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Inorganic pyrophosphate release by rabbit articular chondrocytes in vitro.

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1485
INORGANIC PYROPHOSPHATE RELEASE BY
RABBIT ARTICULAR CHONDROCYTES IN VITRO
A. PIETER A. PRINS, ERN0 KILJAN, ROB J. VAN
Release of inorganic pyrophosphate (PPI) by
rabbit articular chondrocytes in vitro was measured by
a newly developed assay which utilizes radioactive
labeling and anion exchange high
orthophosphate (32Pi)
performance liquid chromatography. Chondrocytes in
monolayer and high density culture failed to release PPi.
Explants (cartilage fragments), however, released newly
formed PPi into the culture medium. Trypsin treatment
of cartilage fragments almost completely blocked the
PPi extrusion. Collagenase treatment had no effect on
PPi extrusion. There was no clear correlation between
proteoglycan synthesis, measured by 35S04 incorporation, and PPi release. Suppression of proteoglycan synthesis with tunicamycin did not influence the PPi release
of the explants.
Calcification of articular cartilage above the
tidemark (chondrocalcinosis),either by calcium pyrophosphate dihydrate (CPPD) crystals or by hydroxyapatite (HA) crystals, is a relatively frequent pathologic finding. Both CPPD crystal deposition and HA
crystal deposition are associated with osteoarthritis
(OA) (1,2). These calcifications may be locally elaborated. CPPD crystal deposition has been found in
humans following meniscectomy (3) and as a postarthritis phenomenon (4), and in animals after trauma
From the Jan van Breemen Institute, Amsterdam, The
Netherlands.
Supported by a grant from the Dutch League Against
Rheumatism.
A. Pieter A. Prins, MD; Erno Kiljan; Rob J. van de Stadt,
PhD; Jan K. van der Korst, MD.
Address reprint requests to A. P. A. Prins, MD, Jan van
Breemen Institute, Centre for Rheumatology and Rehabilitation,
Adm. Helfrichstraat 1, 1056 AB Amsterdam, The Netherlands.
Submitted for publication October 30, 1985; accepted in
revised form June 18, 1986.
Arthritis and Rheumatism, Vol. 29, No. 12 (December 1986)
DE
STADT, and JAN K.
VAN DER
KORST
(5). HA crystals have been found in rabbit knees
following surgical manipulation (6).
High levels of inorganic pyrophosphate (PPi)
have been detected in synovial fluid of patients with
either chondrocalcinosis or osteoarthritis, but plasma
levels of PPi were not increased (7-10). This suggests
that there are local changes in PPi metabolism. Deposition of CPPD crystals has been frequently associated
with various metabolic disorders, such as hypophosphatasia (1 l), which suggests systemic involvement of
PPi metabolism.
Both in vitro and in vivo experiments have been
performed in order to elucidate the details of the PPi
metabolism of articular chondrocytes. Howell et a1
(12) were the first to report the release of PPi into
surrounding medium by fragments of immature, chondrocalcinotic and osteoarthritic articular cartilage;
normal articular cartilage failed to release PPi. In 1981,
Ryan et a1 demonstrated the release of small amounts
of PPi by normal mature articular cartilage (13). Possible PPi release by chondrocytes in monolayer culture
is mentioned in 1 report (14), but other studies found
no PPi release from chondrocytes in monolayer culture or in cell suspension (15). Intracellular PPi levels
of cultured cells may be heightened in some cases of
hereditary chondrocalcinosis (16). Thus, there is ample evidence to suggest a role of PPi metabolism of
articular chondrocytes in CPPD crystal deposition
disease. This might be part of a systemic metabolic
disorder or a locally induced phenomenon.
Extrusion of PPi may be a function of normal
articular chondrocytes in vivo. If this should prove to
be the case, there are 3 main questions to be answered
concerning its activity at the cellular level: How does
PPi leave the cell or cross the plasma membrane? How
1486
PRINS ET AL
is the release of such an energy-rich compound controlled? What is its extracellular function and fate?
Exploration of the question of how the release
of PPi is controlled requires a model, in which the PPi
release from articular chondrocytes can be manipulated. Until now, all in vitro experiments on PPi
release by chondrocytes have used short-term incubations in serum-free media. A cell or tissue culture
system in which cells are metabolically active and
remain so for some time is most appropriate for the
study of control mechanisms of PPi release. A new
assay for PPi, suitable for measuring minute amounts
of newly formed PPi in culture medium, has been
developed and is described elsewhere (17). This report
describes the culture system and certain aspects of PPi
release into the culture medium.
MATERIALS AND METHODS
Ham’s F-12 medium, Dulbecco-Vogt modified Eagle’s medium (DMEM), Gey’s balanced salt solution
(GBSS), and fetal bovine serum (FBS) were all purchased
from Gibco Europe (Hoofddorp, The Netherlands).
Collagenase CLS 2, trypsin, and testicular hyaluronidase for
dissociating the cells, and collagenase CLSP A were obtained from Millipore (Bedford, MA). Inorganic pyrophosphatase, tunicamycin, and proteinase K were from Sigma
Brunschwig Chemie (Amsterdam, The Netherlands).
Bisbenzimide H 33258 was purchased from Hoechst Holland
BV (Amsterdam, The Netherlands). 35s04(10-1,OOO mCi/
mmole sulfur as sodium sulfate), 32Pi(50-1,000 mCi/mmole
Pi as orthophosphoric acid), and 32PPi (1,000-60,000
mCi/mole as Na4P207) were obtained from New England
Nuclear (du Pont de Nemours) (s’Hertogenbosch, The Netherlands). All other chemicals were of analytic grade and
were purchased from Merck Nederland BV (Amsterdam,
The Netherlands).
Seppak Florisil cartridge and high performance liquid
chromatography (HPLC) set-up and columns were from
Waters (Milford, MA), with the exception of the flow cell
scintillator (Isoflo l), which was from New England Nuclear.
Liquid scintillation counting was carried out on a Packard
Tri-Carb 300C using Pico-fluor 15 as liquid scintillation fluid.
Cultures. Monolayer cultures of rabbit articular
chondrocytes were obtained as previously described (18,19).
Six-month-old New Zealand white rabbits, weighing 3-4 kg
each, were used. Tissue was removed from the femoral
condyles, femoral heads, and humoral heads of freshly killed
animals. The margins of the articular surface and the
subchondral bone were carefully avoided. Cells from articular cartilage were dissociated from the matrix by sequential
digestion.
Cartilage fragments were first rinsed for 3 minutes
with 0.05% testicular hyaluronidase at room temperature,
then treated with 0.2% trypsin at 37°C for 30 minutes.
Finally, they were digested with 0.2% clostridial collagenase
(CLS 2) for 90 minutes at 37°C. Cells were grown in Ham’s
F-12 medium supplemented with 10% (volume: volume) FBS
and 0.1% (v :v) penicillin-streptomycin until confluent (7-10
days). The 80-cmZflasks were gassed with 10% COz in air.
Second-passage cells were used for experiments in
monolayer or cell pellet culture. For monolayer culture, 1 x
lo6 cells were seeded in 80-cmZ flasks. For cell pellet
cultures, 6 x 10’ cells suspended in 4 ml of medium were
spun down in plastic centrifuge tubes for 3 minutes at 800
revolutions per minute. The tubes were gassed and left
overnight in the incubator. The next day the pellets were
transferred to bacteriologic petri dishes, 5 cm in diameter,
containing 4 ml of medium. Five or more pellets per dish
were used.
Most experiments were performed on explants.
Chips of articular cartilage, approximately 2 x 2 x 1 mm,
were washed with 0.05% hyaluronidase for 3 minutes at
room temperature and placed in a culture flask with 10 ml of
medium. The following day they were divided among 4 petri
dishes, each of which contained 4 ml of medium. In all
experiments, the culture medium was DMEM supplemented
with 10% FBS. All cultures were kept under the same
conditions with respect to pH (gassed with 10% CO2 in air,
pH was 7.0) and temperature (37°C).
DNA measurement. Samples (100 pl) were taken
from 5 ml of sonicated cell suspension in 0.005% sodium
dodecyl sulfate (SDS), or from the proteinase K digest of
pellets or explants. DNA was measured by the method of
Labarca and Paigen (20). Bisbenzimide H 33258 was used for
fluorescence at 260 nm in a Vitatron spectrophotometer.
3sS04 incorporation. Incorporation of radioactive sulfate into macromolecules (i.e., proteoglycans) was measured
after a 20-hour labeling period with 1.4 pCi of 3SS04/ml
medium. Monolayers, pellets, and explants were washed
thoroughly with GBSS, and part of their suspension or digest
was counted in the liquid scintillation counter. Medium was
dialyzed exhaustively. (Alternatively, the trichloroacetic
acid [TCA] precipitate was washed and resuspended before
counting [see below].)
PPi measurement. PPi in the culture media and the
cell suspension from the monolayer cultures was measured
as previously described (17), and all cultures were labeled
with radioactive orthophosphate. Yeast inorganic pyrophosphatase was used to clear the 32Pilabel of traces of PPi. Cells
were labeled with 5 pCi 32Pi/ml medium for 20 hours. (In
some experiments, double-labeling was used with 35S04and
32Pi.)The cell monolayer was washed 3 times with 10 ml of
GBSS, then scraped off with a rubber policeman. Cells were
spun down in a centrifuge tube and resuspended in 5 ml of
GBSS containing 0.005% SDS. The cell suspension was
clear after sonication and was further treated in the same
way as the culture medium.
Cell suspension and medium were deproteinized by
adding TCA (final concentration 570,v :v); simultaneously,
NaPPi was added (final concentration 0.1 mM). After 10
minutes on ice, the precipitate was spun down in an
ultracentrifuge for 10 minutes at 100,000g at 4°C. In some
experiments, the precipitate was washed and used for measurement of 35S04incorporation.
TCA was extracted from the supernatant with ether.
The extracted sample was neutralized with 4M KOH and
mixed with 20 volumes tetrahydrofuran (THF)/water (3 :1
PPi RELEASE BY RABBIT CHONDROCYTES
v:v). This mixture was passed slowly over a Seppak Florisil
cartridge pretreated with 100% THF. All retained phosphate
compounds were eluted with 4 ml of a buffer containing 50
mmoles H2SO4, 0.2 moles citric acid, and 7 mmoles MgS04.
The pH of this eluent was increased to 2.2.
From this clarified sample, 100-500 pl was applied to
the anion exchange column in the HPLC set-up. The column
used was a RadialPAK-pbondaPAK NH2 column, 18 x 0.8
cm, with 10-pm particles. It was equipped with a C18
guardpack. A gradient of ionic strength and Mg concentration was created using 2 pumps steered by a programmable
system controller. Buffer A (which contained 0.1M citric
acid) and buffer B (which contained 0.2M citric acid, 0.1M
K2SO4, and 0.2M MgS04) were both adjusted to a pH of 2.2
with 4M KOH. Starting with 100% buffer A, a transition to
100% buffer B was made along a straight line in 5 minutes.
The end of the run (10 minutes) was followed by a rapid
switch back to buffer A. The flow was held constant at 2
ml/minute. Cerenkov radiation was measured with an Isoflo
1 scintillator detector; the pulses were processed by an
Apple IIe computer. The chromatogram was monitored and
plotted during the run.
PPi was well separated from Pi and P compounds
(Figure 1). PPi in medium was measured as the percentage of
1487
R
r
250
0
2
4
6
8
time ( m i d
Figure 2. Representative chromatogram, showing intracellular inorganic pyrophosphate (PPi) as well as some mono-, di-, and
triphosphates. Chondrocytes in monolayer culture were labeled
with 3zPi.This chromatogram was made without an elution gradient,
i.e., 1 eluent buffer was used. After 3 minutes of PPase treatment at
room temperature, the PPi peak disappeared.
“PPi counts” out of the total counts. The culture medium
contained 1.27 X lo5 pmoles Pi/l ml. Using 32Pias an internal
standard, PPi could then be calculated. The accuracy of the
quantification depended on the specific activity of 32Pi
reached in the sample and the amount of 32PPi formed.
Assays on standard samples that were comparable with the
experimental samples gave standard deviations well below
10%. The losses of PPi and Pi during sample preparation
were the same (approximately 20%). All samples frqm each
experiment were always completely processed on the day of
harvesting. When double-labeling was used and DNA had to
be measured, only 4 samples could be handled simultaneously.
RESULTS
time
(min)
Figure 1. Chromatographic separation of inorganic pyrophosphate
(PPi) from Pi and other P compounds, using an Mg++ concentration
and an ionic strength gradient over an anion exchange column in a
high performance liquid chromatography system. Small peaks at
EZm nm, which eluted just prior to the monophosphates, and at the
PPi position are buffer change artifacts, which mark the beginning
and the end of the gradient. Countshnterval exceeding 250 are
divided by 10.
In a preliminary experiment, intracellular PPi of
chondrocytes in monolayer culture was measured using the enzymatic assay described by Lust and
Seegmiller (21). The level of intracellular PPi was high
while cells were synthesizing DNA. Once confluency
was reached and the cells ceased to proliferate, the
intracellular PPi level fell off sharply. In the same
experiment, the 35S04incorporation per pg DNA was
PRINS ET AL
1488
J
I
\
PPi
Figure 3. Chromatogram of medium from radioactive orthophosphate (32Pi)-labeled explants, demonstrating the newly formed extracellular inorganic pyrophosphate (PPi). Total surface area =
56,110 counts, PPi peak = 457 counts, amount of PPi/dish =
457/56,110 x 5.08 x lo5 pmoles (Pi in 4 ml medium) = 41 nmoles.
Counts/interval exceeding 250 are divided by 10.
Finally, explants of rabbit articular cartilage
were cultured and labeled according to the same
procedure. The number of cells was estimated by
measurement of DNA after digestion of the explants.
In the first experiments with explants, the amount of
cartilage per dish was kept low so that the amount of
DNA per dish would be the same as in the pellet
cultures.
The number of explants per dish was doubled in
experiments performed to test the influence of various
manipulations. Newly formed 32PPiwas present in the
medium in all explant cultures tested. An example of a
chromatogram of explant medium is shown in Figure
3. In 1 experiment, frozedthawed explants were used
as controls. These dead explants released no 32PPiinto
the medium. 32PPirelease was found not only during
the first days of incubation, but also, in the same
amount, after 1 week of culture. The consistent 32PPi
release by explants contrasted with the absence of
32PPiin the medium of the other culture systems.
To obtain monolayer cultures, the articular
cartilage was digested first by trypsin and then by
collagenase. With this digestion procedure, the chondrocytes seemed to lose their ability to release newly
PPi
200
found to be constant. Since there might be a correlation between intracellular PPi concentration and the
release of PPi in the surrounding medium, monolayer
cultures in all stages of proliferation and confluency
were labeled with 32Piand the media were tested for
the presence of 32PPi.In none of the media could 32PPi
be detected, whereas intracellular 32PPi was readily
detected (Figure 2).
Another procedure for culturing the cells was
developed, in which second-passage cells were
pelleted (6 x lo5 cells/pellet) and grown in small petri
dishes with 4 ml of medium for 8 days. In this culture
system, the cells were surrounded by their own matrix
and their number per ml of medium increased. It had
been anticipated that the matrix would influence the
PPi extrusion. Up to 15 of these cell pellets in 1 dish
were labeled with 32Pifor 20 hours, but no 32PPiwas
found in the surrounding medium. The capacity of the
cells to synthesize proteoglycans (PGs) was checked
by labeling them also with 35S04,which they actively
incorporated into macromolecules. These experiments
were repeated several times.
0CONTROL
B T R Y P S I N TREATED
a
Z
n
a
Z
0 100
cn
<
\
0,
10
\i
m
0
7
X
E
cn
aJ
-0
En
.-C
.-
a
a
a
0
"
m
0
m
Figure 4. Effect of trypsin treatment of explants on inorganic
pyrophosphate (PPi) extrusion and proteoglycan synthesis (expressed by 3sS04incorporation). Values shown are the mean from 2
experiments.
1489
PPi RELEASE BY RABBIT CHONDROCYTES
formed 32PPi,and they did not seem to regain it when
grown in pellets surrounded again by a matrix.
The next experiments were designed to test the
influence of the trypsin and collagenase treatment.
After a 3-minute wash with 0.05% hyaluronidase to
remove adherent cells, explants were treated with
trypsin (180 units/ml) for 1 hour at 37°C. This was done
with the same trypsin used in the first digestion step.
After 1 hour, explants were washed with DMEM-10%
FBS to stop trypsinization.
The trypsinization had no apparent macroscopic effect on the explants, but its effect on 32PPi
release was pronounced. In the medium of the trypsinized explants, 32PPiwas just detectable, and the 32PPi
peaks in the chromatograms were small but distinguishable. The total 32PPi peak counts were about
twice the background counts. There was also some
effect on the incorporation of 35S04into macromolecules: this was reduced in the treated group (Figure 4).
The extent of the effect on PG synthesis was somewhat exaggerated when expressed per pg DNA, however: in the trypsin-treated group, the DNA content
was higher than in the control group, possibly because
of an increase in DNA synthesis.
The influence of collagenase was tested with a
different collagenase from that used for digestion,
since the protease activity of the latter would actually
digest the explants. The highly specific collagenase
CLSP A (400 units/ml) had no effect on either 32PPi
release or PG synthesis. The explants were rinsed with
0CONTROL
n
TUNICAHYCIN
(60% inhibitiond pGsyltheSis\
loo]
I
8o
1
I
0Medium
+Explants
+TrypsinizedExplants
20 hours
Figure 6. Effect of the presence of explants and of trypsinized
explants, under culture cofiditions, on the percentage of inorganic
pyrophosphate (PPi) hydrolyzed after 2 hours and after 20 hours.
Values shown are the mean from 3 experiments.
DMEM-10% FBS after 1.5 hours of collagenase treatment at 37°C (data not shown).
Several authors have suggested the possibility
of a connection between PG synthesis and secretion,
and the release of PPi. Tunicamycin is known to be an
inhibitor of PG synthesis (22) and is only partially
soluble in culture medium. A stock solution of 5 mg
tunicamycin in 10 ml medium was made. After gentle
shaking for 4 hours, the undissolved fraction was
removed by centrifugation. Inhibition of 35S04incorporation was tested using a range of concentrations.
The concentration of tunicamycin at which the PG
synthesis was reduced by 60% was used for the PPi
release experiment. As shown in Figure 5 , the PPi
release was not influenced by tunicamycin-induced
inhibition of PG.
By adding 32PPito the medium, it is possible to
measure the hydrolysis rate of PPi (PPi
2Pi) by
means of the same assay used for PPi detection. When
minute amounts of radioactive PPi (<10-8M) were
added to DMEM alone, nearly all PPi was hydrolyzed
within 1 hour. It was possible to prevent this spontaneous hydrolysis by adding nonradioactive NaPPi
(17). When NaPPi was added to culture medium in
concentrations >O. 1 mM, a visible amorphous precip-
-
Flpre 5. Effect of tunicamycin, in a concentration that inhibited
3’S04 incorporation (i.e., proteoglycan [PG] synthesis) by 60%, on
inorganic pyrophosphate (PPi) extrusion by explants. Values shown
are the mean from 2 experiments.
1490
PRINS ET AL
itate was formed. PPi and Ca-PPi complexes are
known to influence cell metabolism in vitro (23). To
avoid all possible interference in experiments for detection of PPi release, NaPPi was never added during
culture, but always immediately after harvesting.
Hawever, when PPi hydrolysis rates were assayed,
“cold” NaPPi was added in a concentration of lOV4M.
PPi hydrolysis rates (PPase activity) were high
in all cultures, but highest in explant cultures when
expressed as the amount of PPi hydrolzyed per pg
DNA in 20 hours. PPi hydrolysis in the presence of
explants was due largely to PPase activity, as demonstrated by the difference between medium without
explants and medium with explants. Medium was
supplemented with 10% FBS, which probably contained some enzymes with PPase activity. DMEM
without any additive exhibited a PPi hydrolysis rate
somewhat below 15% in 20 hours. Trypsinization of
the explants did not alter the PPase activity found
(Figure 6).
DISCUSSION
It is clear from the present data that normal
adult articular cartilage explants release PPi into surrounding medium in vitro, under “normal” culture
conditions. Since we used a radioactive tracer that had
been cleared of 32PPiwith yeast inorganic pyrophosphatase, there can be no doubt about the origin of the
PPi. The 32PPi found in the culture medium was
generated during the 20-hour labeling period. In the
chromatogram of the intracellular P compounds, 32PPi,
32P-diphosphates, and 32P-triphosphates are distinguishable (Figure 2). Whether all P compounds were
equally labeled cannot be determined from these experiments. The possibility exists that there was undetected release of PPi from an unlabeled pool.
It is still not known how PPi leaves the cell.
Several possibilities have been proposed (see ref. 24
for a recent review). It is also plausible that PPi does
not leave the cell, but is actually produced extracellularly from ntlcleotide triphosphates (NTPs) (25). Cosecretion of PPi with matrix macromolecules
(proteoglycans) is often suggested. In our experiments
there was no clear correlation between the incorporation of 3sS04 into PGs and 32PPirelease. At present,
the extrusion of PPi through matrix vesicles seems to
be the most attractive hypothesis.
Matrix vesicles (MVs) are small, lipid membrane-wrapped packages (26,27). They contain enzymes which are capable of producing PPi from NTPs
and enzymes which can hydrolyze PPi to orthophosphate (28). Whether MVs contain PPi is unknown.
They are certainly capable of “producing” PPi.
Calcium-PPi complexes were formed by MVs in vitro
(29). MVs are believed to be an important mediator of
endochondral calcification. They have been demonstrated in normal articular cartilage (30).
If MVs are indeed responsible for PPi release
by articular cartilage explants, one might speculate on
the effect of trypsin on either the vesicle formation or
the functioning of the MVs. Cultured chick embryo
chondrocytes in monolayer culture were able to form
MVs (31). Monolayer and pellet culture of articular
chondrocytes, as used in the present study, may not
have been favorable for the development of MVs.
Addition of ATP to cartilage from a patient with
CPPD crystal deposition caused an abundant generation of PPi (25). The pyrophosphohydrolases responsible for the PPi generated from NTPs are not confined
to MVs. Pyrophosphohydrolase was found to be an
ecto-enzyme of canine articular chondrocytes. This
ecto-enyzme was extremely sensitive to trypsin treatment (32). PPi generation by this pyrophosphohydrolase fell more than 50% after 5 minutes of trypsin
treatment of the cells. The sharp decrease of PPi
release by explants following trypsinization described
in this report may be due to the same mechanism, i.e.,
inactivation of this ecto-enzyme. This would mean
that PPi is generated outside the cells, from NTPs (33).
However, no 32P-labeled NTPs were detected in the
media in which we found 32PPi.
The function of the extracellular PPi is another
subject for speculation, regardless of the PPi extrusion
mechanisms. One of the most striking features of
normal articular cartilage is the absence of calcification above the tidemark. PPi is known to be a strong
inhibitor of calcium crystal formation. Even small
amounts of PPi released by the chondrocytes could
eventually accumulate to concentrations sufficiently
high to prevent calcification. The function of PPi in
articular cartilage may be to prevent calcification (34).
There may be a flow of PPi from the chondrocytes or
their MVs toward the bony cortex. Hydroxyapatite
crystals, the crystals in bone, bind PPi, and this might
create the gradient for the PPi flow. The tidemark
would then be held in place by this counterflow to the
obtruding calcification front. Remodeling occurs in
osteoarthritis, and this very often means a doubling of
the tidemark, which may be the new calcification front
brought to a halt by an enhanced PPi counterflow (35).
It seems reasonable that an increase in PPi
PPi RELEASE BY RABBIT CHONDROCYTES
release in OA is necessary in order to prevent calcification, mostly HA crystal formation (36). Indeed,
pronounced PPi extrusion from osteoarthritic cartilage
was found by Howell et a1 (37). HA crystals are the
most common crystals in osteoarthritic cartilage (1).
Formation of HA crystals may be due to a low PPi
concentration: the result of too little PPi generation or
excessive hydrolysis of PPi (e.g., too much pyrophosphatase activity). CPPD crystals may be formed only
under special circumstances in which there is a factor
that favors this type of crystal growth (e.g., iron
deposition in hemachromatosis, or abundant presence
of PPi in hypophosphatasia). CPPD crystals are found
around chondrocytes, sometimes especially around
dead cells. This may indicate that MVs play a role as
PPi producers, since degeneration of chondrocytes
stimulates vesiculation (38,39).
In conclusion, calcification of articular cartilage
may be the result of an imbalance of the calcification
prevention system (NTP + PPi + Pi). The type of
calcium crystals formed depends, in part, on the
precise nature of this imbalance. The present model
for the study of the PPi metabolism of articular
chondrocytes should shed more light on the role of
matrix vesicles and the enzymes, i.e., pyrophosphohydrolase and pyrophosphatase, that are likely to be
involved.
ACKNOWLEDGMENTS
We wish to thank Dr. G. P. J. van Kampen and
Dr. R. Kuijer for their advice and valuable comments.
The linguistic expertise of J. M. Cornwell is gratefully
acknowledged.
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