Adenine nucleotides in erythrocytes of horses, Equis cabalus

Comp. Biochem. Physiol., Vol. 67B, pp. 205 to 211

0305-0491/80/0901-0205502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

ADENINE NUCLEOTIDES IN ERYTHROCYTES OF HORSES, EQUIS CABALUS* NANCY S. MAGNUSONt and LANCE E. PERRYMAN Department of Veterinary Microbiology and Pathology, Washington State University Pullman, WA 99164, U.S.A.

(Received 22 February 1980) Abstract--1. Concentrations of adenine nucleotides in erythrocytes from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony) were measured. 2. No significant differences were detected among the various breeds. 3. Erythrocytes from adult horses contained 14.7 + 2.1 nmols ATP and 2.7 + 0.6 nmols ADP/109 cells. AMP was usually not detectable (<0.5 nmols/109 cells). 4. Erythrocytes from foals contained half the concentration of these nucleotides. 5. Effects of phosphate concentration on adenosine and deoxyadenosine metabolism were studied.

were allowed to sit undisturbed for 20 min. At the end of this time, erythrocytes had settled to the bottom of the container following rouleau formation, and the leukocyteand platelet-rich plasma was removed. This procedure removed between 90-99~o of the nucleated cells. Erythrocytes were then washed three times with sterile Hank's Balanced Salt Solution (HBSS). Aliquots of 5.0 ml of packed washed erythrocytes were either extracted for nucleotide analysis or suspended in 45 ml of HBSS or 50 mM phosphate buffer (potassium phosphate, 50mM, pH 7.4, MgSO4 2raM; NaCI, 80mM; and glucose, 10mM) with or without 0.5mM adenosine, and incubated at 30°C in a shaking water bath for 1, 3 or 5 hr. An incubation temperature of 30°C was selected to facilitate comparison with previous work (Parks et al., 1975; Agarwal et al., 1976; Magnuson & Perryman, 1979). Incubation was terminated by washing the cells three times with cold (2°C) incubation buffer followed by extraction of nucleotides for analysis.

INTRODUCTION Altered nucleotide metabolism has been detected in erythrocytes from people with Duchenne and myotonic muscular dystrophy (Solomons et al., 1977), Lesch-Nyhan syndrome (Greene et al., 1970; Brown et al,, 1979), immunodeficiency associated with purine nucleoside phosphorylase deficiency (Cohen et al., 1976; Cohen et al., 1978), and severe combined immunodeficiency (CID) associated with adenosine deaminase deficiency (Agarwal et al., 1976; Mills et al., 1976; Goldblum et al., 1976; Cohen et al., 1978). Recently we obtained evidence of altered purine metabolism in horses with C I D (Perryman et al., 1979; Magnuson & Perryman, 1979a, b), a fatal genetic disorder characterized by an absence of functional B and T lymphocytes (Perryman, 1979). Differences observed in C I D horses included (1) altered distributions of adenine nucleotides in adenosineincubated erythrocytes, and (2) increased sensitivity of PHA-stimulated lymphocytes to the inhibitory effects of adenosine. These observations prompted an analysis of adenine nucleotide concentrations in normal horse erythrocytes and of some experimental parameters which influence these concentrations. MATERIALS AND METHODS

Animals Four breeds of horses (Equis cabalus) were used in these experiments: Shetland ponies, 1 month to 13 yr old; Arabians, 1 month to 16 yr old; Thoroughbreds, 5 to 10 yr old; and Quarter Horses, 5 to 10 yr old.

Isolation oferythrocytes Blood samples were collected in sterile heparin. Samples * Supported in part by PHS Grant No. HD 08886 from the Institute for Child Health and Human Development, PHS Immunology Training Program Grant No. AI 07025, and the Morris Animal Foundation. t To whom reprint requests should be sent.

Extraction of whole blood and erythrocytes for nucleotide analysis by HPLC Prior to extraction an aliquot of cells or whole blood was removed and the number of erythrocytes determined using a Coulter counter, Model 2F. For extraction, 10 ml of whole blood or 5 ml of packed erythrocytes were disrupted in a tissue homogenizer with cold 12Vo trichloroacetic acid (TCA). The supernatant was collected, the TCA removed by the two-phase extraction method described by Khym (1975), lyophilized, and stored at -20°C. For chromatography, the lyophilized extracts were dissolved in 0.4 ml of 0.03 M sodium phosphate buffer, pH 4.0, and filtered through a 0.22/~m millipore filter. Recovery reproducibility for extraction and chromatography procedures was determined on 6 aliquots from the same sample of blood. The coefficient of variation for these samples was 5.6~o. High-pressure liquid chromatography All samples were analyzed on a Waters liquid chromatograph at ambient temperature using a Whatman Partisil SAX 10/25 column, a linear gradient of 0.03-0.75M sodium phosphate, pH 4.0, and a flow rate of 1.0 ml/min. Peaks were identified by cochromatography with known standards, ratios of optical densities at 260 and 280 rim, or by the enzyme peak shift technique (Brown, 1970). Peak areas were quantitated by multiplying the peak height times width at half-height.

205

NANCY S. MAGNUSON and LANCE E. PERRYMAN

206

Internal standard Four hundred nmols of CTP were added to a 5 ml suspension of erythrocytes from an adult horse. Immediately thereafter, TCA was added to the suspension to give a final concentration of 12% and the nucleotides extracted from the erythrocytes as previously described. The lyophilized extract was reconstituted in 0.4 ml 0.03 M sodium phosphate and 20 #1 were chromatographed as previously described. The peak area in the chromatogram resulting from the recovered CTP was compared to the peak area obtained when 20nmols of CTP were chromatographed and the percent recovery determined. It has been previously demonstrated that percent recovery of nucleotides decreases as the complexity of the suspending medium increases (Van Haverbeke & Brown, 1978). For example, nucleotide recovery from erythrocytes is significantly less than is nucleotide recovery from water. Although recoveries will vary depending on the medium complexity, from the same medium there appears to be little difference between the recoveries of pyrimidine and purine nucleotides (Van Haverbeke & Brown, 1978). Therefore, the recovery of CTP in these experiments was assumed to be representative of recovery for all nucleotides.

Chemicals Nucleotides were obtained from Sigma Chemical Co.

Statistical analysis Differences between means were analyzed by Student's t-test for either unpaired or paired (paired t-test) variables (Steel & Torrie, 1960). RESULTS

Nucleotides in erythrocyte extracts The elution pattern for a standard solution of nucleotides is illustrated in Fig. 1. Figure 2A shows the typical pattern of nucleotides extracted from erythrocytes of an adult Arabian horse. Adenosine diphosphate and A T P were easily detected and readily identified by cochromatography with standards and by determining ratios of optical densities at 260 and 280nm. Other peaks present in quantities easily detected occurred in the mono- and diphosphate

1.61 g

g ;

Efficiency of nucleotide extraction To determine the average recovery of nucleotides after procedural losses, CTP was added to erythrocytes of adult horses just prior to extraction with TCA. The recovery of C T P from extracted erythrocytes in six separate experiments was 63.3 + 8.4~o. All subsequent values reported in this study have been corrected for procedural losses and should represent more closely the actual concentration of nucleotides found in equine erythrocytes.

Concentrations of ADP and A TP in, erythrocytes from horses Eight samples collected on different days during a 30 day period from a Shetland pony contained A D P and ATP concentrations that ranged from 2.7 to 4.4 and 12.0 to 19.8 nmols/109 erythrocytes, respectively. The mean values (ADP, 3.4 + 0.7 nmols/109 erythrocytes and ATP, 15.4 + 2.8 nmols/109 erythrocytes) for this pony were not significantly different from those obtained for 10 different Shetland ponies (Table 1). Adenine nucleotide concentrations in erythrocytes have been shown to differ markedly between various species (Brown et al., 1972; Parks et al., 1973). However, in two different but closely related species of baboons, the concentrations were similar (Brown et aL, 1972). To determine if differences exist among

1

ATP

GMP

~,

o

regions of the chromatograms. However, these peaks did not cochromatograph with any of the commercially available nucleotides and, therefore, have not been definitively identified. Adenosine monophosphate was usually present in quantities too small to detect. Figure 2B shows a chromatogram typical for nucleotides extracted from erythrocytes of foals. The nucleotide patterns from adult horses and foals were similar in most respects. However, several peaks were larger in chromatograms from foals and have been indicated in Fig. 2B as a, b, c and CTP. Peaks a, b and c have not been identified.

,b

,'5 2'0

GTP

TP

2'5

3'0 3'5

go

4'5

~'o

5'5 ~o

Minutes

Fig. 1. Chromatogram of standard solutions of mono-, di- and triphosphates of purine and pyrimidine ribosides including the di and triphosphates of 2'-deoxyadenosine. The nucleotides were eluted at room temperature from a partisil SAX column with a linear gradient of 0.03-0.75 M sodium phosphate, pH 4.0: flow rate was 1.0 ml/min.

Nucleotides in erythrocytes of horses

,21

207

ATP

A. Adult

AMP

1

I,o.

E

c

0 ¢; o J~ b. o

'

,~

,~

~

~

,~, ,'5 ;o ~5

~o ~

.16 -

ATP

B. Fool

$

AMP

0

'

5

I

'0

I

'5

I

'

20

ADP

~'5

3.0

.

~

.

4.0.

4.5

.

50

55

Minutes

Fig, 2. Typical chromatograms of nucieotides extracted from horse erythrocytes: adult Arabian horse (A) and Arabian foal (B). Conditions for analysis are described in Fig. 1. breeds of horses we compared the adenine nucleotide concentrations in erythrocytes of four separate breeds. We found that the A D P and A T P concentrations were similar for all four breeds (Table 1). Because no significant difference was detected between sexes, data from both sexes were combined for each breed studied. Variation due to age was also determined (Table 2). The concentrations of A D P and A T P in erythrocytes from adult horses were found to be twice that of foals (P < 0.01). N o significant differences were found in

the concentrations of A D P and A T P between the foals of Shetland ponies and Arabian horses.

Concentrations of ADP and A T P in whole blood Nucleotide profiles have been shown to be characteristic for each of the various cellular elements in human blood (Scholar et al., 1973). Nevertheless, the nucleotide distributions and concentrations of whole blood were shown to be similar to those of erythrocytes (Brown et al., 1972). Because the concentrations of A D P and A T P in erythrocytes from horses are a

Table 1. Concentrations of ADP and ATP in erythrocytes from adult horses of various breeds

Breed

Nucleotide Concentration (nmols/109 cells) ADP ATP

Stetland Pony (n = 10)

3.5 + 1.7 (2.2 _+ 6.9)

16.8 + 4.3 (8.6 + 20.9)

Arabian (n = 5)

2.4 +_ 0.3 (1.9 - 3.0)

12.5 _+ 3.3 (7.6 - 15.6)

Quarter Horse (n = 5)

2.3 +_ 0.5 (2.1 - 3.2)

16.0 +__2.9 (11.9 - 18.7)

Thoroughbred (n = 5)

2.6 + 1.1 (1.9 _+ 4.6)

13.3 +_ 2.9 (10.9 _+ 18.3)

Ages of horses range from 4 to 15 years. Nucleotide concentrations are corrected for procedural losses (see Materials and Methods). AMP was <0.5 nmols/109 ceils. The numbers in parentheses represent range of values.

208

NANCY S. MAGNUSON and LANCE E. PERRYMAN Table 2. Concentrations of ADP and ATP in erythrocytes from foals Nucleotide Concentration (nmols/l 09 cells) ADP ATP

Foals Shetland Pony (n = 6)

1.1 ± 0.5 (0.6 - 1.7)

6.8 ± 1.6 (4.6 - 8.4)

Arabian (n = 3)

1.1 ± 0.1 (0.9 - 1.3)

7.8 ± 0.9 (6.9 - 8.7)

Foals were 1 3 months of age. Nucleotide concentrations are corrected for procedural losses (see Materials and Methods). AMP was <0.5 nmols/109 cells. The numbers in parentheses represent range of values.

factor of 10 less than those found in erythrocytes from h u m a n s (Brown et al., 1972), it was of interest to determine if the concentration a n d distribution of nucleotides of erythrocytes from horses might be altered by the nucleotides contributed from other cellular elements of whole blood. Our results showed that the nucleotide pattern a n d concentrations of whole b l o o d were essentially the same as those found for erythrocytes.

Table 3. Effect of time on concentrations of ADP and ATP in erythrocytes Time (hr) 0 1

2 6 24

Concentrations of ADP and A T P in stored blood samples The stability of adenine nucleotides in equine blood maintained at r o o m temperature was determined. F r o m a sample of heparinized-blood, erythrocytes were p r e p a r e d a n d extracted at 0, 1, 2, 6 a n d 24hr. Only small changes were detected in the concentrations of A D P or A T P during this first 6 hr of storage with a small, but detectable decrease in the ratio of A T P : A D P (Table 3). At the end of 24 hr, however, a substantial decrease in the concentration of A T P and increase in A D P was evident. This change was clearly seen in the decreased ratio of A T P : A D P .

Stability of nucleotide extracts Nucleotide extracts prepared as outlined in Materials a n d M e t h o d s were kept frozen at - 2 0 ° C for over a year without detectable alteration in nucleotide concentrations. Use of an a u t o m a t i c sample injector would allow 8 to 12 samples to be c h r o m a t o g r a p h e d in succession but would require these samples to

Nucleotide Concentration (nmols/109 cells) ADP ATP ATP:ADP 3.5 3.9 3.7 4.3 7.9

18.9 19.0 16.2 17.3 12.4

A blood sample was collected in heparin from an adult Shetland pony and kept at room temperature. At various times (hr) aliquots were taken and the nucleotides extracted. Nucleotide concentrations have been corrected for procedural losses (see Materials and Methods). AMP was usually <0.5 nmols/109 cells. remain at r o o m temperature for 8 - 1 2 h r . Therefore, the effect of maintaining nucleotide extracts at room temperature for extended periods of time was determined. A nucleotide extract was kept at room temperature for 24 hr. During this time, aliquots of the extract were c h r o m a t o g r a p h e d at i, 3, 6, 10 a n d 24 hr. N o detectable change was observed in the nucleotide concentrations during the 24 hr period.

Effect of adenosine on concentrations of ADP and A T P in erythrocytes We found previously that the ratio of A T P : A D P

Table 4. Effect of adenosine on concentrations of ADP and ATP in erythrocytes incubated in HBSS Length of incubation (hr) 1

Adenosine

Nucleotide Concentration (nmols/109 cells) AMP ADP ATP

ATP:ADP

0.5 8.3 ± 5.9

17.3 ± 4.6 16.0 + 5.4

6.9 1.9

-

<0.5

+

4.5 _ 5.1

5.3 4.8 4.4 4.0 1.6

2.5 ±

3

+

<0.5 4.3 ± 3.0 16.0 ± 15.4 13.9 _+ 6.2

17.3 + 8.3 15.7 ± 3.0

4.0 1.1

5

+

<0.5 16.9 ± 15.1

15.2 +_ 6.5 14.4 ± 4.3

5.9 1.0

2.5 ± 1.1 13.8 ± 3.9

The data are expressed as mean + SD of three separate experiments. Incubation of samples was done in a shaking waterbath at 30°C. The concentration of adenosine was 0.5 mM. Nucleotide concentrations'have been corrected for procedural losses (see Materials and Methods).

Nucleotides in erythrocytes of horses

209

Table 5. Effect of adenosine on concentrations of ADP and ATP Length of incubation (hr) 1

Adenosine -

+

Nucleotide Concentration (nmols-109 cells) AMP ADP ATP 0.5 4.1 _+ 0.4

ATP:ADP

2.9 10.8 + 1.6

21.7 15.6 _+ 3.5

7.4 1.4

3

+ +

<0.5 6.9 5.1

2.9 14.6 18.4

22.2 13.0 14.6

7.8 0.9 0.8

5

-

<0.5

-

<0.5

2.5 2.9 17.8 ___5.4

17.8 14.3 16.8 ___6.9

7.0 5.0 0.9

+

10.1 _+ 2.8

The data are expressed as mean + SD of three separate experiments. Incubation of samples was done in a shaking waterbath at 30°C. The concentration of adenosine was 0.5 mM. Nucleotide concentrations ~have been corrected for procedural losses (see Materials and Methods). decreased significantly in erythrocytes from horses after incubation after 4 hr at 30°C with 0.5 m M adenosine in HBSS (Magnuson & Perryman, 1979b). It was of interest, therefore, to examine the influence of other incubation media on the incorporation of adenosine into the adenine nucleotide pool. In a typical experiment whole blood, or erythrocytes prepared from whole blood, were incubated with or without 0.5 m M adenosine. Incubation media for erythrocytes was either HBSS (containing ~ 1 m M phosphate) or 50 m M phosphate buffer. Incubation was carried out for 1, 3 and 5 hr at 30°C in a shaking waterbath. In agreement with our original findings, prolonged incubation (5 hr) resulted in a 1.5-2.0-fold increase in the adenine nucleotides (AMP, A D P and ATP) and a significant decrease in the ratio of A T P : A D P (P < 0.01, Table 4). This response was seen within 1 hr of incubation and reached a maximum at 3 hr. Minimal changes were seen in the concentrations of A D P or A T P when erythrocytes were incubated in HBSS without adenosine. The effect of incubating heparinized whole blood with adenosine was similar to that produced by incubation of erythrocytes with adenosine in HBSS (Table 5). Incubation of erythrocytes with adenosine in the presence of 50 m M phosphate for 1 hr resulted in a doubling of ATP and A D P with no detectable increase in A M P (Table 6). After 3 and 5 hr of incuba-

tion, A D P and A T P increased 4- and 5-fold, respectively. Again, there appeared to be little increase in AMP. In contrast to the low ratios of A T P : A D P obtained with adenosine incubation shown in Tables 4 and 5, the ratio of A T P : A D P for erythrocytes incubated with adenosine in 5 0 m M phosphate buffer remained essentially unchanged.

Effect of incubation with 2'-deoxyadenosine The accumulation of dATP has been demonstrated in the erythrocytes of children with a deficiency of adenosine deaminase (Cohen et al., 1978; Coleman et al., 1978) an enzyme important in purine salvage. Because erythrocytes of normal horses contain little, if any, adenosine deaminase activity (McGuire et al., 1976; Castles et al., 1977) it was of interest to examine changes in adenine nucleotide pools following incubation with 2'-deoxyadenosine. As shown in Fig. 3B, the effect of incubating erythrocytes from a normal Shetland pony with 2'-deoxyadenosine in HBSS was similar to those obtained by incubation with adenosine except that 2'-deoxyadenine nucleotides accumulated. DISCUSSION

Recent findings of altered nucleotide metabolism in erythrocytes from patients with immune deficiency diseases has prompted studies of erythrocyte nucleo-

Table 6. Effect of adenosine on concentrations of ADP and ATP in erythrocytes incubated in 50 mM phosphate buffer Length of incubation (h})

Adenosine

Nucleotide Concentration (nmols/109 cells) AMP ADP ATP

ATP:ADP

+

2.1 _+ 1.5

4.6 _+ 0.8 7.8 +_ 4.9

18.3 _+ 6.7 28.9 _+ 16.3

7.2 3.7

3

+

<0.5 4.8 _+ 2.3

2.7 +_ 0.5 17.8 _+ 5.1

15.1 _+ 4.9 67.8 + 12.1

5.6 3.8

5

+

<0.5 3.5 ___3.0

2.7 + 0.9 15.2 + 4.9 15.7 + 11.7 95.4 + 11.9

5.6 6.1

1

-

<0.5

The data are expressed as mean ___SD of three separate experiments. Incubation of samples was carried out in a shaking waterbath at 30°C. The concentration of adenosine was 0.5 mM. Nucleotide concentrations have been corrected for procedural losses (see Materials and Methods).

210

NANCY S. MAGNUSON a n d LANCE E. PERRYMAN

A. Adeno,,ine Incubation .16 -

AOP

ATP

E

O-

mt~ N o

5

i0

15

20

;,5

30

35

40

45

50

55

B. 2'-deoxyodenollne Incubation i

.ll~"

<

1

dADP ADP1

.b i:, z'o 2'5 3'o ~5 go ,~ ~o 5'5 Minutes Fig. 3. Typical chromatograms of nucleotides extracted from erythrocytes of a Shetland pony after incubation for 5hr in HBSS containing 0.5mM adenosine (A) and 0.5 mM 2'-deoxyadenosine (B). Conditions for analysis are described in Fig. 1. tide metabolism in horses with CID (Agarwal et al., 1976; Mills et al., 1976; Goldblum et al., 1976; Cohen et al., 1978). However, information of nucleotide concentrations for normal horse erythrocytes was sparce and required further evaluation. HPLC was used in this study because it is the most rapid and one of the most sensitive methods for monitoring and quantitat ing nucleotides in cell extracts. From this study we found that total concentrations and relative distributions of nucleotides in erythrocytes from adult horses of different breeds were similar. In spite of the wide range of concentrations reported for adenine nucleotides in erythrocytes of various species of vertebrates (Brown et al., 1972) (from 4.9#mols ADP plus ATP/ml of packed cells for rabbit to <0.2/~mols ADP plus ATP/ml packed cells for cows), we found that within the equine species, the distribution and concentration of nucleotides in erythrocytes were essentially the same. In relation to adenine nucleotide concentrations found in human erythrocytes, horse erythrocytes contain a factor of 10 less ATP and ADP. For direct comparison, values of 151 nmols ATP and 24 nmols ADP/109 erythrocytes have been

recently reported for normal human erythrocytes (Coleman et al., 1978). For normal horse erythrocytes we find 14.7 + 2.1 nmoles ATP and 2.7 + 0.6nmols ADP/109 erythrocytes. Our results also suggest that erythrocytes from agematched horses of any breed could be used as valid controls in studies of purine metabolism. This would be important in studies of CID in horses. Preliminary findings indicate that horses with CID may have a defect in purine metabolism (Perryman et al., 1979; Magnuson & Perryman, 1979a, b) and it is possible that horses heterozygous for the CID trait would have purine metabolic alterations too. At present, it is not possible to differentiate heterozygous from genetically normal horses by biochemical or functional tests. Only by production of affected foals can adult horses be identified as heterozygous for the CID trait. Since the frequency of heterozygotes among the Arabian horse population may exceed 25~o (Poppie & McGuire, 1977) random selection of Arabian horses could result in unsuitable controls for the experiments described here. The total concentration and relative distributions of nucleotides in erythrocytes from foals were different from those of adults (Fig. 2, Table 2). This suggests that purine metabolism in erythrocytes of horses varies with age, but not breed or sex. Selection of age-matched controls for metabolic studies would appear to be more important than selection of horses from the same breed. Our finding that the total concentrations and relative distributions of nucleotides for both whole blood and erythrocytes from horses were the same is in agreement with previous findings for human erythrocytes and whole blood (Brown et al., 1972). These results suggest that the manipulations required to separate erythrocytes from whole blood of horses do not alter nucleotide concentrations. The results also indicate that the contribution of nucleotides from the other cellular elements of the blood to that of erythrocytes is negligible. With human erythrocytes suspended in media containing ~ 1 mM inorganic phosphate (Pi), addition of nucleosides has been shown to decrease intracellular Pi (Planet & Fox, 1976). This depletion was found to be highest with adenosine. Metabolism of adenosine in human erythrocytes may decrease P~ by phosphorylation and phosphoryolysis reactions. In experiments similar to those described in this paper, incubation of human erythrocytes with adenosine under conditions that should have depleted intracellular Pi, did not alter adenine nucleotide concentrations (Magnuson & Perryman, 1979b). The explanation for this observation is that human erythrocytes contain adenosine deaminase, an enzyme which converts adenosine to inosine and removes adenosine from immediate phosphorylation by adenosine kinase. In this instance, Pi levels would not appear to be important for adenosine metabolism with regards to adenine nucleotide concentrations. Horse erythrocytes on the other hand contain very little adenosine deaminase activity (McGuire et al., 1976; Castles et al., 1977). Therefore, when adenosine is taken up by these erythrocytes, it must be phosphorylated to AMP by adenosine kinase. This step would utilize ATP; the by-product of which would be ADP. Conversion of ADP to ATP

Nucleotides in erythrocytes of horses

211

would require Pi, as in the glycolytic step mediated by glyceraldehyde-3-phosphate dehydrogenase. Thus, with adenosine incubation of horse erythrocytes in HBSS or plasma which contain ~ l m M P~ (Oser, 1975), the low Pi level may be limiting phosphorylation of A D P to ATP, since accumulation of A M P and A D P was observed (Fig. 3A, Tables 4 and 5). Indirect evidence for this assumption is that increased adenine nucleotides and maintenance of normal ratios of A T P : A D P were observed in erythrocytes incubated with adenosine in the presence of 50 m M P~ (Table 6). In previous studies, erythrocytes from CID horses were observed to respond to adenosine differently in the presence of low Pi (Magnuson & Perryman, 1979b). C I D erythrocytes were capable of maintaining the A T P : A D P ratio although the increase in the total adenine nucleotide pool was similar to that produced in erythrocytes from normal horses under identical conditions. Increasing the concentration of P~ to 50 m M appears to eliminate the difference in response to adenosine between C I D and normal horse erythrocytes (Magnuson, unpublished observation). The physiological importance of P~ in adenosine metabolism in erythrocytes from CID horses awaits further study.

(1978) Identification and quantitation of adenine deoxynucleotides in erythrocytes of a patient with adenosine deaminase deficiency and severe combined immunodeficiency. J. biol. Chem. 253, 1619-1626. GOLDBLUM R. M., SCHMALSTEIGF. C., GOLDMAN A. S., NELSON J. A. & MONAHANT. M. (1976) Abnormal adenine metabolism in severe combined immunodeficiency (SCID) and normal adenosine deaminase (ADA) activity. Pedlar. Res. 10, 387, (Abstract). GREENE M. L., BOYLEJ. A. & SEEGMILLERJ. E. (1970) Substrate stabilization: genetically controlled reciprocal relationship of two human enzymes, Science 167, 887-889. KHYM J. X. (1975) An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchanges. Clio. Chem. 21, 1245-1252.

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(1976) Abnormal purine overproduction in a patient deficient in purine nucleoside phosphorylase. N. Engl. J. Med. 295, 1449-1454. COHEN A., GUDAS L. J., AMMANN A. J., STAAL G. E. J. & MARTIN D. W. JR (1978) Deoxyguanosine triphosphate as a possible toxic metabolite in purine nucleoside phosphorylase deficiency. J. clin. Invest. 61, 140,%1409. COHEN A., HIRSCHHORN R., HOROWlTZ S. D., RUBINSTEIN

A., POLMARS. H., HONG R. & MARTIN D. W. JR (1978) Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency. Proc. natn. Acad. Sci. U.S.A. 75, 47~476. COLEMAN M. S., DONOFRIOJ., HUTTON J. J. & HAHN L.

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PARKS R. E. JR CRABTREE G. W., KONG C. M., AGARWAL R. P., AGARWAL K. C. 8~ SCHOLAR E. M. (1975) Incorporation of analog purine nucleosides into the formed

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