Proposed Explanation for S-Adenosylhomocysteine
Hydrolase Deficiency in Purine Nucleoside
Phosphorylase and Hypoxanthine-Guanine
Phosphoribosyltransferase-deficient Patients
MICHAEL S. HERSHFIELD, Departments of Medicine and Biochemistry, Division of
Rheumatic and Genetic Diseases, Duke University Medical Center, Durham,
North Carolina 27710
A B S T R A C T We have examined the basis for the recently reported, but unexplained deficiency of S adenosylhomocysteine hydrolase (AdoHcyase) in the
erythrocytes of patients with genetic deficiencies of
purine nucleoside phosphorylase and hypoxanthineguanine phosphoribosyltransferase. We found that a
hemolysate from a patient with purine nucleoside phosphorylase deficiency had only 7% of control AdoHcyase activity, confirming the original observation. Of
the purine nucleosides known to accumulate in nucleoside phosphorylase-deficient patients, inosine
alone caused the phosphate-dependent, irreversible
inactivation of purified huiman placental AdoHcyase,
and of AdoHcyase in intact erythrocytes and cultured
lymphoblastoid cells. Hypoxanthine did not inactivate
purified AdoHcyase, but potentiated the effect of inosine in intact hypoxanthine-guanine phosphoribosyltransferase-deficient human lymphoblastoid cells. This
presumably resulted from the ability ofhypoxanthine to
shift the equilibrium of the nucleoside phosphorylase
reaction, preventing inosine breakdown. This could account for the partial AdoHcyase deficiency reported in
hypoxanthine-guanine phosphoribosyltransferasedeficient patients. We have also demonstrated the
AdoHcyase-catalyzed synthesis of S-inosylhomocysteine from inosine and L-homocysteine, a reaction
which may occur in nucleoside phosphorylasedeficient patients.
phoribosyltransferase (HGPRT) catalyze sequential
steps in the interconversion of purine nucleosides and
reutilization of purine bases. Their respective deficiencies cause a severe combined immunodeficiency
disease (ADA) (1), a selective defect in cell-mediated
immunity (PNP) (2), and the Lesch-Nyhan syndrome
(HGPRT) (3,4), in which marked urate overproduction
is accompanied by a severe neurologic disease. Recent
evidence has suggested some involvement of the enzyme S -adenosylhomocysteine hydrolase (AdoHcyase)
in each of these inborn errors of purine metabolism.
AdoHcyase catalyzes the reversible cleavage of
S-adenosylhomocysteine (AdoHcy) to adenosine and
L-homocysteine (5), a reaction which is essential to prevent the accumulation of AdoHcy, both a product and a
potent inhibitor of S -adenosylmethionine-dependent
transmethylation reactions. We have identified AdoHcyase as the major high affinity adenosine-binding
protein in the cytoplasm (6), and have shown that it may
mediate toxic effects of both adenosine and deoxyadenosine, the nucleosides which accumulate in ADA deficiency. Adenosine, by displacing the equilibrium of
the AdoHcyase reaction, causes AdoHcy accumulation
and inhibition of nucleic acid methylation in ADA-inhibited cultured lymphoid cells (7, 8). Deoxyadenosine
(and the antiviral agent adenine arabinoside) causes the
irreversible inactivation of AdoHcyase (9), such that
erythrocytes of ADA-deficient children have <2% of
normal AdoHcyase activity (10). In cultured human
INTRODUCTION
lymphoblastoid cells, this inactivation causes AdoHcy
Adenosine deaminase (ADA),' purine nucleoside phos- accumulation and contributes to the toxicity of deoxyphorylase (PNP), and hypoxanthine-guanine phos- adenosine (11). These mechanisms may contribute to
Received for publication 28 July 1980 and in revised form
10 October 1980.
'Abbreviations used in this paper: ADA, adenosine
deaminase; AdoHcy, S-adenosylhomocysteine; AdoHcyase,
696
S-adenosylhomocysteine hydrolase; Ara-A, adenine arabinoside; Ara-Hx, arabinosylhypoxanthine; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; InoHcy, S-inosylhomocysteine; PNP, purine nucleoside phosphorylase.
J. Clin. Invest. © The American Society for IClinical Investigation,
0021-9738/81/03/0696/06 $1.00
Volume 67 March 1981 696-701
Inc.
-
the lymphopenia and immune dysfunction in ADA
deficiency.
Recently, Kaminska and Fox (12) reported that PNPdeficient patients had 12-19% of normal erythrocyte
AdoHcyase activity, and Lesch-Nyhan patients,
50-60%, though the researchers were unable to define
the cause of this deficiency. They reported that none
of the purines that accumulate in PNP- and HGPRTdeficient patients affected AdoHcyase activity, and
suggested that their AdoHcyase deficiency might result
from some mechanism other than the type of inactivation caused by deoxyadenosine. We have reexamined
this question, both because of our interest in the unusual properties of AdoHcyase and because understanding the cause of its deficiency is necessary to assess its possible role in prodtucing the immunologic or neurologic abnormalities in these patients.
METHODS
Materials. Radioactive chemicals were obtained from
Amersham Corp., Arlington Heights, Ill.; nonradioactive compounds from Sigma Chemical Co., St. Louis, Mo., or P-L
Biochemicals, Inc., Milwaukee, Wise.; cellulose thin-layer
plates from Eastman Kodak, Rochester, N. Y. The nonspecific
ADA from Aspergillus oryzeae was purified (13) from Sanzyme-R (Calbiochem-Behring Corp., American Hoechst
Corp., San Diego, Calif.), and was a gift from Dr. N. Kredich.
AdoHcyase was puirified from human placenta by a procedure
to be described in detail elsewhere.2 The enzyme was
homogeneous by the criteria of sodium dodecylsulfateacrylamide gel electrophoresis and analytical ultracentrifugation, and contained 1 mol tightly bound NAD+/mol of subunit (48,000 mol wt), as described for beef liver AdoHcyase (14).
Cell growth an(l preparation of extracts. Clone 35-1, an
HGPRT-deficient mutant (15) of the WI-L2 human splenic
lymphoblastoid cell line, was grown as described (16), except
that 10o horse serum was tused instead of fetal calf serum.
For measuring AdoHcyase in extracts, the cells were harvested by brief centrifugation at 4°C, and washed once with
phosphate-btuffered NaCl; the cell pellet was frozen and
thawed three times in 0.1 ml of Tris-HCl, pH 7.4, 1 mM
Na2EDTA. After centriftugation for 2 min in a Beckman microfuge (Beckman Instruiments Inc., Ftullerton, Calif), 0.1 ml of
stupernate was passed over Sephadex G-25 as described
(11). Hemolysates prepared as described (10) were treated
similarly, and the excltuded fractionis from the G-25 columns
were assayed for AdoHcyase.
Assays. AdoHcyase was assayed in the direction of
AdoHcy synthesis from ['4C]adenosiine and L-homocysteine,
using a thin-layer chromatographic method (6, 9). In the assay
of S-inosylhomocysteine (InoHcy) synthesis by purified placental AdoHcyase, [8-'4C]inosine (15 cpm/pmol) replaced
['4C]adenosine, aind nonradioactive InoHcy (prepared by
quantitative deaminiation of AdoHey by nionspecific A.
oryzeae ADA) was tused as ai thin-layer mlarker instead of
AdoHcy. The prodict ofthe reactioni was also shown to coeltute
with InoHcy by high-press;ure liquidi chromntatography on a C,8
uBondapak reversed-phase column (Waters Associates, Inc.,
Milford, Mass.), eltuted with 50 mM soditum acetate, pH 3.9,
2
Hershfield, M. S., and W. C.
preparation.
Smiall.
Mantuscript
in
containing 3.0% methanol, flow rate 2 ml/min. Absorbance was
monitored at 280 and 254 nm with a Waters model 440 detector. In this system, InoHcy eluted at 7.2 min, inosine at 8.1
min, AdoHcy at 14.4 min, and adenosine at 20.4 min.
RESULTS
A hemolysate from a PNP-deficient patient was kindly
provided to us by Dr. S. K. Wadman, Utrecht Universiteitskinderklineik, Utrecht, The Netherlands. It
had normal levels of ADA and adenine phosphoribosyltransferase activities, but its AdoHcyase activity, 0.27
nmol/h/mg protein, was only 6- 7% of our control population erythrocyte activity (10). This confirmed the result reported by Kaminska and Fox (12), who found
values of -15% of controls. In studying possible explanations for this deficiency, they evaluated the effects
of inosine, deoxyinosine, guanosine, deoxyguanosine
(PNP substrates), and of hypoxanthine and guanine
(PNP products, HGPRT substrates) on AdoHcyase activity in hemolysates of normal individuals. To prevent
breakdown of the nucleosides by PNP present in these
hemolysates, they chose to conduct these studies in
phosphate-free buffer. Under these assay conditions,
they found no effect of any ofthe above mentioned compouinds (0.5 mM, 20- and 60-min incubations) on AdoHcyase, but did observe inactivation by deoxyadenosine, used as a "positive control."
In our published studies of AdoHcyase inactivation
we have used phosphate-containing buffers (9-11).
Furthermore, we have found that the rate of inactivation of homogeneous placental AdoHcyase by several
purines is phosphate dependent (17). Fig. 1 compares
the rates of inactivation of homogeneous human placental AdoHcyase, in the presence and absence of 10
mM phosphate, by 0.05 mM adenine arabinoside
(Ara-A), 0.2 mM arabinosylhypoxanthine (Ara-Hx), and
by 0.2 mM inosine, demonstrating the phosphate dependence of the rates of inactivation by Ara-A and inosine. No inactivation was observed with 0.2 mM inosine in the absence of phosphate, and Ara-Hx did not
inactivate even in the presence of phosphate. In similar
kinetic experiments performed in the presence of phosphate, we observed no appreciable inactivation with
0.25 mM hypoxanthine, guanine, deoxyinosine,
guanosine, or deoxyguianosine (Table I). The rate of
inactivation by inosine is pseudo-first order and saturable (Fig. 2). From these studies we determined a
K, (18) for inosine as an inactivator of 5.85 mM, with a
Vmax of 0.16 min-'. In experiments not shown, we also
found that prolonged dialysis (up to 68 h) did not restore activity to inosine-treated AdoHcyase (dialyzed,
untreated enzyme lost no activity), and that low concentrations of adenosine blocked inactivation of the
enzyme by inosine. Thus the inactivation by inosine is
irreversible and is active site directed, as we previously
showed with deoxyadenosine and adenine arabinoside
Int Vivo Inactivation of S-Adenosylhomocysteine Hydrolase by Inosine
697
z 100
(Z
E
w
z
< 50
uJ
50
?: 10
25
\
c
25
a
4 5
z
u
10 <
2.5
w
0-
5
z
w
MINUTES
(K
2.5_
w
40 -B KI =5.9mM
0
20
40
60 80
MINUTES
100
120
E-
30
0
F
8 20
FIGURE 1 Phosphate dependence of the rate of inactivation
.
rof AdoHcyase by purine nucleosides. Purified placental Ado10
Hcyase was dialyzed for 22 h at 4°C against 5,000 vol of bufC
fer containing 25 mM Tris-HCl, pH 8.5, 1 mM dithiothreitol,
and 1 mM disodium EDTA. Aliquots of dialyzed enzyme (1.24
0.2
0.4
0.6 0.8 1.0
jig) were preincubated at 37°C with the indicated concentra1/ [INOSINE], mM
tions of nucleosides, in 0.05-ml mixtures containing dialysis
buffer with (solid symbols) or without (open symbols) addition FIGURE 2 (A) Kinetics ofinactivation of placental AdoHcyase
of 10 mM potassium phosphate, pH 7.0. At the times indicated, by inosine. Purified placental AdoHcyase (0.9 ug) was incu0.005-ml aliquots were transferred to 0.05-ml assay mixtures bated with the indicated concentrations of inosine in 25 mM
(9, 10) for measuring the residual capacity to synthesize potassium phosphate, pH 7.0, 1 mM dithiothreitol, and 1 mM
AdoHcy. The control (no nucleoside during preincubation) disodium EDTA. At the indicated times 0.005-ml aliquots
had a specific activity of 3.49 ,umol/min per mg preincubated were transferred to AdoHcyase assay mixtures, and capacity
without phosphate, and 3.32 preincubated with phosphate. to synthesize AdoHcy was measured as described in Fig. 1.
Open symbols, no phosphate; solid symbols, with phosphate. (B) Reciprocal plot of first-order rate constants for inactivation
Triangles, Ara-Hx, 0.2 mM; circles, inosine, 0.2 mM; squiares, (kinactivation) calculated from (A) vs. inosine concentration.
Ara-A, 0.05 mM.
K, (18) is an index of the equilibrium constant for the enzymeinosine complex from which inactivation proceeds. The maximal rate of inactivation at saturating inosine, estimated from
(9). We have also found that inosine can replace adeno- the y intercept, is -0.16/min for the experiment shown.
sine as a substrate for human AdoHcyase, combining
with L-homocysteine to form inosylhomocysteine
(InoHcy) (Fig. 3). However, this is a very inefficient reaction, with a Km of 9.3 mM, compared with a Km
for adenosine of 1 ,uM (6). More detailed studies of the
mechanism of inactivation by inosine and effects of
InoHcy on cells will be presented elsewhere.
In other experiments (not shown), we found that exposure to 0.25 mM inosine for 2 h caused a 50% reduction of AdoHcyase activity in intact normal erythrocytes. More precise studies of inactivation kinetics in
intact erythrocytes were not attempted because of the
inability to control the phosphorolytic cleavage of inosine to hypoxanthine and ribose-l-PO4 by PNP. The
equilibrium for the PNP reaction is known to favor nucleoside formation (19), but in the intact cell the reaction is "pulled" in the direction of cleavage by removal of hypoxanthine, suggesting an explanation for
the 50-60% (12) deficiency of AdoHcyase in the
erythrocytes of HGPRT-deficient patients: higher
steady-state concentrations of guanine or hypoxanthine
(particularly the latter in the allopurinol-treated pa-
698
M. S. Hersh field
TABLE I
Effect of PNP and HGPRT Substrates on
Purified AdoHcyase
Purine (250
AuM)
AdoHcyase activity
% control
Inosine
Deoxyinosine
Guanosine
Deoxyguanosine
Hypoxanthine
Guanine
36.9
104
90.9
105.2
111.2
108.3
Purified placental AdoHcyase was dialyzed as described in
Fig. 1, and 1.4 ,ug was incubated at 37°C with the indicated
purines, 250 ,mM, in preincubation mixtures (0.05 ml) containing 25 mM potassium phosphate, pH 7.0, 1 mM dithiothreitol,
and 1 mM Na2 EDTA. At various times aliquots were removed
as described in Fig. 1 for measuring the residual capacity
to synthesize AdoHcy. Only the results from the last (90 min)
time point are shown. The control (no purine during preincubation) had a specific activity of 2.2 ,umol/min per mg.
TABLE II
0
E
Inactivation ofAdoHcyase in HGPRT-deficient Lymphoblasts
E 1.2 -A
a 1.0
u.1
AdoHcyase
X 0.8
r
0
z
remaining after
Purine (0.5 mM)
0.4
0.2
6h
24 h
% control
1
1
60
40
20
MINUTES
Hypoxanthine
Inosine
Hypoxanthine plus inosine
92
30
17
120
83
45
30-ml cultures (4.5 x 105/ml) of HGPRT-deficient human
lymphoblastoid cells were grown as described in Methods,
with no purines (control) or with 0.5 mM hypoxanthine and/or
inosine, for 6 or 24 h, at which time cells were harvested and
washed, and lysates were prepared for assays of AdoHcyase
activity as described in Methods. Cell densities increased by
2.05-2.2-fold in all cultures in 24 h. Results are expressed as
percent activity in the control lysate after 6 and 24 h.
E
-c
E
I
0
z
E
0
N.i
3
1/ [INOSINE], mM
1
2
FIGURE 3 (A) Time course of InoHcy synthesis. Purified placental AdoHcyase (1.25 ug) was incubated at 37°C in a reaction mixture (0.10 ml) containing 25 mM potassium phosphate,
pH 7.0, 1 mM dithiothreitol, 1 mM Na2 EDTA, 2.3 mM [8-14C]inosine (15 cpm/pmol), and 5 mM L-homocysteine. At the indicated times, aliquots were removed for measurement of
[14C]InoHcy formation by thin-layer chromatography (Methods). (B) Reciprocal plot of the velocity of InoHcy synthesis
(V) vs. concentration of [8-14C]inosine. Reaction mixtures
(as described in [A]) containing [8-14C]inosine at final concentrations of 0.3, 0.6, 1.2, 3, 4.5, 9, or 15 mM, were incubated
at 37°C for 30 min. [14C]InoHcy was measured as in (A). Km
= 9.3 mM. Vmax = 0.69 ,umol/min per mg.
tient) could increase the steady-state concentration of
intracellular inosine and cause a slow but progressive
inactivation of AdoHcyase activity in long-lived
erythrocytes. We have tested this hypothesis in an experiment employing cultured HGPRT-deficient human lymphoblastoid cells (Table II). Hypoxanthine
alone had little if any effect on intracellular AdoHcyase
activity, but clearly potentiated the effect of inosine.
The increase in AdoHcyase activity in the inosinetreated cultures between 6 and 24 h of incubation probably results from synthesis of new enzyme, which cannot occur in mature erythrocytes.
DISCUSSION
We have confirmed the finding of a significant (8090%) decrease in erythrocyte AdoHcyase in PNP deficiency reported by Kaminska and Fox (12), and we are
able to offer a reasonable explanation for its occurrence.
We have shown that there is a clearly demonstrable
inactivation of purified human placental AdoHcyase by
inosine in the presence of phosphate, and that inosine
inactivates intracellular AdoHcyase in cultured human
lymphoblasts and intact erythrocytes. Although inosine
is a weak inactivator (KI = 5.9 mM compared with 66
,uM for deoxyadenosine and 24 uM for adenine
arabinoside [9]), the concentration of plasma inosine in
PNP-deficient patients has been reported in the 40-100
,uM range (20, 21), whereas deoxyadenosine in the
plasma of ADA-deficient children is usually <2 AM
(10, 22). Slow, irreversible inactivation by inosine in
long-lived erythrocytes, which are incapable of new enzyme synthesis, seems the most likely explanation for
the observed deficiency of AdoHcyase. The ability of
hypoxanthine to displace the equilibrium of the
PNP reaction toward inosine formation can explain its
potentiation of the effect of inosine on AdoHcyase in
HGPRT-deficient human lymphoblasts (Table II), and
probably accounts for the lower erythrocyte AdoHcyase
deficiency that has been found in Lesch-Nyhan
patients.
Could the observed deficiencies of AdoHcyase
contribute to the clinical manifestations of HGPRT
or PNP deficiency? Abnormalities in neurotransmitter
methylation might conceivably play some role in producing the motor disturbance or self-mutilation that
occurs in the Lesch-Nyhan syndrome. However, since
more extensive AdoHcyase inactivation in both ADA
and PNP deficiency does not result in any neurologic
disease, this seems unlikely. More consideration
needs to be given to the possibility that interaction of
intracellular AdoHcyase with inosine contributes to the
lymphopenia and defect in cell-mediated immunity
in PNP deficiency. Both lymphopenia and AdoHcyase
inactivation do occur in ADA deficiency, and selec-
In Vivo Inactivation of S-Adenosylhomocysteine Hydrolase by Inosine
699
tive lymphopenia has also recently been reported to
occur in Ara-A-treated patients (23), in whose blood
cells AdoHcyase inactivation has been observed (24,
25, and our unpublished studies). However, inosine is
not likely to produce as complete inactivation of
AdoHcyase in cells capable of protein synthesis as
either deoxyadenosine or Ara-A.
We have shown that inosine, unlike deoxyadenosine
or Ara-A, can also act as a nucleoside substrate in the
formation of a thioether bond with L-homocysteine.
Though the Km for inosine is nearly four orders of magnitude higher than that of adenosine, there has been a
report of the detection of trace amounts of InoHcy in
mouse lymphoblasts exposed to 150 ,uM inosine (26).
Although some accumulation of InoHcy in cells of PNPdeficient patients may occur, InoHcy is probably much
less effective than AdoHcy as an inhibitor of methylation reactions. Inosine is virtually nontoxic to PNP-deficient mouse lymphoblasts (27), in marked contrast to
the AdoHcy-mediated toxicity of adenosine to ADAinhibited mouse (7) and human (8) lymphoblasts. LHomocysteine greatly potentiates adenosine toxicity
by augmenting AdoHcy synthesis catalyzed by
AdoHcyase. In preliminary experiments we have found
that 1 mM inosine did not inhibit growth of WI-L2
lymphoblasts, alone or in the presence of 1 mM Lhomocysteine.
Although additional studies in PNP-deficient
lymphoid cells would be uiseful, it presently seems unlikely that inosine produces sufficient AdoHcyase inactivation to cause significant accumulation of AdoHcy,
or that its conversion to InoHcy by AdoHcyase is sufficiently toxic to contribute to the selective T cell defect
in PNP deficiency. The more severe defects in both T
and B cell development in ADA deficiency may, however, be attributable in part to the far more potent interactions of both adenosine and deoxyadenosine with
AdoHcyase, a hypothesis under investigation.
ACKNOWLE DGM E NTS
We appreciate the expert technical assistance of W. Curtis
Small and Joan Fetter. We are grateful to Dr. S. K. Wadman for
providing us with blood from a PNP-deficient patient.
Dr. Hershfield is a recipient of a Research Career Development Award AM 00434 and was supported by Research Grant
AM 20902 and by a Basil O'Connor Starter Research Grant
from the National Foundation-March of Dimes.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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701