i z i n g cells as c o n s t a n t as p o s s i b l e during incubation.
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Opossum Red Cell dATP and ADA
Vol. 44, No. 14, 1989
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C h a n g e s in h e m a t o l o g i c p a r a m e t e r s of o p o s s u m RBC incubated in a h y p e r t o n i c buffer (460 mosM) at 1 h (open) and at 2 h (hatchEd bars) from those at 0 time (RBC, 1.72 x 10V/ul; HGB, 3.8 g/dl; HCT, 15%; MCV, 68.3 fl; MCH, 22 uug; MCHC, 32 g/dl). All values are the means of six d e t e r m i n a t i o n s . Discussion The finding of d A T P in h a l f - m i l l i m o l a r q u a n t i t i e s in o p o s s u m RBC was s u r p r i s i n g . S i m i l a r dATP levels in u n t r e a t e d h u m a n s have so far only been o b s e r v e d in cases h a v i n g a type of inherited d e f i c i e n c y of ADA w h i c h results in severe i m m u n o d e f i c i e n c y d i s e a s e c h a r a c t e r i z e d by m a r k e d l y m p h o p e n i a and loss of both T and B l y m p h o c y t e f u n c t i o n s (12-18), or f o l l o w i n g t h e r a p e u t i c inh i b i t i o n of ADA with d e o x y c o f o r m y c i n as a t r e a t m e n t m o d a l i t y of lymphoproliferative malignancy. In the reported cases, inhibition of ADA in-vivo has been shown to result in a d r a m a t i c and p r o g r e s s i v e rise of RBC dADP and dATP c o n c o m i t a n t with a prec i p i t o u s d e c l i n e of RBC ATP and m u l t i p l e organ s y s t e m t o x i c i t y (19-21). The o p o s s u m s kept in our animal care facility cons i s t e n t l y had m o d e s t l y m p h o c y t o s i s and did not reveal any clinical signs s u s p i c i o u s of an i m m u n e - d e f i c i e n t state d u r i n g a 1218 m o n t h span of o b s e r v a t i o n . We o b s e r v e d RBC ATP and d A T P levels to remain fairly c o n s t a n t in individual animals on rep e a t e d s a m p l i n g of blood. Our p r e l i m i n a r y o b s e r v a t i o n s suggest that RBC of this species r e a d i l y "trap" d A T P b e c a u s e of a. low a c t i v i t y RBC ADA and b. kinase(s) m u c h more e f f i c i e n t than those p r e s e n t in human h e m a t o p o i e t i c tissues (22). The o b s e r v e d increase in MCV of o p o s s u m RBC s u s p e n d e d in h y p e r t o n i c b u f f e r s (17, this s t u d y and in p r e p a r a t i o n ) is in c o n t r a s t to the dehyd r a t i o n human cells e x p e r i e n c e in a similar e n v i r o n m e n t (23,24) and remains an u n e x p l a i n e d p h e n o m e n o n at present. Competition in d e o x y a d e n o s i n e - t r e a t e d A D A - i n h i b i t e d human RBC of d A D P with
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Opossum Red Cell dATP and ADA
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ADP for p h o s p h a t e and g l y c o l y t i c i n t e r m e d i a t e s has been d e m o n strated (14). The e x t e n t to w h i c h d A T P p a r t i c i p a t e s in cation and GSSG t r a n s p o r t in e r y t h r o c y t e s and the full c o n s e q u e n c e s of d A T P on the e n e r g y m e t a b o l i s m of o p o s s u m RBC now require further d e t a i l e d study. P r e c i s e d e f i n i t i o n of the k i n e t i c s of g l y c o lytic and of p u r i n e m e t a b o l i z i n g e n z y m e s of the o p o s s u m m a y provide for these insights. Acknowled@ements This r e s e a r c h was w h o l l y s u p p o r t e d by grant 80/650 from the D e p a r t m e n t of C l i n i c a l I n v e s t i g a t i o n , FAMC. We thank Drs. J. W h i t e and P. O'Barr for m a n y helpful d i s c u s s i o n s . Special tribute is due to Ms. C. M o n t o y a for typing the m a n u s c r i p t and to Ms. K. W y a t t for the p r e p a r a t i o n of the figures. "The views of the authors do not p u r p o r t to reflect the p o s i t i o n s of the A r m y or the D e p a r t m e n t of D e f e n s e . " References I. 2. 3. 4. 5. 6.
7. 8. 9. 10. ii. 12.
13. 14.
15. 16. 17. 18.
P.R. BROWN, R.P. AGARWAL, J. GELL and R.E. PARKS, JR., Comp. Biochem. Physiol. 43B 891-904 (1972). M. MCKEAG, P.R. BROWN, and J.D. SALLIS, Comp. Biochem. Physiol. 70B 541-547 (1981). N.C. B E T H L E N F A L V A Y , M.R. W A T E R M A N , J.E. LIMA and T. W A L D R U P , Comp. Biochem. Physiol. 75A 635-639 (1983). N.C. B E T H L E N F A L V A Y , M.R. W A T E R M A N , J.E. LIMA and T. W A L D R U P , Comp. Biochem. Physiol. 73B 591-594 (1982). N. SPENCER, D.A. H O P K I N S O N and H. HARRIS, Ann. Hum. Genet. 32 9-14 (1968). V. STOCCHI, L. C U C C H I R I A N I , M. MAGNAMI, L. C H I A R A N T I N I , P. PALMA and G. C R E S C E N T I N I , Anal. Biochem. 146 118-124 (1985). H.K. W E B S T E R and J.M. WHAUN, J. Chromat. 209 283-292 (1981). F. AREZZO, Anal. Biochem. 160 57-64 (1987). G.S. MORRIS and H.E. SIMMONDS, J. Chromat. 344 101-113 (1985). C. G A R R E T T and D.V. SANTI, Anal, Biochem. 99 268-273 (1979). G.J. PETERS, A. O O S T E R H O F and J.H. V E E R K A M P , Int. J. Biochem. 13 445-455 (1981). M.S. H E R S H F I E L D , R.H. BUCKLEY, M.L. G R E E N B E R G , A.L. MELTON, R. SCHITT, C. HARTEN, J. KURTZBERG, M.L. MARKERT, R.H. K O B A Y A S H I , A.L. K O B A Y A S H I and A. A B U C H O W S K I , N. Eng. J. Med. 316 589-596 (1987). O. PERRETT, A. SHOBOTA, H.A. SIMMONDS and K. H U G H - J O N E S , Biosci. Rep. 1 933-944 (1981). C.A. KOLLER, E.P. O R R I N G E R , L.R. B E R K O W I T Z and A.T. MULHERN, Progress in Clin. and Biol. Res. 165 p. 227 (1984). A. HAYS JR. and F.M. LOEW, J . A . V . M . A . 153 800-802 (1968). E.H. T I M M O N S and P.A. MARGUES, Lab. Anlm. Care 19 342-344 (1969) . N.C. B E T H L E N F A L V A Y , J.E. LIMA, E. C H A D W I C K and I. STEWART, Comp. Biochem. Physiool. 89A 119-124 (1988). D. PERRETT, A. SHOBOTA, H.A. S I M M O N D S and K. H U G H - J O N E S , Biosci. Rep. 1 933-944 (1981).
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19. 20. 21.
22. 23. 24.
Opossum Red Cell dAPT and ADA
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M.F.E. SIAW, B.S. M I T C H E L L , C.A. KOLLER, M.S. C O L E M A N and J.J. HUTTON, Proc. Nat. Acad. Sci. USA 77 6 1 5 7 - 6 1 6 1 (1980). C.A. K O L L E R and B.S. M I T C H E L L , Cancer Res. 43 140-1414 (1983). A.S. SPIERS, D. MOORE, P.A. C A S S I L E T A , D.P. H A R R I N G T O N , F.J. C U M M I N G S , R.S. NEIMAN, J.M. B E N N E T T and M.J. O ' C O N N E L L , N. Eng. J. Med. 316 825-830 (1987). D.A. CARSON, J. KAYE and J.E. S E E G E M I L L E R , Proc. Nat. Acad. Sci. (USA) 74 5 6 7 7 - 5 6 8 1 (1977). E. PONDER, H e m o l y s i s and related p h e n o m e n a . Grune and S t r a t t o n , New York (1948). N. M O H A N D A S and S.B. SHOHET, C u r r e n t T o p i c s in Hematol. 1 71-125 (1978).
Pergamon Press
STUDIES ON THE ENERGY M E T A B O L I S M OF O P O S S U M D I D E L P H I S V I R G I N I A N A ERYTHROCYTES-IV. RED CELLS HAVE LOW A D E N O S I N E D E A M I N A S E A C T I V I T Y AND HIGH LEVELS OF D E O X Y A D E N O S I N E N U C L E O T I D E S N.C.
Bethlenfalvay,
Department
E. C h a d w i c k *
and J.E.
Lima*
of Primary Care and Clinical I n v e s t i g a t i o n F i t z s i m o n s Army Medical Center Aurora, C o l o r a d o 80045 (Received in final form February 3, 1989) Summary
A l k a l i n e extracts of adult o p o s s u m red cells were used to d e t e r m i n e t r i p h o s p h a t e s of adenosine, d e o x y a d e n o s i n e and g u a n o s i n e by anion e x c h a n g e HPLC. Mean (nm/g Hg) ATP content of e r y t h r o c y t e s was 3713 and that of dATP 1913 (n = 12). S o n i c a t e s of red cells d e a m i n a t e d a d e n o s i n e (ADO) at a rate of 1.55 nm/mg Hg/h and d e o x y a d e n o s i n e (dADO) at 1.82 nm/mg Hg/h. dATP s y n t h e s i s from provided dADO was one order of m a g n i t u d e greater in o p o s s u m than in human e r y t h r o c y t e s at both low and high d A D O and Pi concentrations. During our current studies of the salvage and d e - n o v o s y n t h e s i s of purine n u c l e o t i d e s by o p o s s u m e r y t h r o c y t e s (in preparation) we have o b s e r v e d a s t r i k i n g l y altered HPLC pattern of red cell extracts, c h a r a c t e r i z e d by well d e f i n e d d o u b l e t s at p o s i t i o n s where a d e n o s i n e d i p h o s p h a t e (ADP) and a d e n o s i n e t r i p h o s p h a t e (ATP) usually elute. Such e r y t h r o c y t e purine n u c l e o t i d e pattern has, to date, not been d e s c r i b e d in either e u t h e r i a n (I) or meta t h e r i a n (2) species. Here we present e v i d e n c e of the p r e s e n c e of d e o x y a d e n o s i n e t r i p h o s p h a t e (dATP) in h a l f - m i l l i m o l a r quantities in o p o s s u m e r y t h r o c y t e s . We further show that red cells, but not white cells of this species have low a d e n o s i n e d e a m i n a s e (ADA) a c t i v i t y and p o s t u l a t e the e x i s t e n c e of high a f f i n i t y / h i g h a c t i v i t y kinase(s) which, in concert, provide for s p e c t a c u l a r a c c u m u l a t i o n of dATP in red cells. Materials
and M e t h o d s
O p o s s u m blood (15ml) was obtained as p r e v i o u s l y d e s c r i b e d (3). Nucleotides, d e o x y n u c l e o t i d e s , ribo and d e o x y r i b o n u c l e o sides, bases and deoxy c o f o r m y c i n were obtained from Sigma C h e m i c a l Co. (St. Louis, Mo). D e o x y a d e n o s i n e [8-~H], (18 Ci/mmole) was p u r c h a s e d from ICN R a d i o c h e m i c a l s (Irvine, Ca). ADO and dADO t r i p h o s p h a t e s : E r y t h r o c y t e s sedimented from whole blood were washed thrice in saline, removing the buffy coat each time. Cells (20-30%) were suspended in an electrolyte solution (basic buffer) c o n t a i n i n g (mmoles/liter) : 150 NaCI, 5 KCI, 1.5 MgCI2, 10 D - g l u c o s e and 100 TRIS-HCI buffer (pH 7.4). A l i q u o t s of these s u s p e n s i o n s were used for the 0024-3205/89 $3.00 + .00
964
Opossum Red Cell dATP and ADA
Vol. 44, No. 14, 1989
d e t e r m i n a t i o n of cell count, h e m o g l o b i n , and b a s e l i n e n u c l e o t i d e t r i p h o s p h a t e d e t e r m i n a t i o n s w i t h i n 45 min of c a r d i o c e n t h e s i s . ADA a c t i v i t y : E n z y m i c a c t i v i t y was i n v e s t i g a t e d by isoe l e c t r i c focusing (IEF) in red cell (RBC) and p e r i p h e r a l blood m o n o n u c l e a r cell (MNC) s o n i c a t e s and by r e v e r s e phase HPLC in RBC s o n i c a t e s p r o v i d e d to a known a m o u n t of substrate. After s e d i m e n t a t i o n in F i c o l l - D e x t r a n , RBC and MNC were washe~ thrice and then r e s u s p e n d e d in saline (RBC = 50%, MNC = 6-7x10" c e l l s / ml). The s u s p e n s i o n s were s o n i c a t e d and c e n t r i f u g e d for lh at Ixl0 b RPM and IEF was p e r f o r m e d as p r e v i o u s l y d e s c r i b e d (4)~ 20ui of s o n i c a t e s c o r r e s p o n d i n g to 1-1.5 mg Hg and i-1.2x10 MNC w e r e focused and the gel stained for ADA a c t i v i t y as d e s c r i b e d (5). For HPLC, the r e a c t i o n m i x t u r e (I ml) c o n t a i n e d ADO or d A D O (100 nmoles), EDTA (i umole) TRIS-HCI buffer (100 u m o l e s pH 7.4) and RBC s o n i c a t e c o r r e s p o n d i n g to 10-15 mg h e m o g l o b i n . After i n c u b a t i o n for 30 min at 37 ° C, the r e a c t i o n was term i n a t e d by i m m e r s i o n of the vessel into b o i l i n g water for 4 min, and u n r e a c t e d ADO or d A D O in the c l e a r e d s u p e r n a t a n t d e t e r m i n e d as d e s c r i b e d below. P r o d u c t i o n of d A T P from dADO: Both short (10 min) and long (120 min) i n c u b a t i o n s were p e r f o r m e d . For short incubations, p a c k e d RBC (200 ul) were s u s p e n d e d in 1 ml of basic buffer supp l e m e n t e d to c o n t a i n 1 u m o l e Pi and 0.55 n m o l e s [8-JH] d A D O (10 uCi). For the 2h i n c u b a t i o n , 200 ul of packed RBC were added to 800 ul of basic buffer s o l u t i o n s u p p l e m e n t e d to c o n t a i n 20 umoles of Pi and 180 nmoles of [8-~H] d A D O (167 u C i / u m o l e ) . Identical cell s u s p e n s i o n s w i t h o u t r a d i o l a b e l l e d dADO were used for the d e t e r m i n a t i o n of h e m a t o l o g i c p a r a m e t e r s at h o u r l y intervals. C e l l s were p e l l e t e d after the a p p r o p r i a t e period of inc u b a t i o n , w a s h e d thrice and r e s u s p e n d e d to original volume (i ml) in the b a s i c buffer s o l u t i o n and i m m e d i a t e l y e x t r a c t e d . Extracts not c h r o m a t o g r a p h e d i m m e d i a t e l y were kept at -70 ° C until analyzed. HPLC/Radiochromato@raphy: A l k a l i n e e x t r a c t s (6) were analyzed. The c h r o m a t o g r a p h i c s y s t e m (Waters) c o n s i s t e d of an 840 c h r o m a t o g r a p h y station, three model 510 pumps, a digital prof e s s i o n a l model 350 solvent p r o g r a m m e r , a model 710-B s a m p l e p r o c e s s o r , a TCM t e m p e r a t u r e control m o d u l e and a No. 490 programmable multi-wavelength detector. R a d i o a c t i v i t y was m e a s u r e d using a Flo-one beta model I-C flow d e t e c t o r e q u i p p e d with a 2500 ul flow-cell ( R a d i o m a t i c I n s t r u m e n t s , Addison, Ii, USA). The d e t e c t o r unit was c o n n e c t e d to the HPLC system in series e n a b l i n g the column e f f l u e n t to pass from the UV d e t e c t o r dir e c t l y to the r a d i o a c t i v i t y d e t e c t o r . M i n i - s c i n t (Radiomatic Instruments) liquid s c i n t i l l a t o r fluid was used for all a n a l y s e s in a ratio r e c o m m e n d e d by the m a n u f a c t u r e r . A p p a r e n t flow cell c o u n t i n g e f f i c i e n c i e s were c a l c u l a t e d as d e s c r i b e d e l s e w h e r e (7). S e p a r a t i o n of n u c l e o t i d e di and t r i p h o s p h a t e s was d o n e in a B e c k m a n U l t r a s i l AX (4.6x250mm) anion e x c h a n g e c o l u m n with 0.2M p h o s p h a t e buffer, pH 6.4, in an i s o c r a t i c m o d e at 19 ° C as d e s c r i b e d (8). Ribo and d e o x y r i b o n u c l e o s i d e s were s e p a r a t e d in a r e v e r s e - p h a s e W a t e r s u B o n d a p a k phenyl (3.6x300mm) c o l u m n with a m o b i l e phase c o n s i s t i n g of an a m m o n i u m a c e t a t e - m e t h a n o l a c e t o n i t r i l e t e t r a h y d r o f u r a n g r a d i e n t at 23 ° C as d e s c r i b e d (9). Peak i d e n t i t i e s were c o n f i r m e d by r e t e n t i o n time of and c o e l u tion with s t a n d a r d s , by e n z y m a t i c p e a k - s h i f t t e c h n i q u e s and by
Vol. 44, No. 14, 1989
Opossum Red Cell dATP and ADA
965
resistance of d e o x y n u c l e o t i d e s to o x i d a t i v e d e s t r u c t i o n by p e r i o d a t e (10). Total area integration of r a d i o a c t i v i t y (CPM) was p o s s i b l e using the Flo-9~e system m i c r o c o m p u t e r which can simultaneously quantitate [ ~ C ] and [3H] r a d i o a c t i v i t y . Results Nucleotide triphosphate (NTP) c o n t e n t of RBC: Anione x c h a n g e HPLC a n a l y s i s revealed a 2:1 ratio of ATP : dATP. Of 12 adult o p o s s u m s control values of NTP were (mean ± SEM nmoles/g Hg): 3713 ~ 46 ATP, 1913 + 71 dATP, and 1116 ~ 20 GTP. No dATP was seen in extracts of MNC (data not shown). ADA a c t i v i t y in RBC and MNC: At s u b s t r a t e concentration of 100 uM, ADA a c t i v i t y in sonicates of RBC d e t e r m i n e d . b y reversephase HPLC were (nM ADO or dADO d e a m i n a t e d / m g Hg/h ~ S E M ) : 1.54 ~ 0.09 ADO, 1.82 ~ 0.24 dADO (n=5). 10uM d - c o f o r m y c i n fully a b o l i s h e d ADA activity. These values are s l i g h t l y higher than those reported in horse (0.2nm), but an order of m a g n i t u d e below the a c t i v i t y (60nm) d e t e c t e d in human RBC (ii). Fig. 1 shows IEF patterns of s o n i c a t e s of human (H) o p o s s u m (O) RBC and MNC stained for ADA a c t i v i t y for 15 min.
Fig.
1
I s o e l e c t r i c focusing of human (H), o p o s s u m (O), red (RBC), and m o n o n u c l e a r (MNC) cell s o n i c a t e s followed by staining for ADA activity. A c t i v i t i e s in (H) RBC and MNC are similar, staining is less intense in (O) MNC and can barely be seen in (O) RBC. Hg = hemoglobin. pl of ADA is a p p r o x i m a t e l y 4.5.
966
Opossum Red Cell dATP and ADA
Vol. 44, No. 14, 1989
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A representative radiochroma t o g r a m (n = 3) of human (A) and o p o s s u m (B) RBC incubated with 1 umgle Pi and 0.55 nmoles [8-~H] dADO (10 uCi) per ml suspension for 10 min. N u c l e o s i d e and d e o x y n u c l e o side t r i p h o s p h a t e s from an extract (50ui) c o r r e s p o n d i n g to 0.5 mg Hg were separated isocratically. Flow cell (2500 ul) counting e f f i c i e n c y is 4.5 - 4.8% under the cond i t i o n s used.
4'o
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ADA a c t i v i t y is c l e a r l y visible in s o n i c a t e s of both (H) and (O) MNC and of (H) RBC. O p o s s u m RBC sonicate on IEF reveals b a r e l y v i s i b l e ADA band.
a
P r o d u c t i o n of dATP from dADO in RBC: Fig. 2 depicts the inc o r p o r a t i o n of 0.55 nmoles of [8-3 H] dADO (10uCi)/ml cell susp e n s i o n into (A) human and (B) o p o s s u m RBC in 10 min. No detectable r a d i o a c t i v i t y is seen i n c o r p o r a t e d in the human material. This o b s e r v a t i o n c o n f i r m s data reported by others (12,13) that in human RBC s i g n i f i c a n t dATP formation in-vitro occurs only at h i g h s u b s t r a t e c o n c e n t r a t i o n and at u n p h y s i o l o g i c a l Pi levels. As shown in Fig. 3, at high (20 mM) Pi and (180 uM, 167 uCi/umole) [8-~H] dADO c o n c e n t r a t i o n , o p o s s u m RBC i n c o r p o r a t e d dADO into dATP one order of m a g n i t u d e greater than did human RBC (784 vs. 73 nmoles/g Hg/h [n=3], compare Fig. 3D and 3C). While ATP in human RBC (Fig. 3A and C) remains e s s e n t i a l l y u n c h a n g e d d u r i n g the 2 h incubation, ATP in o p o s s u m RBC d e c l i n e d to 23%, w h e r e a s dATP rose to 177% of control value (Fig. 3B and D). The above results o b t a i n e d using RBC of this m a r s u p i a l species having inherent low ADA a c t i v i t y are similar to those reported by others using in-vitro ADA inhibited human RBC (14). In that study a similar d e c l i n e of ATP a c c o m p a n i e d by the synthesis of d A T P was seen at high (20 mM) Pi and (0.2 - 2.0 mM) dADO concentrations. O p o s s u m RBC whose ADA was inhibited with deoxycoformycin (10 uM) were able to synthesize dATP (dADO 180 uM, Pi 20 mM) 1209 nmoles/g Hg/h compared to the synthesis of 310 nmoles/g Hg/h in ADA inhibited human RBC (values are the means of 3 d e t e r m i n a t i o n s ) .
Vol. 44, No. 14, 1989
Opossum Red Cell dATP and ADA
967
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A representative (n=3) r a d i o c h r o m a t o g r a m of human (A,C) and o p o s s u m (B,D) RBC incubated for 2 h with 20 umoles Pi and 180 nmoles [8-3H] dADO (167 uCi/uM) per ml suspension. ADO, dADO and GUO di and t r i p h o s p h a t e s from an extract (75 ul) c o r r e s p o n d i n g to 0.85 mg Hg were s e p a r a t e d isocratically. Flow cell (2500 ul) c o u n t i n g e f f i c i e n c y is 4.5 - 4.8% under the c o n d i t i o n s used. Hematolo@~: H e m a t o l o g i c p a r a m e t e r s obtained in whole blood, plasma e l e c t r o l y t e c o m p o s i t i o n and o s m o l a r i t y agreed c l o s e l y with values reported e l s e w h e r e (15-16)~ MNC in cell suspensions were c o n s i s t e n t l y below 8.7 x 10=/ul and p l a t e l e t s were not observed. C o n s i d e r a b l e lysis of o p o s s u m RBC occurs when incubated for 3 or more hours in isotonic media (17). To m a i n t a i n c o n s t a n c y of the number of m e t a b o l i z i n g RBC in suspensions, the use of a h y p e r t o n i c buffer was n e c e s s a r y (17). As Fig. 4 indicates, o p o s s u m RBC, HGB and MCH remain stable in such a buffer (460 mosM) w h e r e a s mean c o r p u s c u l a r volume (MCV) and h e m a t o c r i t (HCT) increase to 111% of control value. A corr e s p o n d i n g d e c l i n e to 89% of control value is seen to occur in mean c o r p u s c u l a r h e m o g l o b i n c o n c e n t r a t i o n (MCHC) after a 2 h incubation. Cells incubated for 2 h in a buffer (34g mosM) c o m p a r a b l e to plasma o s m o l a r i t y behaved as those s u s p e n d e d in a h y p e r o s m o l a r buffer, but with loss of cells. For the sake of c o m p a r a b i l i t y of the c o n d i t i o n s used in this study, and that d e s c r i b i n g the salvage and of the d e - n o v o synthesis of p u r i n e n u c l e o t i d e s requiring a 4 h incubation of red cells (in preparation), a h y p e r t o n i c buffer was used to m a i n t a i n the number of m e t a b o l i z i n g cells as c o n s t a n t as p o s s i b l e during incubation.
968
Opossum Red Cell dATP and ADA
Vol. 44, No. 14, 1989
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MCHC
4
C h a n g e s in h e m a t o l o g i c p a r a m e t e r s of o p o s s u m RBC incubated in a h y p e r t o n i c buffer (460 mosM) at 1 h (open) and at 2 h (hatchEd bars) from those at 0 time (RBC, 1.72 x 10V/ul; HGB, 3.8 g/dl; HCT, 15%; MCV, 68.3 fl; MCH, 22 uug; MCHC, 32 g/dl). All values are the means of six d e t e r m i n a t i o n s . Discussion The finding of d A T P in h a l f - m i l l i m o l a r q u a n t i t i e s in o p o s s u m RBC was s u r p r i s i n g . S i m i l a r dATP levels in u n t r e a t e d h u m a n s have so far only been o b s e r v e d in cases h a v i n g a type of inherited d e f i c i e n c y of ADA w h i c h results in severe i m m u n o d e f i c i e n c y d i s e a s e c h a r a c t e r i z e d by m a r k e d l y m p h o p e n i a and loss of both T and B l y m p h o c y t e f u n c t i o n s (12-18), or f o l l o w i n g t h e r a p e u t i c inh i b i t i o n of ADA with d e o x y c o f o r m y c i n as a t r e a t m e n t m o d a l i t y of lymphoproliferative malignancy. In the reported cases, inhibition of ADA in-vivo has been shown to result in a d r a m a t i c and p r o g r e s s i v e rise of RBC dADP and dATP c o n c o m i t a n t with a prec i p i t o u s d e c l i n e of RBC ATP and m u l t i p l e organ s y s t e m t o x i c i t y (19-21). The o p o s s u m s kept in our animal care facility cons i s t e n t l y had m o d e s t l y m p h o c y t o s i s and did not reveal any clinical signs s u s p i c i o u s of an i m m u n e - d e f i c i e n t state d u r i n g a 1218 m o n t h span of o b s e r v a t i o n . We o b s e r v e d RBC ATP and d A T P levels to remain fairly c o n s t a n t in individual animals on rep e a t e d s a m p l i n g of blood. Our p r e l i m i n a r y o b s e r v a t i o n s suggest that RBC of this species r e a d i l y "trap" d A T P b e c a u s e of a. low a c t i v i t y RBC ADA and b. kinase(s) m u c h more e f f i c i e n t than those p r e s e n t in human h e m a t o p o i e t i c tissues (22). The o b s e r v e d increase in MCV of o p o s s u m RBC s u s p e n d e d in h y p e r t o n i c b u f f e r s (17, this s t u d y and in p r e p a r a t i o n ) is in c o n t r a s t to the dehyd r a t i o n human cells e x p e r i e n c e in a similar e n v i r o n m e n t (23,24) and remains an u n e x p l a i n e d p h e n o m e n o n at present. Competition in d e o x y a d e n o s i n e - t r e a t e d A D A - i n h i b i t e d human RBC of d A D P with
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ADP for p h o s p h a t e and g l y c o l y t i c i n t e r m e d i a t e s has been d e m o n strated (14). The e x t e n t to w h i c h d A T P p a r t i c i p a t e s in cation and GSSG t r a n s p o r t in e r y t h r o c y t e s and the full c o n s e q u e n c e s of d A T P on the e n e r g y m e t a b o l i s m of o p o s s u m RBC now require further d e t a i l e d study. P r e c i s e d e f i n i t i o n of the k i n e t i c s of g l y c o lytic and of p u r i n e m e t a b o l i z i n g e n z y m e s of the o p o s s u m m a y provide for these insights. Acknowled@ements This r e s e a r c h was w h o l l y s u p p o r t e d by grant 80/650 from the D e p a r t m e n t of C l i n i c a l I n v e s t i g a t i o n , FAMC. We thank Drs. J. W h i t e and P. O'Barr for m a n y helpful d i s c u s s i o n s . Special tribute is due to Ms. C. M o n t o y a for typing the m a n u s c r i p t and to Ms. K. W y a t t for the p r e p a r a t i o n of the figures. "The views of the authors do not p u r p o r t to reflect the p o s i t i o n s of the A r m y or the D e p a r t m e n t of D e f e n s e . " References I. 2. 3. 4. 5. 6.
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M.F.E. SIAW, B.S. M I T C H E L L , C.A. KOLLER, M.S. C O L E M A N and J.J. HUTTON, Proc. Nat. Acad. Sci. USA 77 6 1 5 7 - 6 1 6 1 (1980). C.A. K O L L E R and B.S. M I T C H E L L , Cancer Res. 43 140-1414 (1983). A.S. SPIERS, D. MOORE, P.A. C A S S I L E T A , D.P. H A R R I N G T O N , F.J. C U M M I N G S , R.S. NEIMAN, J.M. B E N N E T T and M.J. O ' C O N N E L L , N. Eng. J. Med. 316 825-830 (1987). D.A. CARSON, J. KAYE and J.E. S E E G E M I L L E R , Proc. Nat. Acad. Sci. (USA) 74 5 6 7 7 - 5 6 8 1 (1977). E. PONDER, H e m o l y s i s and related p h e n o m e n a . Grune and S t r a t t o n , New York (1948). N. M O H A N D A S and S.B. SHOHET, C u r r e n t T o p i c s in Hematol. 1 71-125 (1978).