5 24

PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY Biochemical Properties and Heterogeneity in Two Families ROBERT L. WORTMANN, CATHERINE ANDRES, JANICE KAMINSKA, EDWIN MEJIAS, ERWIN GELFAND, WILLIAM ARNOLD, KENNETH RICH, and IRVING H. FOX The biochemical features of two families with purine nucleoside phosphorylase deficiency are compared. Laboratory studies and an evaluation of kinetic and physical properties of erythrocyte purine nucleoside phosphorylase give evidence that a) the degree of abnormality in uric acid and nucleoside concentrations in plasma and urine reflect the severity of the enzymatic deficiency and b) structural alterations of the mutant enzymes result from structural gene mutations and demonstrate genetic heterogeneity in the disease purine nucleoside phosphorylase deficiency.

Purine nucleotide degradation is an intricately regulated enzymatic sequence. The relevance of these enzyme reactions to clinical disorders of immune function was first noted when adenosine deaminase deficiency was found associated with combined B- and T-cell dysfunction (1). The observations that purine nucleoside phosphorylase deficiency occurs with cellular immune dysfunction (2-8) and ecto-5'-nucleotidase From the Human Purine Research Center, Departments of Internal Medicine and Biological Chemistry, University of Michigan, AM Arbor, Michigan. Robert L. Wortmann, MD: University of Michigan; Catherine Andres, MD: University of Michigan; Janice Kaminska, MD: University of Michigan; Edwin Mejias, MD: University of Michigan; Erwin Gelfand, MD: Hospital for Sick Children, Toronto, Canada; William Arnold, MD: Lutheran General Hospital, Chicago; Kenneth Rich, MD: Children's Memorial Hospital, Chicago; Irving H. Fox, MD: University of Michigan. Supported by a grant from the United States Public Health Service AM 19674 and 5MOIRR42 and from the American Heart Foundation 77-849 Address reprint requests to Dr. I. H. Fox, Clinical Research Center, University of Michigan Medical Center, Ann Arbor, Michigan, 48 109. Submitted for publication November 2, 1978; accepted January 9, 1979. Arthritis and Rheumatism, Vol. 22, No. 5 (May 1979)

deficiency occurs with agammaglobulinemia (9) further emphasized this relationship. Purine nucleoside phosphorylase is an integral part of the nucleotide degradation pathway. A deficiency of this enzyme blocks this reaction sequence and results in an immune disorder (2-8). The enzyme deficiency and the concomitant clinical syndrome may result from structural gene mutations (10,ll). We have examined the clinical features and biochemical properties of the enzyme from 3 patients in two families with purine nucleoside phosphorylase deficiency. The data indicate that the laboratory changes observed in the patients reflect the degree of enzyme deficiency and that altered properties of the deficient enzymes provide evidence for a structural gene alteration and genetic heterogeneity in the disease purine nucleoside phosphorylase deficiency.

MATERIALS AND METHODS Dithiothreitol, adenosine, inosine, guanosine, parachloromercuribenzoic acid, cytidine, uridine, bovine serum albumin, alpha-chymotrypsinogen, yeast alcohol dehydrogenase, cytochrome C, DNP-alanine, deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, calf thymus, DNA template, and DNA polymerase from Micrococcus lysodeiklicus were purchased from Sigma Chemical Company, St. Louis, Missouri. Hypoxanthine was purchased from Calbiochem, San Diego, California and (8-I4C) inosine (60 mCi/mM) was purchased from Amersham Corporation, Arlington Heights, Illinois. Ampholine, pH 5.0-7.0,was obtained from LKB instruments, Rockville, Maryland and ['HI-thymidine triphosphate (19.7 Ci/mM) was purchased from New England Nuclear Corporation, Boston, Massachusetts. Mercuric chloride was purchased from Fisher Scientific Company, Pittsburgh, Pennsylvania. Sephadex G150 and dextran blue were purchased from Pharmacia Incorporated, Piscataway, New Jersey. Erythro-9-(2-hydroxy1-3nonyl) adenine (EHNA), an inhibitor of adenosine deaminase, was a gift from Dr. G. B. Elion of Burroughs Wellcome

PURINE NUCLEOSIDE PHOSPHORYLASE

Co., Research Triangle Park, North Carolina. All other reagents were of the highest quality commercially available. Blood for studies was obtained from normal volunteers and members of two families known to be deficient in purine nucleoside phosphorylase. Family one includes 2 brothers, ages 9 and 1 1 years, with recurrent infections, lymphopenia, and abnormal cellular immunity with intact humoral immunity (6,7). In family two there is a 5-year-old boy with similar abnormalities (8). Enzyme assays, kinetic studies, isoelectric focusing, and Stokes radius determinations were performed on hemolysate. Erythrocytes were obtained by collecting fresh blood in heparin-treated tubes. Blood was centrifuged at IOOOg at 4°C for 5 minutes. The plasma was removed and stored at -20°C. The erythrocytes were washed twice with cold 150 mM sodium chloride and frozen at -70°C for later use. Prior to use hemolysate was dialyzed in 1.0 mM Tris-HCI pH 7.4 with 0.1 mM EDTA for 6 to 18 hours. Purine nucleoside phosphorylase activity was determined by a radiochemical assay that quantitated the rate of hypoxanthine formation from inosine (10). Dialyzed hemolysate was diluted with 50 mM potassium phosphate pH 7.4 by 1:1500 for normal, 1:19 for family one, and 1:9 for family two. Substrate competition studies were performed to determine whether the deficient enzymes had properties corresponding to purine nucleoside phosphorylase. Hemolysates from normal subjects and each mutant were assayed as described above with or without the addition of 1 mM guanosine, inosine, cytidine, uridine, or adenosine plus 7.5 pM EHNA. Only those compounds that were substrates for the enzyme studies would substantially inhibit the amount of radiolabeled product formed. Kinetic studies with hemolysate were performed using initial velocity experiments. Dialyzed normal hemolysate diluted 1:6000 was incubated for 10 minutes at 37°C with (8I4C) inosine 3.3 to 20.0 pM.Dialyzed enzyme deficient hemolysates were similarly studied using a 1:19 dilution for family one and 1.9 dilution for family two and 60-minute incubations. The values of the apparent Michaelis constants were estimated from the slope of Eadie-Hofstee plots. Activity of the proteins over a pH range was determined by diluting dialyzed hemolysate 1:1500 for the normal enzyme and 1:9 for the deficient enzymes with 4.0 mM potassium phosphate pH 7.4. Diluted extracts were incubated with 50 mM sodium acetate pH 4.5 to 6.0 and 50 mM Tris-HC 1 pH 6.5 to 8.5 as described above. Isoelectric focusing was performed according to the method of Vesterberg and Svensson with an LKB 8100 ampholine electrofocusing column ( 12) as previously described (13). Hemolysates (0.5 ml) were dialyzed for 20 hours against 1% glycine and mixed in a I 10 ml linear gradient containing 0 to 40% sucrose and 1% ampholine, pH 5.0 to 7.0. The column was eluted from below with a Buchler multistatic pump at a rate of 60 ml/hour. The pH of every other fraction (1.0 ml) was measured at 4°C with a Radiometer PHM 64 Research pH Meter. Protein was quantitated by reading the absorbance at 280 nm with a Beckman DU Spectrophotometer. Enzyme activity was quantitated as described above.

525

Gel filtration was performed using hemolysate from normal subjects and family one at 4°C on a Sephadex G-150 column, 60 by 1.6 cm, previously equilibrated with 50 mM Tris-HC1 pH 7.4. Samples of 1.0 ml were applied and 1 ml fractions were withdrawn using an LKB 7000 Ultrarac Fraction Collector. The column was calibrated with 2.0 mg/ml of dextran blue, 1.0 mg/ml of yeast alcohol dehydrogenase, 10 mg/ml of yeast alcohol dehydrogenase, 10 mg/ml of bovine serum albumin, 5 mg/ml of alpha-chymotrypsinogen, 3 mg/ ml of cytochrome C, and 0.25 mg/ml of DNP-alanine in 20% sucrose solutions. The void volume, as quantitated by dextran blue elution, was 53 ml. A protein solution, prepared for chromatography by diluting 300 pI of hemolysate to 1.5 ml in the equilibrating buffer and centrifuging at 2000g for 10 minutes at 4"C, was applied to the column. Elution of standard peaks was recorded by measuring absorbance at 630 nm for dextran blue, 280 nm for bovine serum albumin and alpha-chymotrypsinogen, 410 nm for cytochrome C, and 360 nm for DNPalanine on a Cary 15 Spectrophotometer. Yeast alcohol dehydrogenase was assayed by determining the formation of NADH by absorbance at 340 nm. The eluted fractions were assayed for purine nucleoside phosphorylase activity as described above. The procedure was repeated after equilibrating the column with 2 mM inosine in 50 mM Tris-HC1 pH 7.4. The Stokes radius was estimated from the distribution coefficient (Kd) for the standard proteins and the normal and deficient purine nucleoside phosphorylase (14). The apparent molecular weight was estimated from a plot of elution volume versus log molecular weight (15). The effect of mercuric chloride and parachlormercuribenzoic acid, sulfhydryl oxidizing agents, on purine nucleoside phosphorylase activity was assessed in hemolysate from a normal subject and from family one. Partially purified enzyme protein was obtained from the eluant from the isoelectric focusing described above, diluted (l:9 for normal; 1:2 for family one) with 50 mM potassium phosphate pH 7.4, and incubated with 0 to 5 pM mercuric chloride or 0 to 10 pM parachloromercuribenzoic acid at 37°C for 20 minutes. Samples were cooled to 4°C and then assayed for purine nucleoside phosphorylase activity. Serum urate, urine uric acid, urinary oxypurines, plasma and urinary inosine and guanosine levels were quantitated by enzymatic spectrophotometric methods (16-18). Creatinine was measured by an automated modified Jaffe reaction (19). Inosine and deoxyinosine and guanosine and deoxyguanosine are measured as the ribonucleoside derivatives only. Protein was quantitated by the method of Lowry et al using bovine serum albumin as a standard (20). Deoxyguanosine triphosphate concentrations were determined by a DNA polymerase assay according to Solter and Handschumacher (21). Erythrocyte extract for the assay was prepared as follows: One ml of packed red blood cells was washed with 10 mM potassium phosphate pH 7.4 in normal saline and stored at -2OOC until use. The erythrocytes were mixed with one ml of 2.0 M cold perchloric acid and centrifuged at 10,OOOg. The supernatant was neutralized with 5.0 M potassium hydroxide and recentrifuged. The supernatant was lyophyllized, resuspended in 400 pI of distilled water, and assayed for deoxyguanosine triphosphate.

WORTMANN ET AL

526

RESULTS Enzyme activity Erythrocyte purine nucleoside phosphorylase was deficient in the 3 members of the two families. The erythrocyte enzyme from 2 brothers in family one had 0.45% of normal activity whereas a 4-year-old boy in family two had an erythrocyte enzyme with only 0.07% normal activity (Table 1). Substrate competition studies indicated that the deficient enzymes studied were purine nucleoside phosphorylase. The deficient and normal enzymes appeared to have identical substrate specificity. The enzyme activity for each hemolysate was substantially inhibited by inosine and guanosine, but not by cytidine, uridine, or adenosine plus EHNA.

Block in purine catabolism Each patient demonstrated a block in purine catabolism. In family one the plasma urate levels had a normal value of 2.6 mg/dl for each patient, and the plasma inosine concentrations were markedly elevated at 41.2 and 45.2 pM. Urinary uric acid excretion was normal at 454 and 527 mg/gm creatinine, whereas the urinary inosine excretion of 5.1 and 4.7 millimoles/gm creatinine was elevated. Deoxyguanosine triphosphate levels were elevated at 1140 picomoles/ml packed RBC (Table 1). The patient in family two shows a more severe block in purine catabolism (Table 1). He is hypouricemic with a serum urate of 1.8 mg/dl and has an elevated plasma inosine level of 38 pM.Urinary uric acid excretion is reduced to 73 mg/gm creatinine, and urinary excretion of inosine and guanosine is elevated at 14.8 and 4.8 millimoles/gm creatinine, respectively.

Furthermore, deoxyguanosine triphosphate levels were markedly elevated at 2225 picomoles/ml packed RBC.

Properties of the deficient erythrocyte enzymes The finding of patients with a partial deficiency of erythrocyte purine nucleoside phosphorylase activity provided a unique opportunity for investigation. Studies were undertaken to assess for structural alterations of the mutant enzyme protein. Kinetic properties Initial velocity studies. Initial velocity studies were performed to compare the apparent Michaelis constants for inosine in normal and enzyme deficient hemolysates. Although all enzymes demonstrated hyperbolic kinetics, the apparent Km values for inosine for the mutant enzymes were substantially higher than those for normal enzymes. The apparent Km values estimated from Eadie-Hofstee plots were 370 and 343 pM for the enzymes from the 2 patients in family one and 110 pM for the enzyme from the patient in family two (Figure 1). These are tenfold and three- to fourfold higher than the values of 25 to 37 pM obtained using normal enzymes. These findings agree with values previously reported for normal purine nucleoside phosphorylase (22,23). p H curvex The mutant enzymes were also compared to normal enzymes by examining their activity from pH 4.5 to 8.5. The enzyme activity from normal hemolysate is maximal at pH 6.0 and near optimal at pH 7.4. The enzyme activity from family two was similar to the normal enzyme, but the enzyme activity from family one clearly had less than near optimal activity at pH 7.4 despite maximal activity at pH 6.0 (Figure 2). Physical properties. The enzyme protein from family one was further studied to assess for additional evidence of structural alteration. The very low activity

Table 1. Laboratory findings in purine nucleoside phosphorylase deficiency

Plasma urate (mg/dl) inosine (pM) Urine creatinine (mg/24 hr) uric acid (mg/gm creatinine) inosine (millimoles/gm creatinine) guanosine (millimoles/gm creatinine) Erythrocyte deoxyguanosine triphosphate (picomoles/ml packed RBC) Erythrocyte enzyme activity (nanomoles/hr/mg protein)

Family 1

Family 2

Normal

2.6, 2.6 41.2, 45.2 441,355 454,527 5.06, 4.73 1.39, 1.83 I140

1.8 38 232 13 14.78 4.78 2225

3.7 f 1.07 Undetectable

10.6, 10.6

1.6

Undetectable Undetectable < 20 2368 rt 6 14

PURINE NUCLEOSIDE PHOSPHORYLASE

I

I

I

,

I

A. Normal Nucleoside Phosphorylase

--

I

527 ,

I

6. Mutant Nucleoside Phosphorylase

12.0-

6.0-

10.0-

5.0-

C. Mutant Nucleoside Phosphorylose Fomily 2

0 ul

8.0-

4.0-

-2 6.0-

3.0-

0

o)

.-

f

0

x*

4.0-

2.0 -

2.0 -

1.0-

%

I

I

1

0

.2

.4

.8

.6

.01

0

.02

.03

.04 .05 .004 .008 ,012

I

.016

,020

each brother from family one was diminished compared to normal and ranged between 5.09 and 5.26. Isoelectric focusing of normal hemolysate after incubation with 0.1 mM parachlormercuribenzoic acid lowered the isoelectric pH to 5.19. This was within the range of values observed for the enzyme from family one (Figure 3). Apparent molecular weight and Stokes radius. The apparent molecular weight and Stokes radius for the normal hemolysate enzyme and for the enzyme from I I I I I I [ I family one were estimated by gel filtration (Table 2). NORMALS U 100 IFAMILY I M These were found to be identical for each hemolysate FAMILY 2 W with values of 84,000 daltons and 36.6 di. These values agree with previously published results in which purified enzyme was used (24,25). A possible conformational change has been suggested by the observation that inosine protects normal but not deficient purine nucleoside phosphorylase against thermal inactivation (10). The Stokes radius was not altered for either enzyme when preincubated with 2 mM inosine and run in a column equilibrated with 2 I I 1 I I 1 I I mM inosine (Table 2). 45 50 55 60 65 70 75 80 85 PH Efect of sulfiydryl oxidation. Two sulfhydryl Figure 2. Effect of pH on erythrocyte purine nucleoside phosphoryoxidizing agents, mercuric chloride and paralase. Purine nucleoside phosphorylase activity is expressed as a perchlormercuribenzoic acid, can inhibit normal purine centage of maximum activity at pH 6.0 over a p h range of 4.5 to 8.5. Hemolysates were from normal subjects (U family ), one (M), nucleoside phosphorylase activity (22,26). While the normal and mutant enzyme proteins were equally inand family two ( A-A ).

of the enzyme from family two prevented the further evaluation of that patient’s enzyme protein. Electrical charge. Isoelectric focusing was employed to assess the electrical charge properties of the mutant erythrocyte enzyme from family one compared to the enzyme from normal erythrocytes (Figure 3). The isoelectric pH for normal hemolysate ranged from 5.4 to 5.8. These values agree with values reported for the crystallized normal enzyme (22). The isoelectric pH for I

I

I

WORTMANN ET AL

5 28

hibited by similar concentrations of parachloromercuribenzoic acid, a 20-fold higher concentration of mercuric chloride was required to cause a similar 50% inhibition of mutant enzyme from family one as compared to normal enzyme (Table 3).

I

I

I

I

i

In an effort to increase enzyme activity, the hemolysate from family one was assayed after incubation with up to 10 mM dithiothreitol, a sulfhydryl reducing agent. This failed to increase the purine nucleoside phosphorylase activity.

I

I

I

5t

C. NORMAL + P C M B

6 4 2

'1

Ip

b 6 4

,2 I

0'

10

1

20

1

30

L

0

FRACTION NUMBER Figure 3. Isoelectric focusing of erythrocyte purine nucleoside phosphorylase. Hemolysate from (A,B) the 2 brothers in family one, (C) a normal subject and preincubated with parachloromercuribenzoic acid and (D) a normal subject were purine nucleoside phosphorylase activity U. studied as described in Materials and Methods. pH M,

PURINE NUCLEOSIDE PHOSPHORYLASE

Table 2. Gel filtration studies of purine nucleoside phosphorylase

Normal Family one

Stokes radius (angstroms)*

Apparent molecular weight (daltons)*

36.6 36.6

84,000 84,000

~~

* Values were identical in the presence of 2 mM inosine.

DISCUSSION Purine nucleoside phosphorylase catalyzes the phosphorolysis of purine nucleosides to the corresponding base in the reversible reactions; inosine (and guanosine) +Pi ++ hypoxanthine (or guanine) ribose-lphosphate; deoxyinosine (or deoxyguanosine) +Pi t* hypoxanthine (or guanine) deoxyribose-I-phosphate. The enzyme is normally distributed throughout the body with significant activity greater than 10.0 nanomoles/min/mg protein in thymus, spleen, brain, kidney, liver, lung, small intestine, heart, peripheral lymphocytes, and granulocytes (27). As an integral part of the purine catabolic pathway, purine nucleoside phosphorylase is necessary for uric acid formation. The exact physiologic role of this enzyme is unclear. It appears to influence purine salvage, since a deficiency of the enzyme leads to purine overproduction (6,8.28,29). Inosine, deoxyinosine, guanosine, and deoxyguanosine are the end-products of purine catabolism for patients deficient in purine nucleoside phosphorylase. Consequently, the purine bases, guanine and hypoxanthine, are not formed and are unavailable for conversion to nucleotides by hypoxanthine-guanine phosphoribosyltransferase. The immune dysfunction observed in the enzyme deficiency state may result from high concentrations of deoxyguanosine triphosphate accumulated from the elevated deoxyguanosine levels (27.30). A block of purine nucleoside degradation is indicated by the accumulation of nucleosides in purine nucleoside phosphorylase deficiency. Plasma inosine levels and urinary inosine and guanosine excretion are markedly elevated for each patient studied (Table I). Furthermore, the patient in family two is hypouricemic and excretes small quantities of uric acid and large amounts of nucleosides in urine. The low normal serum urate levels in the 2 brothers in family one, the normal urinary uric acid excretion, and the relatively lower urinary nucleoside excretion reflect the fact that their enzyme deficiency is less severe than that of family two.

+

+

529

Patients with a complete deficiency of this enzyme have a serum urate concentration less than 1.0 mg/dl (2,28). Thus there is a correlation between the degree of enzyme deficiency and the pattern of plasma purine concentrations and urinary purine excretions. Additional evidence that the severity of the block in purine catabolism reflects the severity of the enzyme deficiency is found in the deoxyguanosine triphosphate concentrations. While deoxyguanosine triphosphate concentration is elevated in each family, it is significantly higher in family two where the enzyme activity is lower. A structural alteration of purine nucleoside phosphorylase has been suggested by kinetic and electrophoretic studies (2,3,10, I I ) . We have observed altered values for the apparent Michaelis constants, the variations of pH optima, the diminished isoelectric pH, the lack of protection by inosine against thermal lability, and the altered sensitivity to inhibition by mercuric chloride. The data support the existence of a structural gene mutation and genetic heterogeneity in purine nucleoside phosphorylase deficiency. The possibility that the structural alteration of purine nucleoside phosphorylase from family one involved a modification of a sulfhydryl moiety was considered. This was based upon the isoelectric pH of the normal enzyme heated with parachloromercuribenzoic acid resembling the mutant enzyme and the relative resistance of the mutant enzyme to inactivation by mercuric chloride. The PI and Km values of the mutant enzyme are the same as the PI and Km values of the normal enzyme treated with parachloromercuribenzoic acid in previous studies (22,23). If an oxidized sulfhydry1 group is responsible for the enzyme deficiency, then potential therapy might result from incubation with a sulfhydryl reducing substance. Accordingly, the hemolysate from family one was assayed after incubation with dithiothreitol, which is known to restore normal purine nucleoside phosphorylase activity following parachloromercuribenzoic acid inactivation (26). Table 3. Inhibition of purine nucleoside phosphorylase by sulfhydryl oxidizing compounds

Concentration causing 50% inhibition of activity

Normal Family one

I .oo 1.00

* PCMB, parachloromercuribenzoic acid

t HgCI,,

mercuric chloride.

0.06 1.15

WORTMANN ET AL

5 30

However, this failed to increase the activity of the mutant hemolysate. The data presented indicate that the degree of abnormality in plasma and urinary purines reflects the severity of nucleoside phosphorylase deficiency. The altered kinetic and physical properties of purine nucleoside phosphorylase from two families with a deficiency of this enzyme provide evidence for structural alterations of the enzyme protein. This supports the occurrence of a structural gene mutation and genetic heterogeneity in this disease. The evidence for structural gene mutations in purine nucleoside phosphorylase deficiency strengthens the hypothesis that a block at this enzyme is the cause of the immune dysfunction, rather than a consequence of a deletion of two closely linked genes coding for enzyme synthesis and immune regulation.

ACKNOWLEDGMENTS The authors wish to thank Sally Jones for determining serum and urinary purines and Sharon Conley and Kathy Flaherty for typing the manuscript.

REFERENCES 1. Giblett ER, Anderson JE, Cohen F, Pollara B, Meuwissen

2.

3.

4.

5.

6.

7.

HJ: Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet 2: 1067- 1069, 1972 Giblett ER, Ammann AJ, Wara DW, Sandman R, Diamond LK: Nucleoside-phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet 1:1010-1013, 1975 Stoop JW, Zegers BJM, Hendricks GFM, Siegenbeek van Heukelom LH, Staal GEJ, DeBree PK, Wadman SK, Ballieux RE: Purine nucleoside phosphorylase deficiency associated with selective cellular immunodeficiency. N Engl J Med 296:651-655, 1977 Stoop JW, Eijsvoogel VP, Zegers BJM, Blok-Schut B, van Bekkum DW, Ballieux RE: Selective severe cellular immunodeficiency. Effect of thymus transplantation and transfer factor administration. Clin Immunol Immunopathol 6:289-298, 1976 Virelizier JL, Hamet M, Ballet JJ, Reinert P, Griscelli C: Impaired defense against vaccinia in a child with T-lymphocyte deficiency associated with inosine phosphorylase defect. J Pediatr 92:358-362, 1978 Edwards NL, Gelfand EW, Biggar D, Fox IH: Partial deficiency of purine nucleoside phosphorylase: studies of purine and pyrimidine metabolism. J Lab Clin Med 9 11736-749, I978 Gelfand EW, Dosch HM, Biggar DW, Fox 1H: Partial pu-

rine nucleoside phosphorylase deficiency studies of lymphocyte function. J Clin Invest 61:1071-1080, 1978 8. Rich K, Arnold WJ, Palella T, Fox IH: Cellular immune deficiency with autoimmune hemolytic anemia in purine nucleoside phosphorylase deficiency. Am J Med, in press 9. Edwards NL, Magilavy DB, Cassidy JT, Fox IH: Lymphocyte ecto-5'-nucleotidase deficiency in agammaglobulinemia. Science 201:628-630, 1978 10. Fox IH, Andres CM, Gelfand EW, Biggar D: Purine nucleoside phosphorylase deficiency: altered kinetic properties of a mutant enzyme. Science 197:1804-1806, 1977 1 I . Osborne WRA, Chen SH, Giblett ER, Biggar WD, Ammann AJ, Scott CR: Purine nucleoside phosphorylase deficiency. Evidence for molecular heterogeneity in two families with enzyme-deficient members. J Clin Invest 60:74 1-746, 1977 12. Vesterberg 0, Svensson H: Isoelectric fractionation, analysis, and characterization of ampholytes in natural pH gradients. IV. Further studies on the resolving power in connection with separation of myoglobins. Acta Chem Scand 20:820-834, 1966 13. Fox IH, Dwosh IL, Marchant PJ, Lacroix S, Moore MR, Omura S, Wyhofsky V: Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout. J Clin Invest 56:1239-1249, 1975 14. Siege1 LM, Monty KJ: Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Applications to crude preparations of sulfite and hydroxylamine reductases. Biochem Biophys Acta I 12:346362, 1966 15. Andrews P: Estimation of the molecular weights of proteins by sephadex gel-filtration. Biochem J 91:222-233, I964 16. Liddle L, Seegmiller JE, Laster L: The enzymatic spectrophotometric method for determination of uric acid. J Lab Clin Med 54:903-913, 1959 17. Klinenberg JR, Goldfinger S, Bradley KH, Seegmiller JE: An enzymatic spectrophotometric method for the determination of xanthine and hypoxanthine. Clin Chem 13~834-846,1967 18. Andres C, Fox IH: Measurement of inosine and guanosine in body fluids by enzymatic spectrophotometric methods. In preparation 19. Technicon Corporation: Technicon auto analyzer R method file, method N- 116, Tarrytown, New York, 1969 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ:Protein measurement with the Fohn phenol reagent. J Biol Chem 193~265-275,1951 21. Solter AW, Handschumacher RE: A rapid quantitative determination of deoxyribonucleotide triphosphates based on the enzymatic synthesis of DNA. Biochem Biophys Acta 174:585-590, 1969 22. Agarwal KC, Agarwal RP, Stoeckler JD, Parks RE Jr: Purine nucleoside phosphorylase. Microheterogeneity and

PURINE NUCLEOSIDE PHOSPHORYLASE

23.

24.

25.

26.

27.

comparison of kinetic behavior of the enzyme from several tissues and species. Biochem 14:79-84, 1975 Kim BK, Cha S, Parks RE Jr: Purine nucleoside phosphorylase from human erythrocytes. 111. Kinetic analysis and substrate binding studies. J Biol Chem 243: 17711776, 1968 Agarwal RP, Parks RE Jr: Purine nucleoside phosphorylase from human erythrocytes. IV. Crystallization and some properties, J Biol Chem 244:644-647, 1969 Zannis V, Doyle D, Martin DW Jr: Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits. J Biol Chem 283504-5 10, 1978 Agarwal RP, Parks RE Jr: Purine nucleoside phosphorylase from human erythrocytes. Content and behavior of sulfhydryl groups. J Biol Chem 246:3763-3768, 197 1 Carson DA, Kaye J, Seegmiller JE: Lymphospecific toxicity in adenosine deaminase deficiency and purine nucle-

53 1

oside phosphorylase deficiency: possible role of nucleoside kinase(s). Proc Natl Acad Sci USA 745677-5681, 1977 28. Cohen A, Doyle D, Martin DW Jr, Ammann AJ: Abnormal purine metabolism and purine overproduction in a patient deficient in purine nucleoside phosphorylase. N Engl J Med 2951449-1454, 1976 29. Siegenbeek Van Heukelom LH, Akkerman JWN, Staal GEJ, DeBruyn CHMM, Stoop JW, Zegers BJM, DeBree PK, Wadman SK: A patient with purine nucleoside phosphorylase deficiency: enzymological and metabolic aspects. Clinica Chimica Acta 74:27 1-279, 1977 30. Cohen A, Gudas LJ, Ammann AJ, Staal GEJ, Martin DW Jr: Deoxyguanosine triphosphate as a possible toxic metabolite in the immunodeficiency associated with purine nucleoside phosphorylase deficiency. J Clin Invest 61:1405-1409, 1978