Vol. 87, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 380487
March 30, 1979
INCREASED PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE ACTIVITY IN FIBROBLASTS OF HYPOXANTHINE-GLJANINE PHOSPHORIBOSYL TRANSFERASE DEFICIENT PATIENTS B. Marina
Torrelio
and Mercedes
A. Paz
Departments of Orthopaedic Surgery and Oral Hospital Medical Center and Harvard School Boston, Massachusetts 02115
Children's Received
February
Biology, of Dental
Medicine
5,1979
SUNMARY: An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This excessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.
The Lesch-Nyhan
INTRODUCTION:
ciated
with
a severe
thine-guanine terized
by spastic
partial
cerebral
deficiency
somatic
opment;
heterozygous
is
loci
(6).
for
activity
(7).
has generally
As a result
normal
In these
cell
hemolyzates
between
In the excessive of purine
nucleotides,
phosphoribosyl-1 Abbreviations:
0006-291 Copyright All rights
red blood
the role
pyrophosphate
of uric
@ 1979
According
fibroblasts having
from
normal selection.
deficiency state, as in gout (9), HGPRT activities
(PP-ribose-P),
have been
found
intracellular the precursor
devel-
of x-linked
deficient
as a consequence
with to
of embryonic
of cell
of increased
380
stage
population
acid
A
in each fe-
cell
HGPRT, hypoxanthine-guanine PP-ribose-P, 5-phosphoribosyl-1 APRT, adenine phosphoribosyl
by Academic Press. Inc. in any form reserved.
(2).
as a result
the partial metabolic
characcompulsive
acid
shown two populations
and the other
X/79/060380-08$01.00/0 of reproduction
have
is
in some patients
to the expression of skin
22% and 75% of normal
production
of uric (3,4).
at an early
cultures
asso-
choreoathetosis,
been described
respect
and probably
other hand, in heterozygotes for of l-2% of patients with primary
it
mammals one x-chromosome
HGPRT activity (8)
disorder
enzyme hypoxan-
Clinically,
excretion
HGPRT deficiency
heterozygotes
been found
(1).
abnormalities
inactivated with
recessive pathway
retardation,
eutherian
mosaics
the severe
one having
mental
neurological
randomly thus
salvage
(HGPRT)
enzyme has also
(5),inmost
are
an x-linked
and excessive
apparent
cell
females
heterozygotes cells,
palsy,
of this
without
the Lyon Hypothesis male
transferase
hyperuricemia,
gout
is
of the purine
phosphoribosyl
self-mutilation, severe
syndrome
deficiency
obligate of HGPRT activity On the the case in red
(10). of overproduction
concentration
of 5-
of the ribose-phosphate
phosphoribosyl transferase; pyrophosphate; transferase.
BIOCHEMICAL
Vol. 87, No. 2, 1979
moiety
of purine,
pyrimidine
number
of studies
(11,X).
PP-ribose-P
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and pyridine
an activated
sugar
nucleotides
phosphate
has been emphasized
compound which
in a
is synthesized
from ATP and ribose-5-phosphate in a reaction which is magnesium and inorganic phosphate dependent and catalyzed by the enzyme PP-ribose-P synthetase (E-C. 2.7.6.1).
The PP-ribose-P
purine
synthesis
transferase in which
catalyzed
as well purine
are
An HGPRT deficient by two mechanisms: purine
(b)
decrease
tide
(IMP,
level is
ciated
with
an altered
a Lesch-Nyhan synthetase is
not
and his is found
seen in fibroblast
first
in reactions
catalyzed adenine
purine
overproduction
, purine
level; step
of -de and
--de novo synthesis; due to decrease in nucleohand,
a depletion
analogs
of the
or pyrimidines
of both
--de novo and salvage pathway of regulatory role of PP-ribose-P in these in the rate
synthetase
mother,
in which
synthesis
increased phosphate
asso-
(15,16).
of the disorder
in fibroblasts
of inorganic derived
of purine
of PP-ribose-P feature
system
at low levels cultures
nucleotides
rate-limiting
On the other
concentration
carrier
purine
of the PP-ribose-P
stimulates
a new and additional
PP-ribose-P
patient activity
the
by purines
activity
intracellular
to promote
of the same step
by the alterations
reveal
for
and the autosomal-linked
elevation
The important
(15,16).
altered
for
(13,14).
decreased
is emphasized
Our studies namely
its
pathway
and elevation
PP-ribose-P
with
of --de novo amido-
2.4.2.6).
considered
inhibition
GMP) concentrations
synthesis
pathways
this
hence
of intracellular
purine
is
(E.C.
as the substrate
of feedback
associated
2.4.2.7)
(APRT) state
synthesis,
reaction
to form nucleotides
(a) accumulation
serves
salvage
re-utilized
transferase
compound
in the first
by the enzyme phosphoribosylpyrophosphate
enzyme HGPRT (E.C.
phosphoribosyl
noto
a substrate
as in the so-called
bases
by the x-linked
this
is
cells,
derived
from both
PP-ribose-P concentration;
from normals.
AND METHODS: Materials: Ribose 5-phosphate and ATP were purchased -from Boehringer-Mannheim, yeast orotate phosphoribosyl transferase and phosphoribosyl pyrophosphate were from Sigma Chemical Co. The radioactive compounds were obtained from New England Nuclear. The NCS (trademark) tissue solubilizer and Spectrafluor liquid scintillator were from Amersham-Searle.
MATERIALS
Skin fibroblasts from a 12-year-old boy with the Lesch-Nyhan syndrome (GM 2227) and his 45-year-old mother (GM 2226) were obtained from the Institute for Medical Research, Camden, New Jersey. Control skin fibroblasts (GM 730) were also obtained from the same source as well as from a skin biopsy performed in our laboratory (C.A.). The primary explant was cultured in a modified Eagle's Minimum Essential Medium (17) supplemented with 2mM glutamine, 2OmM NaHC03, 15% fetal calf serum and penicillin and Streptomycin. All the cells were subcultured at confluency in the same medium with 10% fetal calf serum and without antibiotics. To determine the number of population doublings the cells were counted using an hemocytometer after each trypsinization and were always seeded at the same cell density. The cells were examined for mycoplasma contamination by the procedures of Levine (18), Chen (19) and by culturing in different media.
381
Vol. 87, No. 2, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The functional activities of the enzymes HGPRT and APRT radiochemically in intact fibroblasts by measuring the rate of [3H]-hypoxanthine (5 Ci/mmol) and [3H]-adenine (20.2 Ci/mmol) phosphorylated products. In addition the incorporation of the bases into nucleic acids was measured.
were determined conversion of to their ribolabelled purine
PP-ribose-P synthetase in dialyzed cell-free extracts was determined radiochemically at saturating concentrations of the respective substrates. The procedure used is a modification of the method described by Reem (20) which is based on the assay by Kornberg et al. (21). The PP-ribose-P generated from ATP and ribose-5-phosphate is measured by determining [14C]CO2 release from [14C-carboxyl]-orotic acid (41.25 mCi/mmol). Orotidylate and uridylate synthesis were catalyzed by the addition of a partially purified extract of yeast orotate phosphoribosyl transferase (orotidine-5'-phosphate: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.10, and oritidine-5'-phosphate decarboxylase, E.C. 4.11.23). This method is suitable for measuring PP-ribose-P synthetase activity in a one-step assay. The assays were carried out in Warburg flasks containing in the center well a roll of Whatman #3 filter paper (1.6 x 3.3 cm) wetted with 130 ul of NCS tissue solubilizer. One hour prior to harvesting the cells the medium was replaced with fresh medium containing 10% dialyzed calf serum. The medium was separated from the cells, which were washed three times in cold phosphatebuffered saline, pH 7.3 and were harvested by scraping in 1 ml of 1 mM phosphate buffer, pH 7.3, containing 1 mM EDTA and 1 r&l B-mercaptoethanol. After the cells were harvested they were sonicated for 20 seconds using a Biosonik III at 20% output, and an aliquot was removed for protein determination. For PP-ribose-P determination the suspension was immediately heated to 95°C for 60 seconds, chilled at 4°C and centrifuged to remove proteins. To measure PPribose-P synthetase the suspension after centrifugation was dialyzed for 3 hours against three changes of 1 mM phosphate buffer, pH 7.3 containing 1 mM EDTA and 1 ti S-mercaptoethanol. The reaction mixture for the determination of PP-ribose-P synthetase was incubated at 37'C at pH 7.3 in a total volume of 500 ul and contained various concentrations of inorganic phosphate ranging from 1 to 50 mM, 2.5 mM ATP, 2.5 mM ribose-5-phosphate, 2.5 mM MgC12, 1.0 mg of yeast orotate phosphoribosyl transferase and 0.25 pCi in a volume of 10 ul of [14C-carboxyl]erotic acid. The reaction was started by adding 50A of the cell extract placed in the side arm to the reaction mix at the bottom of the Warburg flask which had been warmed at 37°C. At the end of the incubation period the reaction was stopped by placing the Warburg flasks in ice. The paper rolls were immediately removed and placed in a scintillation vial containing 0.2 ml of H20. After the addition of 15 ml of a toluene-based scintillation fluid the vials were counted in a Beckman Scintillator counter with 94% efficiency. The reaction was dependent on ribose-5-P, ATP and Mg and was stimulated by inorganic phosphate. The reaction was linear up to 30 minutes and proportional to protein added. The amount of protein added in 50 ul volume was maintained practically constant in all the determinations. Suitable blanks containing the same amount of heat inactivated enzyme and for each inorganic phosphate concentration were run in parallel. For the determination of PP-ribose-P content the ATP and ribose-5-phosphate were omitted from the reaction mixture and 50X of the heated cell extract was used. The amount of PP-ribose-P was calculated from standard solutions of PP-ribose-P which were run in parallel; the results were corrected for the blank values obtained without added PP-ribose-P. Assays were linear with PP-ribose-P concentrations ranging from 0.05 mM to 10 mM and in 15 min incubation recovery of PP-ribose-P was complete. All the studies were carried out on freshly harvested cells. For each cell extract the results represent the average of at least five determinations. The method of Lowry et al. (22) was used for protein determination.
382
BIOCHEMICAL
Vol. 87, No. 2, 1979
RESULTS:
The incorporation
studies
fibroblasts
in the logarithmic
Practically
no [3H]-hypoxanthine
cleic
acids
2226)of
after
2 hrs.
of
phase
patient
and nucleic
acids
than
the incorporation of
skin
fibroblasts pathway
provides (23,24).
level,
These
ferent ribose-P
levels
mM inorganic
phosphate doublings vity
in
It
the Lesch-Nyhan
of the PP-ribose-P logarithmic
phase
phase.
Similar
observations
maximum activity cultures
were
was generally
in skin
after
As shown in Figure
of the fibro-
at 50 mM inorganic of PP-ribosepopulation The acti-
was higher
during
and late
the
stationary
and Maler
(25).
The
seeding,
but
in some
seeding.
2, fibroblast
at difthe PP-
the normal
fibroblasts.
after
synthetase or its might contribute
the
at 5 mM and 50
increasing
in early
by Martin
24 hours
gested an abnormality in PP-ribose-P which catalyzed PP-ribose-P formation patient.
carrier
48 hours
in
for
that
the activities
fibroblasts
reported
evident fibroblasts
than
with
and decreased
observed
the maximum was observed
Lesch-Nyhan
that
and heterozygous
of growth
fibroblasts
at 5 mM than
increased
synthetase
early
normal is
in the
carrier
is noteworthy
Pi concentrations
It
investi-
levels
synthetase
are higher
the heterozygous
is much more marked
concentrations. at both
of the PP-ribose-P
in
doublings
activity
as for 1.
by other
population
synthetase as well
concentrations and in
The difference
P synthetase
fibroblasts
observed
of the salvage
has been found at different
are shown in Figure
and the activity
patient
to what
The
The use of intact
doublings.
repeated
in
Never-
fibroblasts.
the range
These
results
sug-
regulation; this enzyme to the defect in the extracts
from
the Lesch-
Nyhan patient, in the presence of saturating amounts of ATP and ribose-5phosphate and at inorganic phosphate concentrations from 1 to 50 mM, were found to have higher PP-ribose-P synthetase The fibroblast extracts from the heterozygous level activity patient tested
between
normal
fibroblasts
of the PP-ribose-P compared with the and indeed
and her
synthetase controls is
activity carrier
Lesch-Nyhan
than normal fibroblasts. showed intermediate son.
The difference
in fibroblasts from the Lesch-Nyhan clearly seen at all Pi concentrations
even more significantly
in the physiological
383
(GM
of approx-
doublings.
of the functioning
and PP-ribose-P
doublings
phosphate
Lesch-Nyhan
appraisal were
and heterozygous
population
blasts.
content
individuals
son's
cases was within
similar
studies
in her
mother
of f3H]-hypoxanthine
of normal
population
an accurate
similar results. The PP-ribose-P
Lesch-Nyhan
in all
at similar
at the cellular
gators with
[3H]-adenine
or in nu-
the heterozyqous
incorporation
than
of HGPRT.
nucleotides
the same population
was much higher
fibroblasts
from
lower
GM 2227
deficiency
in soluble
fibroblasts
the same age and approximately
in Lesch-Nyhan
the extreme
Fibroblasts
theless
incorporation
confirmed
showed
imately
normal
[3H]-hypoxanthine
was incorporated
incubation.
the Lesch-Nyhan
nucleotides
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
range.
in
BIOCHEMICAL
Vol. 87, No. 2, 1979
NORMAL SKIN FISROBLASTS _. P .;
1007
3
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LESCH-NYHAN PATIENT
HETEROZYGOUS CARRIER
I
l-l'
go
rroo
90 902 E 70 3 60 .c s 50;
4o E 302 20, t
&IIi
10
2.9 ’14.2 131I POPULATlON
DOUBLINGS
Figure 1: Catalytic activities of PP-ribose-P synthetase 5 mM and 50 mM Pi and PP-ribose-P content in normal skin Nyhan and heterozygous carrier skin fibroblasts.
in the presence of fibroblasts, Lesch-
Pi (mM) Figure 2: Activity of PP-ribose-P synthetase in normal skin fibroblast tracts (A---A), Lesch-Nyhan patient (m) and his mother (G--V fibroblast extracts as a function of added inorganic phosphate (Pi).
DISCUSSION:
The stimulatory
has been demonstrated ies (26,27) and could purine
--de nova in extensive pharmacological, clinical and kinetic studbe a major part of the biochemical defect underlying
overproduction.
has been ascribed
effect
The increased to both
excessive
of PP-ribose-P
cellular PP-ribose-P
384
on purine
ex) skin
concentration synthetase
synthesis
of PP-ribose-P activity
(20,25,
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
utilization
by HGPRT, the purine
Vol. 87, No. 2, 1979
28,29)
and to decreased
has been considered The Lesch-Nyhan both
deficient
may be responsible
in the patient's
was reported with
to extents
equal
selected
that
tase
activity
reduced
is no direct
Graf
(29)
induced
mutant than
PP-ribose-P
This
observation type
in erythrocytes
observed
of activity
studies
deficiency
in
en-
of --de novo cells
of the --de novo pursynthe-
PP-ribose-P
the presence
of saturating
at low phosphate PP-ribose-P
concen-
levels
were is
gouty
in
an obligate for
both
enzyme less
responsive
phosphate.
If, for
reported
by deVries
synthetase
was only
phosphate
of the Lesch-Nyhan
heterozygous
with
Based on the hypothesis
and codes
at all
patients with our patients,
catalytic
a marked
that
and regulatory
to metabolic
control
as has been proposed HGPRT, is x-linked,
of HGPRT mutations.
via
concentration.
and the other, of the
HGPRT gene is bifunc(29),
might also synthetase
the concentration
(35,36),
the PP-ribose-P also
385
and Sperling observed
deficiency
the possibility
The inability
inorganic
syndrome
functions
of HGPRT catalytic activity this gene has on PP-ribose-P
study,
normal HGPRT and one having a severe
partial
the x-linked
(33)
from one of
was apparent
inorganic
on hyperuricemic in contrast with
et al.
the case in the present
patient
of erythrocyte
by Becker
and in fibroblasts
of the PP-ribose-P
range
that causes a deficiency regulatory function that
effects
acid
of HGPRT and an associated
as is
activity
of HGPRT and the stigmata
that
rate
hepatoma
rate
been found
brothers
In the
This
same enzyme.
like
levels.
the complete
rat
to the increased
cells,
synthetase
activity
(33,34)
mother,
tional
found
of the patient's PP-ribose-P synthegreater than normal at all the phos-
has also
patients'
concentrations. increased
APRT activities. his
with
is more marked
may relate
PP-ribose-P
the physiological
Both
the effect
of two hyperuricemic
increased
phosphate in
en-
and uric
an enhanced
the enhanced
In our study
of mutation
In these
the patients.
the
Both
fibroblasts.
A similar
(34)
have
the degree
acid
purine
was due to increased
to the loss
tested;
between and uric
HGPRT deficient
that
cells
utilization.
concentrations
correlation
than many patients
et al.
They proposed
in these
the patient's
the
here
of PP-ribose-P
of HGPRT overproduce
in chemically
rather
trations.
activity.
levels
of purines
amounts of ATP and ribose-5-P the activity tase in the fibroblast extracts was found phate
(30).
studied
synthetase
the increased
to or even greater
in culture.
synthesis
there
deficiencies
(31,32).
synthesis
ine
for
of PP-ribose-P
fibroblasts
PP-ribose-P
and the overproduction
partial
zyme deficiency purine
consumer
carrier
enzyme that
fibroblasts.
of HGPRT deficiency Patients
intracellular
and heterozygous
HGPRT and increased
zyme defects It
to be a major
patient
salvage
exists
of the PP-ribose-P
a mutation
alter the making the of inorganic synthetase of polar synthetase
gene, genetic en-
BIOCHEMICAL
Vol. 87, No. 2, 1979
zyme, derived al levels
of inorganic
synthetase Lesch-Nyhan purine
from cells from normal cells
which
with
HGPRT deficiency,
phosphate, cells,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the behavior
results
in
can contribute
to be inhibited seen with
increased
significantly
levels
by physiologic-
the PP-ribose-P of PP-ribose-P
to augmented
in
--de novo
synthesis.
ACKNOWLEDGEMENTS: We thank Dr. Paul M. Gallop for helpful discussions and critical reading of the manuscript and Kathleen S. Pearson and Elizabeth Erickson for expert technical assistance. Supported by NIA Research Grant AG-376-04. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Seegmiller, J.E., Rosenbloom, F.M., and Kelley, W.N. (1967) Science 152, 1682-1684. Nyhan, W.L., Oliver, W.J., and Lesch, M. (1965) J. Pediat. 67, 257-263. Kelley, W.N., Rosenbloom, F.M., and Henderson, J.F. (1967) Proc. Natl. Acad. Sci. USA 57, 1735-1739. Emmerson, B.T., and Wyngaarden, J.B. (1969) Science 166, 1533-1535. Lyon, M.F. (1961) Nature (London) 190, 372-373. Beutler, E., Yeh, M., Fairbanks, U.F. (1962) Proc. Natl. Acad. Sci. USA 48, 9-16. Migeon, B.R., der Kaloustian, W.M., Nyhan, W.L., Young, W.S., and Childs, B. (1968) Science 160, 425-427. Nyhan, W.L., Bakay, B., Connor, J.D., Marks, J.F., and Keele, D.K. (1970) Proc. Natl. Acad. Sci. USA 65, 214-218. Yu, T.G., Balis, M.E., Krenitsky, T.A., Dancis, J., Silvers, D.N., Elion, G.B., and Gutman, A.B. (1972) Ann. Intern. Med. 76, 255-264. Emerson, B.T., Thompson, C.J., and Wallace, D.C. (1972) Ann. Intern. Med. 76, 285-287. Rosenbloom, F.M., Henderson, J.F., Caldwell, J.C., Kelley, W.N., and Seegmiller, J.E. (1968) J. Biol. Chem. 243, 1166-1173. Becker, M.A. (1976) J. Clin. Invest. 57, 308-318. Holmes, E.W., Wyngaarden, J.B., and Kelley, W.N. (1973) J. Biol. Chem. 248, 6035-6040. Greene, M.L. and Seegmiller, J.E. (1969) Arthr. Rheum. 12, 666-667. Boyle, J.A., Raivio, K.O., Becker, M.A., and Seegmiller, J.E. (1972) Biochim. Biophys. Acta 269, 179-183. Kelley, W.N., Fox, I.H., and Wyngaarden, J.B. (1970) Biochim. Biophys. Acta 215, 5120. Cristofalo, V.J. and Sharf, B.B. (1973) Exp. Cell Res. 76, 419-427. Levine, E.M. (1974) in Methods in Cell Biology, Volume 8, D.M. Prescott, Ed. (Academic Press, London and New York, 1974), pp. 229-248. Chen, T.R. (1977) Exp. Cell Res. 104, 255-262. Reem, G.H. (1975) Science 190, 1098-1099. Kornberg, A., Lieberman, I., and Simms, E.S. (1955) J. Biol. Chem. 215, 389-402. Lowry, O.H., Rosebrough, N.H., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275. Holland, M.J.C.; DiLorenzo, A.M., Dancis, J., Balis, M.E., Yu, T.F., and Cox, R.P. (1976) J. Clin. Invest. 57. 1600-1605. Friedman, T., Seegmiller, J.E., Subak-Sharpe, J.H. (1969) Exp. Cell Res. 56, 425. Martin, D.W., Jr., and Maler, A. (1976) Science 193, 408-411. Kelley, W.N., Fox, I.H., Beardmore, T.D., and Meade, J.C. (1971) Ann. N.Y. Acad. Sci. 179, 588-595. Becker, M.A., and Seegmiller, J.E. (1974) Ann. Rev. Med. 25, 15-28.
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28. 29. 30. 31. 32. 33. 34. 35. 36.
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sperling, O., Boer, P., Per-sky-Brosh, S., Kanarek, E., and devries, A. (1972) Rev. Eur. Etud. Clin. Biol. 17, 703-706 (1972). Graf, L.H., Jr., McRoberts, J.A., Harrison, T.M., and Martin, D.W., Jr. (1976) J. Cell Physiol. 88, 331-342. Gordon, R.B., Thompson, L., and Emmerson, B.T. (1974) Metabolism 23, 921-927. Emmerson, B.T., and Thompson, C.J. (1973) Quart. J. Med. 42, 423-440. Kogut, M.D., Donnell, E.N., Nyhan, W.L., and Sweetman, L. (1970) Am. J. Med. 48, 148-161. Becker, M.A., Meyer, L.J., Wood, A.W., and Seegmiller, J.E. (1973) Science 179, 1123-1126. de Vries, A., and Sperling, 0. (1973) Urologe A. 12, 153-157. Zoref, E., de Vries, A., and Sperling, 0. (1977) Hum. Hered. 27, 73-80. Yen, R.C.K., Adams, W.B., Lazar, C., and Becker, M.A. (1978) Proc. Natl, Acad. Sci. USA 75, 482-485.
387
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 380487
March 30, 1979
INCREASED PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE ACTIVITY IN FIBROBLASTS OF HYPOXANTHINE-GLJANINE PHOSPHORIBOSYL TRANSFERASE DEFICIENT PATIENTS B. Marina
Torrelio
and Mercedes
A. Paz
Departments of Orthopaedic Surgery and Oral Hospital Medical Center and Harvard School Boston, Massachusetts 02115
Children's Received
February
Biology, of Dental
Medicine
5,1979
SUNMARY: An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This excessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.
The Lesch-Nyhan
INTRODUCTION:
ciated
with
a severe
thine-guanine terized
by spastic
partial
cerebral
deficiency
somatic
opment;
heterozygous
is
loci
(6).
for
activity
(7).
has generally
As a result
normal
In these
cell
hemolyzates
between
In the excessive of purine
nucleotides,
phosphoribosyl-1 Abbreviations:
0006-291 Copyright All rights
red blood
the role
pyrophosphate
of uric
@ 1979
According
fibroblasts having
from
normal selection.
deficiency state, as in gout (9), HGPRT activities
(PP-ribose-P),
have been
found
intracellular the precursor
devel-
of x-linked
deficient
as a consequence
with to
of embryonic
of cell
of increased
380
stage
population
acid
A
in each fe-
cell
HGPRT, hypoxanthine-guanine PP-ribose-P, 5-phosphoribosyl-1 APRT, adenine phosphoribosyl
by Academic Press. Inc. in any form reserved.
(2).
as a result
the partial metabolic
characcompulsive
acid
shown two populations
and the other
X/79/060380-08$01.00/0 of reproduction
have
is
in some patients
to the expression of skin
22% and 75% of normal
production
of uric (3,4).
at an early
cultures
asso-
choreoathetosis,
been described
respect
and probably
other hand, in heterozygotes for of l-2% of patients with primary
it
mammals one x-chromosome
HGPRT activity (8)
disorder
enzyme hypoxan-
Clinically,
excretion
HGPRT deficiency
heterozygotes
been found
(1).
abnormalities
inactivated with
recessive pathway
retardation,
eutherian
mosaics
the severe
one having
mental
neurological
randomly thus
salvage
(HGPRT)
enzyme has also
(5),inmost
are
an x-linked
and excessive
apparent
cell
females
heterozygotes cells,
palsy,
of this
without
the Lyon Hypothesis male
transferase
hyperuricemia,
gout
is
of the purine
phosphoribosyl
self-mutilation, severe
syndrome
deficiency
obligate of HGPRT activity On the the case in red
(10). of overproduction
concentration
of 5-
of the ribose-phosphate
phosphoribosyl transferase; pyrophosphate; transferase.
BIOCHEMICAL
Vol. 87, No. 2, 1979
moiety
of purine,
pyrimidine
number
of studies
(11,X).
PP-ribose-P
is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and pyridine
an activated
sugar
nucleotides
phosphate
has been emphasized
compound which
in a
is synthesized
from ATP and ribose-5-phosphate in a reaction which is magnesium and inorganic phosphate dependent and catalyzed by the enzyme PP-ribose-P synthetase (E-C. 2.7.6.1).
The PP-ribose-P
purine
synthesis
transferase in which
catalyzed
as well purine
are
An HGPRT deficient by two mechanisms: purine
(b)
decrease
tide
(IMP,
level is
ciated
with
an altered
a Lesch-Nyhan synthetase is
not
and his is found
seen in fibroblast
first
in reactions
catalyzed adenine
purine
overproduction
, purine
level; step
of -de and
--de novo synthesis; due to decrease in nucleohand,
a depletion
analogs
of the
or pyrimidines
of both
--de novo and salvage pathway of regulatory role of PP-ribose-P in these in the rate
synthetase
mother,
in which
synthesis
increased phosphate
asso-
(15,16).
of the disorder
in fibroblasts
of inorganic derived
of purine
of PP-ribose-P feature
system
at low levels cultures
nucleotides
rate-limiting
On the other
concentration
carrier
purine
of the PP-ribose-P
stimulates
a new and additional
PP-ribose-P
patient activity
the
by purines
activity
intracellular
to promote
of the same step
by the alterations
reveal
for
and the autosomal-linked
elevation
The important
(15,16).
altered
for
(13,14).
decreased
is emphasized
Our studies namely
its
pathway
and elevation
PP-ribose-P
with
of --de novo amido-
2.4.2.6).
considered
inhibition
GMP) concentrations
synthesis
pathways
this
hence
of intracellular
purine
is
(E.C.
as the substrate
of feedback
associated
2.4.2.7)
(APRT) state
synthesis,
reaction
to form nucleotides
(a) accumulation
serves
salvage
re-utilized
transferase
compound
in the first
by the enzyme phosphoribosylpyrophosphate
enzyme HGPRT (E.C.
phosphoribosyl
noto
a substrate
as in the so-called
bases
by the x-linked
this
is
cells,
derived
from both
PP-ribose-P concentration;
from normals.
AND METHODS: Materials: Ribose 5-phosphate and ATP were purchased -from Boehringer-Mannheim, yeast orotate phosphoribosyl transferase and phosphoribosyl pyrophosphate were from Sigma Chemical Co. The radioactive compounds were obtained from New England Nuclear. The NCS (trademark) tissue solubilizer and Spectrafluor liquid scintillator were from Amersham-Searle.
MATERIALS
Skin fibroblasts from a 12-year-old boy with the Lesch-Nyhan syndrome (GM 2227) and his 45-year-old mother (GM 2226) were obtained from the Institute for Medical Research, Camden, New Jersey. Control skin fibroblasts (GM 730) were also obtained from the same source as well as from a skin biopsy performed in our laboratory (C.A.). The primary explant was cultured in a modified Eagle's Minimum Essential Medium (17) supplemented with 2mM glutamine, 2OmM NaHC03, 15% fetal calf serum and penicillin and Streptomycin. All the cells were subcultured at confluency in the same medium with 10% fetal calf serum and without antibiotics. To determine the number of population doublings the cells were counted using an hemocytometer after each trypsinization and were always seeded at the same cell density. The cells were examined for mycoplasma contamination by the procedures of Levine (18), Chen (19) and by culturing in different media.
381
Vol. 87, No. 2, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The functional activities of the enzymes HGPRT and APRT radiochemically in intact fibroblasts by measuring the rate of [3H]-hypoxanthine (5 Ci/mmol) and [3H]-adenine (20.2 Ci/mmol) phosphorylated products. In addition the incorporation of the bases into nucleic acids was measured.
were determined conversion of to their ribolabelled purine
PP-ribose-P synthetase in dialyzed cell-free extracts was determined radiochemically at saturating concentrations of the respective substrates. The procedure used is a modification of the method described by Reem (20) which is based on the assay by Kornberg et al. (21). The PP-ribose-P generated from ATP and ribose-5-phosphate is measured by determining [14C]CO2 release from [14C-carboxyl]-orotic acid (41.25 mCi/mmol). Orotidylate and uridylate synthesis were catalyzed by the addition of a partially purified extract of yeast orotate phosphoribosyl transferase (orotidine-5'-phosphate: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.10, and oritidine-5'-phosphate decarboxylase, E.C. 4.11.23). This method is suitable for measuring PP-ribose-P synthetase activity in a one-step assay. The assays were carried out in Warburg flasks containing in the center well a roll of Whatman #3 filter paper (1.6 x 3.3 cm) wetted with 130 ul of NCS tissue solubilizer. One hour prior to harvesting the cells the medium was replaced with fresh medium containing 10% dialyzed calf serum. The medium was separated from the cells, which were washed three times in cold phosphatebuffered saline, pH 7.3 and were harvested by scraping in 1 ml of 1 mM phosphate buffer, pH 7.3, containing 1 mM EDTA and 1 r&l B-mercaptoethanol. After the cells were harvested they were sonicated for 20 seconds using a Biosonik III at 20% output, and an aliquot was removed for protein determination. For PP-ribose-P determination the suspension was immediately heated to 95°C for 60 seconds, chilled at 4°C and centrifuged to remove proteins. To measure PPribose-P synthetase the suspension after centrifugation was dialyzed for 3 hours against three changes of 1 mM phosphate buffer, pH 7.3 containing 1 mM EDTA and 1 ti S-mercaptoethanol. The reaction mixture for the determination of PP-ribose-P synthetase was incubated at 37'C at pH 7.3 in a total volume of 500 ul and contained various concentrations of inorganic phosphate ranging from 1 to 50 mM, 2.5 mM ATP, 2.5 mM ribose-5-phosphate, 2.5 mM MgC12, 1.0 mg of yeast orotate phosphoribosyl transferase and 0.25 pCi in a volume of 10 ul of [14C-carboxyl]erotic acid. The reaction was started by adding 50A of the cell extract placed in the side arm to the reaction mix at the bottom of the Warburg flask which had been warmed at 37°C. At the end of the incubation period the reaction was stopped by placing the Warburg flasks in ice. The paper rolls were immediately removed and placed in a scintillation vial containing 0.2 ml of H20. After the addition of 15 ml of a toluene-based scintillation fluid the vials were counted in a Beckman Scintillator counter with 94% efficiency. The reaction was dependent on ribose-5-P, ATP and Mg and was stimulated by inorganic phosphate. The reaction was linear up to 30 minutes and proportional to protein added. The amount of protein added in 50 ul volume was maintained practically constant in all the determinations. Suitable blanks containing the same amount of heat inactivated enzyme and for each inorganic phosphate concentration were run in parallel. For the determination of PP-ribose-P content the ATP and ribose-5-phosphate were omitted from the reaction mixture and 50X of the heated cell extract was used. The amount of PP-ribose-P was calculated from standard solutions of PP-ribose-P which were run in parallel; the results were corrected for the blank values obtained without added PP-ribose-P. Assays were linear with PP-ribose-P concentrations ranging from 0.05 mM to 10 mM and in 15 min incubation recovery of PP-ribose-P was complete. All the studies were carried out on freshly harvested cells. For each cell extract the results represent the average of at least five determinations. The method of Lowry et al. (22) was used for protein determination.
382
BIOCHEMICAL
Vol. 87, No. 2, 1979
RESULTS:
The incorporation
studies
fibroblasts
in the logarithmic
Practically
no [3H]-hypoxanthine
cleic
acids
2226)of
after
2 hrs.
of
phase
patient
and nucleic
acids
than
the incorporation of
skin
fibroblasts pathway
provides (23,24).
level,
These
ferent ribose-P
levels
mM inorganic
phosphate doublings vity
in
It
the Lesch-Nyhan
of the PP-ribose-P logarithmic
phase
phase.
Similar
observations
maximum activity cultures
were
was generally
in skin
after
As shown in Figure
of the fibro-
at 50 mM inorganic of PP-ribosepopulation The acti-
was higher
during
and late
the
stationary
and Maler
(25).
The
seeding,
but
in some
seeding.
2, fibroblast
at difthe PP-
the normal
fibroblasts.
after
synthetase or its might contribute
the
at 5 mM and 50
increasing
in early
by Martin
24 hours
gested an abnormality in PP-ribose-P which catalyzed PP-ribose-P formation patient.
carrier
48 hours
in
for
that
the activities
fibroblasts
reported
evident fibroblasts
than
with
and decreased
observed
the maximum was observed
Lesch-Nyhan
that
and heterozygous
of growth
fibroblasts
at 5 mM than
increased
synthetase
early
normal is
in the
carrier
is noteworthy
Pi concentrations
It
investi-
levels
synthetase
are higher
the heterozygous
is much more marked
concentrations. at both
of the PP-ribose-P
in
doublings
activity
as for 1.
by other
population
synthetase as well
concentrations and in
The difference
P synthetase
fibroblasts
observed
of the salvage
has been found at different
are shown in Figure
and the activity
patient
to what
The
The use of intact
doublings.
repeated
in
Never-
fibroblasts.
the range
These
results
sug-
regulation; this enzyme to the defect in the extracts
from
the Lesch-
Nyhan patient, in the presence of saturating amounts of ATP and ribose-5phosphate and at inorganic phosphate concentrations from 1 to 50 mM, were found to have higher PP-ribose-P synthetase The fibroblast extracts from the heterozygous level activity patient tested
between
normal
fibroblasts
of the PP-ribose-P compared with the and indeed
and her
synthetase controls is
activity carrier
Lesch-Nyhan
than normal fibroblasts. showed intermediate son.
The difference
in fibroblasts from the Lesch-Nyhan clearly seen at all Pi concentrations
even more significantly
in the physiological
383
(GM
of approx-
doublings.
of the functioning
and PP-ribose-P
doublings
phosphate
Lesch-Nyhan
appraisal were
and heterozygous
population
blasts.
content
individuals
son's
cases was within
similar
studies
in her
mother
of f3H]-hypoxanthine
of normal
population
an accurate
similar results. The PP-ribose-P
Lesch-Nyhan
in all
at similar
at the cellular
gators with
[3H]-adenine
or in nu-
the heterozyqous
incorporation
than
of HGPRT.
nucleotides
the same population
was much higher
fibroblasts
from
lower
GM 2227
deficiency
in soluble
fibroblasts
the same age and approximately
in Lesch-Nyhan
the extreme
Fibroblasts
theless
incorporation
confirmed
showed
imately
normal
[3H]-hypoxanthine
was incorporated
incubation.
the Lesch-Nyhan
nucleotides
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
range.
in
BIOCHEMICAL
Vol. 87, No. 2, 1979
NORMAL SKIN FISROBLASTS _. P .;
1007
3
4
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LESCH-NYHAN PATIENT
HETEROZYGOUS CARRIER
I
l-l'
go
rroo
90 902 E 70 3 60 .c s 50;
4o E 302 20, t
&IIi
10
2.9 ’14.2 131I POPULATlON
DOUBLINGS
Figure 1: Catalytic activities of PP-ribose-P synthetase 5 mM and 50 mM Pi and PP-ribose-P content in normal skin Nyhan and heterozygous carrier skin fibroblasts.
in the presence of fibroblasts, Lesch-
Pi (mM) Figure 2: Activity of PP-ribose-P synthetase in normal skin fibroblast tracts (A---A), Lesch-Nyhan patient (m) and his mother (G--V fibroblast extracts as a function of added inorganic phosphate (Pi).
DISCUSSION:
The stimulatory
has been demonstrated ies (26,27) and could purine
--de nova in extensive pharmacological, clinical and kinetic studbe a major part of the biochemical defect underlying
overproduction.
has been ascribed
effect
The increased to both
excessive
of PP-ribose-P
cellular PP-ribose-P
384
on purine
ex) skin
concentration synthetase
synthesis
of PP-ribose-P activity
(20,25,
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
utilization
by HGPRT, the purine
Vol. 87, No. 2, 1979
28,29)
and to decreased
has been considered The Lesch-Nyhan both
deficient
may be responsible
in the patient's
was reported with
to extents
equal
selected
that
tase
activity
reduced
is no direct
Graf
(29)
induced
mutant than
PP-ribose-P
This
observation type
in erythrocytes
observed
of activity
studies
deficiency
in
en-
of --de novo cells
of the --de novo pursynthe-
PP-ribose-P
the presence
of saturating
at low phosphate PP-ribose-P
concen-
levels
were is
gouty
in
an obligate for
both
enzyme less
responsive
phosphate.
If, for
reported
by deVries
synthetase
was only
phosphate
of the Lesch-Nyhan
heterozygous
with
Based on the hypothesis
and codes
at all
patients with our patients,
catalytic
a marked
that
and regulatory
to metabolic
control
as has been proposed HGPRT, is x-linked,
of HGPRT mutations.
via
concentration.
and the other, of the
HGPRT gene is bifunc(29),
might also synthetase
the concentration
(35,36),
the PP-ribose-P also
385
and Sperling observed
deficiency
the possibility
The inability
inorganic
syndrome
functions
of HGPRT catalytic activity this gene has on PP-ribose-P
study,
normal HGPRT and one having a severe
partial
the x-linked
(33)
from one of
was apparent
inorganic
on hyperuricemic in contrast with
et al.
the case in the present
patient
of erythrocyte
by Becker
and in fibroblasts
of the PP-ribose-P
range
that causes a deficiency regulatory function that
effects
acid
of HGPRT and an associated
as is
activity
of HGPRT and the stigmata
that
rate
hepatoma
rate
been found
brothers
In the
This
same enzyme.
like
levels.
the complete
rat
to the increased
cells,
synthetase
activity
(33,34)
mother,
tional
found
of the patient's PP-ribose-P synthegreater than normal at all the phos-
has also
patients'
concentrations. increased
APRT activities. his
with
is more marked
may relate
PP-ribose-P
the physiological
Both
the effect
of two hyperuricemic
increased
phosphate in
en-
and uric
an enhanced
the enhanced
In our study
of mutation
In these
the patients.
the
Both
fibroblasts.
A similar
(34)
have
the degree
acid
purine
was due to increased
to the loss
tested;
between and uric
HGPRT deficient
that
cells
utilization.
concentrations
correlation
than many patients
et al.
They proposed
in these
the patient's
the
here
of PP-ribose-P
of HGPRT overproduce
in chemically
rather
trations.
activity.
levels
of purines
amounts of ATP and ribose-5-P the activity tase in the fibroblast extracts was found phate
(30).
studied
synthetase
the increased
to or even greater
in culture.
synthesis
there
deficiencies
(31,32).
synthesis
ine
for
of PP-ribose-P
fibroblasts
PP-ribose-P
and the overproduction
partial
zyme deficiency purine
consumer
carrier
enzyme that
fibroblasts.
of HGPRT deficiency Patients
intracellular
and heterozygous
HGPRT and increased
zyme defects It
to be a major
patient
salvage
exists
of the PP-ribose-P
a mutation
alter the making the of inorganic synthetase of polar synthetase
gene, genetic en-
BIOCHEMICAL
Vol. 87, No. 2, 1979
zyme, derived al levels
of inorganic
synthetase Lesch-Nyhan purine
from cells from normal cells
which
with
HGPRT deficiency,
phosphate, cells,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the behavior
results
in
can contribute
to be inhibited seen with
increased
significantly
levels
by physiologic-
the PP-ribose-P of PP-ribose-P
to augmented
in
--de novo
synthesis.
ACKNOWLEDGEMENTS: We thank Dr. Paul M. Gallop for helpful discussions and critical reading of the manuscript and Kathleen S. Pearson and Elizabeth Erickson for expert technical assistance. Supported by NIA Research Grant AG-376-04. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
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Sperling, O., Boer, P., Per-sky-Brosh, S., Kanarek, E., and devries, A. (1972) Rev. Eur. Etud. Clin. Biol. 17, 703-706 (1972). Graf, L.H., Jr., McRoberts, J.A., Harrison, T.M., and Martin, D.W., Jr. (1976) J. Cell Physiol. 88, 331-342. Gordon, R.B., Thompson, L., and Emmerson, B.T. (1974) Metabolism 23, 921-927. Emmerson, B.T., and Thompson, C.J. (1973) Quart. J. Med. 42, 423-440. Kogut, M.D., Donnell, E.N., Nyhan, W.L., and Sweetman, L. (1970) Am. J. Med. 48, 148-161. Becker, M.A., Meyer, L.J., Wood, A.W., and Seegmiller, J.E. (1973) Science 179, 1123-1126. de Vries, A., and Sperling, 0. (1973) Urologe A. 12, 153-157. Zoref, E., de Vries, A., and Sperling, 0. (1977) Hum. Hered. 27, 73-80. Yen, R.C.K., Adams, W.B., Lazar, C., and Becker, M.A. (1978) Proc. Natl, Acad. Sci. USA 75, 482-485.
387
