вход по аккаунту


Ntp pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilagesome biochemical characteristic.

код для вставкиСкачать
I. Some Biochemical Characteristics
Nucleoside triphosphate pyrophosphohydrolase
activity was first detected in articular cartilage in previous studies at our laboratory. In this report, the enzyme
is partially characterized with respect to its pH optimum
and Km. The enzyme was metal-dependent and was
active in the presence of 1 mM Ca++. It was inhibited by
several substances, including cysteine and dithiothreitol. Its activity was not inhibited by tetramisole at
concentrations which inhibited 100% of the pyrophosphatase activity in the same extracts. It functioned most
effectively on ATP, but also on UTP, CTP, and GTP. A
role for scavenging nucleotides and production of pyrophosphate in osteoarthritic and chondrocalcinotic cartilage is postulated.
Previous studies have favored the view that
pyrophosphate ion is generated in both normal and
From the Department of Medicine, University of Miami
School of Medicine and the Veterans Administration Hospital,
Miami, Florida.
Supported in part by the Research Service, US Veterans
Administration, and Grant AM08662 from the National Institutes of
Health, the Medical Research Council of Canada, Canadian Arthritis Society Fellowship Awards, and the Kroc Foundation.
Ofelia Muniz, DSc: Assistant Research Professor, Department of Medicine, University of Miami School of Medicine; JeanPierre Pelletier, MD: Assistant Professor, Rheumatic Disease Unit,
University of Montreal, HBpital Notre-Dame, Montreal, Canada;
Johanne Martel-Pelletier, PhD: Research Associate, Rheumatic
Disease Unit, University of Montreal, HBpital Notre-Dame, Montreal, Canada; Sara Morales, BSc: Research Assistant, Department
of Medicine, University of Miami School of Medicine; David S.
Howell, MD: Professor of Medicine, Chief of Arthritis Division,
Medical Investigator, U S Veterans Administration.
Address reprint requests to David S. Howell, MD, Department of Medicine (D26), University of Miami School of Medicine,
PO Box 016960, Miami, FI, 33101.
Submitted for publication March 7, 1983; accepted in
revised form September 27, 1983.
Arthritis and Rheumatism, Vol. 27, No. 2 (February 1984)
diseased articular cartilage, but particularly in osteoarthritic and chondrocalcinotic cartilage (1,2). In seeking
a mechanism for local extracellular pyrophosphate
elaboration to explain Ca2P207 * 2H20 deposition, we
have shown that pyrophosphate ions can be generated
from ATP using extracts of articular cartilage as an
enzyme source (3,4). Furthermore, Ryan et a1 ( 5 )
showed that in incubates of articular cartilage slices,
radiolabeled ATP generated pyrophosphate. With regard to other cartilages, a similar enzyme activity was
studied first in sheep growth cartilage by Cartier and
Picard (6) and recently, in embryonal epiphyseal cartilage by Hsu (7).
The enzyme ATP pyrophosphohydrolase (EC has previously been characterized partially in
liver, muscle, and gut wall (8-12). Flodgaard and
Torp-Pedersen postulated that the generation of pyrophosphate by this enzyme was associated with energy
transfers needed for Ca++ transport through plasma
membranes in a thermodynamically optimal manner
(12). The recent evidence that it is an ecto-enzyme
would, at least in cartilage, indicate an alternative
possibility, i.e., that this enzyme may serve to scavenge nucleotides liberated via inadvertent exocytotic
functions during either cell division or cell injury
commonly seen in osteoarthritic tissues (13). More
information on the nature of this enzyme was sought
by studying diseased tissues, to be compared later
with normal tissues needed for studying the purified
Subjects. Nineteen patients undergoing knee surgery
at Jackson Memorial Hospital or the VA Medical Center,
Miami during the years 1979-82 were entered in the study.
Ten of the patients had osteoarthritis, and 9 had chondrocalcinosis. Complete medical history was taken on all patients,
and all received a thorough physical examination.
There was an equal sex distribution for osteoarthritic
and chondrocalcinotic patients. Their mean age was 68 2
10.2 for osteoarthritis patients and 72 5 5.8 for chondrocalcinosis patients (Table 1). The chondrocalcinosis patients all
had radiologic evidence of the disease and fulfilled McCarty’s criteria for definite diagnosis (14).
Roentgenograms of all operated knees were evaluated and were designated by the criteria of Kellgren and
Lawrence (15): knees of 3 of the osteoarthritic patients were
designated radiologic class I or 2, and knees of 7 of the
osteoarthritic patients and all 9 chondrocalcinotic patients
were designated radiologic class 3 or 4.
Articular cartilage samples were obtained from the
normal knees of 4 accident victims, for use as controls.
Tissue preparations and histologic studies. The tibial
plateaus of patients undergoing orthopedic surgery were the
source of fresh cartilage. Control cartilage was obtained
from the same topographic site in knees of the accident
victims. Experimental tissues were obtained from margins
0.5-1 cm wide, surrounding the erosions in weight-bearing
sites on the medial and lateral tibial plateaus, as well as the
femoral condyles. The osteoarthritic cartilage appeared fibrillated. Portions of each sample were fixed, embedded,
cut, and stained with hematoxylin and eosin as well as
Safranin 0. Histologic examination of the osteoarthritis
(OA) cartilage revealed deep fissures and cell cloning and
met Mankin’s criteria for grade 7-1 I OA (16).
Only a small portion of the chondrocalcinosis (calcium pyrophosphate deposition disease, CPDD) tissue was
used for morphologic analysis. These samples showed degenerative changes similar to those in OA cartilage, and
there were minerals in some of the samples but not others.
This could not have been a result of hemachromatosis, gout,
or hyperparathyroidism, since patients with these diagnoses
had been excluded from the study.
After surgery, tissue samples were promptly frozen
and stored. After freezing storage, the dissected cartilages
were thawed, weighed wet, snap frozen in a stream of C 0 2
and cut into 20p slices in a cryostat, and placed in 5 volumes
weight:volume 1 mVlOO ml 0.05M Tris pH 7.5, triton X-100
(Sigma Chemical Co., St. Louis, MO). The cartilage triton
X-100 mixtures were then homogenized by using an all-glass
motor-driven dual tissue grinder (Kontcs Glass Co., Vineland, NJ). The triton homogenate was stirred for 4 hours and
Table 1. Characteristics of patients from whom cartilage was
OA radiologic class 1-11
* Mean
* 10.2
2 5.8
SD. Difference between the 2 groups was not significant
(P> 0.09).
then centrifuged at 23,OOOg for 20 minutes. Routinely, the
residue was reextracted with 1% triton X-100 under the same
conditions, and the extracts were combined and used for the
measurements of enzyme activity. Pilot data indicated that a
third extraction was not necessary because all (>95%)
enzyme activity was recovered in the first 2 extractions.
Materials. Unlabeled ATP, ADP, AMP, adenosine,
UTP, UDP, UMP, uridine, CTP, CDP, CMP, cytidine, GTP,
GDP, GMP, and guanosine were from P-L Biochemicals,
Inc. (Milwaukee, WI), Cleland’s reagent (dithiothreitol) and
cysteine were from Calbiochem Corp. (San Diego, CA).
Tetramisole hydrochloride and bromotetramisole oxalate
were from Aldrich Chemical Co. (Milwaukee, WI). Threeisobutyl- I-methylxanthine was from Sigma Chemical Co.
Radioactive substrates adenosine S’triphosphate, tetrasodium salt (8-I4C) 54 mCi/mmole; uridine S’triphosphate,
tetrasodium salt 2-14C) 47 mCi/mmole; cytidine 5’ triphosphate, tetrasodium salt I4C(U) 519 mCi/mmolc; guanosine
5’ triphosphate, tetrasodium salt (8-I4C) 57 mCi/mmole;
adenosine S‘triphosphate tetra (triethylammonium) salt
(d2P), 6.33 Ci/mmole were obtained from New England
Nuclear Corp. (Boston, MA).
Marker enzyme assays. Alkaline phosphatase and
S’nucleotidase were assayed by classic methods described in
a previous publication (3). and by the method of Dixon and
Purdom (17), respectively.
Measurement of NTP pyrophosphohydrolase using
(y3’P)-ATP as substrate. NTP pyrophosphohydrolase was
measured after pyrophosphate (PPi) and AMP were identified as the enzyme’s products. All tests were run in duplicate; variation in duplicate ranged 2-3%.
The PPi product was characterized by two different
thin-layer chromatographic methods using (dZP)-ATP as
substrate. The results were calibrated with a third method of
measuring pyrophosphate, the spectrophotometric method
previously reported (1). This spectrophotometric method
depended on measurement of NADPH generated in a 3-step
enzymatic procedure (data not presented). The incubation
was performed with (?*P)-ATP, 1 mM, with a specific
activity of 2 mCi/mmole in the presence of Tris-HC1 buffer
O.lM, pH 9, and calcium 0.1 mM. Cartilage extract was
added and incubations carried out at 37°C for 2.5 hours with
agitation. The reaction was stopped by boiling for 3 minutes
in a water bath. Tests on the possible effect of boiling 3
minutes, on several concentrations from ~ o - ~toM10-94 of
NazP207,were performed and revealed no hydrolysis.
The boiled samples were centrifuged for 2 minutes in
a Beckman microfuge and radioactive spots separated using
the thin-layer chromatographic technique as follows. Twenty-microliter samples and cold markers of ATP, PPi, and Pi
were spotted in MN cellulose 300 thin-layer sheets and
chromatograms developed in n-propylacetate/90% formic
acidwater, 11/5/3 volume/volume, twice in the same direction with intermediate drying. The phosphate marker spots
were localized by wetting the chromatograms with a mixture
containing 1 gm ammonium molybdate, 3 ml concentrated
HCI, 3 ml of 70% perchloric acid, and 8 ml HzO diluted to
100 ml with acetone, then exposed to an ultraviolet lamp for
10 minutes; the resulting blue spot was stabilized by contact
with NH3 vapor (18). The PPi spot was located halfway
between Pi and ATP. The spots were cut out and counted in
10 ml of aquasol in a Packard tricarb liquid scintillation
In the second technique, the samples and cold markers were spotted in 300 PEI cellulose thin-layer sheets
(previously washed for 5 minutes in a 10% NaCl solution
and twice washed in distilled water). The chromatograms
were then developed in 1M LiCl until the solvent front
advanced 6 cm. The next development was continued in
1.5MLiCl until the solvent front advanced 13 cm. The spots
were then dried. Next, the chromatograms were immersed in
1.5MLiCl containing 0.2M sodium acetate and 4M urea. The
ATP spot by now was at the front, Pi in the middle, and PPi
behind. This method, originally reported by Fullerton and
Finch (19), was modified to provide better separation for
current purposes. The PPi spots were localized, cut, and
counted as described above.
Measurement of NTP pyrophosphohydrolase using (8I4C)-ATPas substrate. With this method, the incubation was
carried out as before at 37°C for 2.5 hours with agitation; an
aliquot of the triton extracts was mixed with 100 pI of
substrate mixture containing 0.1M sodium bicarbonate-carbonate buffer pH 9.8, CaClz at 0.1 mM and (8-I4C)-ATPat
1.0 mM with a specific activity of 2 mCi/mmole. After
stopping the reaction by boiling for 3 minutes in a water
bath, 50 nmole each of AMP, cyclic AMP, ADP, and
adenosine were added in a volume of 10 pl. The 20-pl
samples were spotted on cellulose 300 PEI thin-layer sheets,
washed as described above, and the chromatograms developed first in water for 60 minutes with intermediate drying,
and then in 0.8M LiCl for 90 minutes. The spots of ADP,
AMP, CAMP, and adenosine were localized by an ultraviolet
lamp and cut and counted as described before.
ADP and cyclic AMP were well separated from AMP
and adenosine. These separations mitigated against any
spurious counts from products of adenyl cyclase or alkaline
phosphatase action appearing in the extracts tested. The
AMP and adenosine counts were proportional to the amount
of enzyme and were linear with time for at least 4 hours. It
was found that 100% of substrate could be hydrolyzed to
AMP and adenosine, with excess of enzyme and in the
presence of S’nucleotidase. All experiments using (8-’4C)ATP were repeated using as inhibitors 1 mM 3-isobutyl-lmethylxanthine and, separately, 1 mM bromotetrdmisole, to
exclude effects of adenyl cyclase and alkaline phosphatase,
Determination of the radioactive background. Radio-
active background was determined by counting areas of the
thin-layer chromatogram corresponding to the location of
the products AMP, adenosine, Pi, and PPi, after boiled
substrate mixture was chromatographed in each experiment.
This procedure was carried out in every experiment and
accounted for any hydrolysis of substrate not due to enzyme
action. It also eliminated the possibility that observed activity was merely due to tailing of the labeled substrate.
Kinetic experiments for the enzymes were carried out
according to the method of Michaelis and Mcnton (20).
Protein determinations were made according to the
method of Lowry et al (21). Triton X-100 interfered in the
color reaction at high concentration. Therefore, all protein
solutions were diluted to approximately 3 mg/ml triton X-100
and run with a series of triton standard solutions.
Addition of metals and various compounds. These
were added to the assays using (8-l4C)-ATP substrate to
observe their effects on the activity of the enzyme studies.
Substrate specificity. The nucleotides GTP, CTP, and
UTP were tested in regard to their ability to replace ATP.
The incubation mixtures were the same as described above,
and radiolabeled substrates were substituted for (8-“C)ATP. All samples were spotted with corresponding internal
cold markers. All the chromatograms were developed in the
same solvent except for GTP, which required IM instead of
0.8M LiCI. The same specific activities at 2 mCi/mmole were
used with these other substrates.
DNA. DNA content was determined by the method
described in a previous publication (l), i.e., the modified
method of Ceriotti (22).
Statistical analysis. Statistical analysis was carried
out using Student’s nonpaired t-test.
T h e overall profile of phosphohydrolases was
studied t o characterize t h e cartilage samples. As in
samples from previous series of patients (3), w e typically found a moderately elevated level of alkaline
phosphatase in the osteoarthritic cartilages compared
with normal cartilages (Table 2). Also as in previous
studies, w e found S’nucleotidase levels to b e elevated
3-4-fold in chondrocalcinosis patients compared with
normal or osteoarthritic patients. As in previous nor-
Table 2. Total activity of NTP-pyrophosphohydrolase and other hydrolases based on (8-I4C)-ATP
assay, pH 9.8*
Normal (n = 4)
OA (n = 10)
CPDD (n = 8)
1.3 +- 2
52.8 10
27.6 f 9.5
140 2 41
158 42
20 i 12
16 ? 7
18 5 10
* Values, except for DNA, are in units per gm wet weight, starting sample of cartilage, one unit = 1
nmole product formed per minute. DNA values are gg/gm wet weight of starting sample. OA =
osteoarthritis; CPDD = chondrocalcinosis.
t P < 0.001, OA versus CPDD.
$ P = 0.02, OA versus CPDD; P < 0.001, CPDD versus normal; P < 0.003, OA versus normal.
ma1 patients we have reported, S'nucleotidase from
the 4 normal patients in this study was in the same
range as that from osteoarthritic patients (Table 2).
Thus these data, as well as histologic grade and DNA
content of the tissues used here, are similar to those in
our past studies for characterization of alkaline phosphatase and pyrophosphatase (3). NTP pyrophosphohydrolase activity appears significantly elevated in
CPDD and OA patients compared with controls.
The products resulting from incubating triton
X-100 extracts of the cartilage with both (8-I4C)-ATP
and (g2P)-ATP are compared in Table 3. First, it is
seen that there is a very small yield of split hydrolytic
products (<1% of starting ATP) for extracts of normal
adult cartilage. In diseased cartilages, in contrast, 910% of the ATP was hydrolyzed in 2% hours, at
concentrations required to produce linear kinetics of
enzyme activities (data not shown).
Another important finding was that PPi was
generated by all cartilage samples, with 9-fold higher
levels by CPDD than by OA or normal cartilages, and
2-fold higher levels by CPDD than by OA plus inhibitor. A most significant point is that by the (8-14C)-ATP
assay, concentrations of AMP plus adenosine almost
equaled PPi production by the ( g2P)-ATP experiments in the OA plus tetramisole and the CPDD
groups. These findings can only be explained by a
predominant conversion of ATP to PPi, and the resultant AMP being converted partially to adenosine, with
release of phosphate by S'nucleotidase. AMP + adenosine in the OA without inhibitor group is generated
not only by NTP pyrophosphohydrolase, but also by
alkaline phosphatase in the extract. Thus, PPi genera-
tion was substantial only if native pyrophosphatase
level registered by alkaline phosphatase was low in
starting samples, or if the tetramisole inhibitor was
used. Finally, the extremely low cAMP level relative
to other products indicated no major turn-on of the
adenyl cyclase enzyme at the time of study (Table 3).
Table 4 shows the pH optimum of the enzyme
as it operated free in the synthetic lymph. Activity was
measured between pH 7 and 11, with an optimum
between 9.5 and 9.8. Accordingly, most further measurements were made at the pH optimum of 9.8 in
order to facilitate comparison with other data. The
physiologic pH around neutral of triton X-100 extracts
also showed some activity. Cartilage slices at neutral
pH in organ culture, assumed to be present in vivo,
also showed some substantial activity in the studies of
Ryan et a1 (5); however, no activity of the bovine
enzyme cartilage at pH 8.0 was shown (7).
Table 5 displays the effect of metal ions and
chelators on the enzyme activity. In comparison with
untreated controls, experimental samples had severe
inhibition of enzyme activity in the presence of either
EDTA or EGTA. In contrast, a normal or slightly
above-normal activity was obtained with 1 mM Ca++
or 0.1 mM Ca++.Mg++ Ca++at 1 and 0.1 mM levels
was slightly less active. Na+ and K' supported the
enzymes if they were not previously treated with
chelators, indicating the probable presence of divalent
cations already attached to the isolated enzymes. For
the bovine enzyme, an absolute requirement for zinc
has been established (7).
Table 6 reveals the effect of various substances
on the NTP pyrophosphohydrolase and an alkaline
Table 3. Products of various phosphohydrolase activities found after incubation of I 0 0 nmole ATP.
pH 9.0, with triton X-100extracts of OA, CPDD, and normal cartilages*
Starting substrate (8-'4C)-ATP
Starting substrate
( Q?P)-ATP
(nmole/2.5 hours)
(nmole/2.5 hours)
Group studied
OA (n = 4)
OA tetramisole
(n = 4)$
CPDD (n = 4)
Normal (n = 4)
0 . 8 2 0.4
3.5 ? 0.3
0.8 2 0.2
4.620.08 0.29f0.12
0.7 f 0.01 0.12 f 0.05
0.4 t 1.5
1.7 & 0.1
4.1 ? 0.7
2.3 2 0.1
3.5 50.04
0.2 f 0.02
9.2 2 0 . 9
0.8 t 0.3
f 0.2
* Incubation was for 2% hours at 37°C. Five milligrams wet cartilage provided extract for each sample
in 0.1 ml incubation mixture. Numbers shown are mean 2 SD. OA = osteoarthritis; CPDD =
chondrocalcinosis; PPi = pyrophosphate.
t P < 0.001, OA versus OA + tetramisole; P < 0.001, OA versus CPDD.
$ 1.0 mM tetramisole needed for this group of OA cartilages; CPDD samples had negligible alkaline
phosphatase levels and, since corresponding tetramisole experiment gave about the same values, data
are omitted. See Patients and Methods.
Table 4. Activity of NTP pyrophosphohydrolase as a function of
Table 6. Effect of various compounds on enzymic activities of
alkaline phosphatase and NTP pyrophosphohydrolase
% enzymic activity after
9% activity
addition of inhibitor
* The reaction mixtures were prepared as described in Patients and
Methods using (8-I4C)-ATP as substrate, and the pH of the buffer
was varied from 7.5 to 11. Tris-HCI buffer pH 7.5 to 9.5 and
bicarbonatexarbonate buffer pH 9.8 to 11 were used.
phosphatase control preparation assay. It can be seen
that the NTP pyrophosphohydrolasc is strongly inhibited by dithiothreitol, cysteine, and sodium etidronate,
to about the same extent as alkaline phosphatase is.
However, the former enzyme activity was not inhibited detectably by bromotetramisole, tetramisole, or
levamisole in contrast to alkaline phosphatase which,
at the concentration used, was totally blocked by these
three inhibitors. cAMP did not accumulate with or
without isobutyl-I-methylxanthine,indicating a very
low level of adenyl cyclase in the triton extracts.
Figure I depicts a Lineweaver-Burk plot (20),
which shows Km values under experimental conditions described for (8-CI4)-ATP, for OA and CPDD
cartilage extracts. The plot shows no difference between the slopes for OA and CPDD cartilages. The
Km of various samples tested was 2.3 ? 0.52 x
10-'M. These Km values were within a range of 1.4 X
to 3 x 1O-'M for ATP. In contrast, bovine
epiphyseal enzyme Km was previously found to be
1.8 x 10-4M (7). For a liver cell membrane derived
enzyme, the Km with ATP as substrate at pH 8.0 was
previously determined at 0.32 p M , and Ca++ 10-4M
(23). In another report the Km was 780 p M , using ATP
NTP pyrophosphohyd r o I a se
(% of control
(% of control
(1 mmole)
Na etidronate
1 I7
* A phosphodiesterase inhibitor. No cAMP accumulation was demonstrated.
Table 5. Effect of metal ions on enzymic activities, with (8-"C)ATP used as substrate
% activity
EDTA, 1 mM
EGTA, 1 mM
C a + + , I mM
Ca+', 0.1 mM
Mg++, 1 mM
Mg+', 0.1 mM
K + , 30 mM
Na', 30 mM
Figure 1. Lineweaver-Burk plots of NTP (ATP) pyrophosphohydrolase activity in the presence of IO-'M tetramisole. Open circles
represent an extract from cartilage of,a patient with osteoarthritis.
Closed circles represent an extract from cartilage of a patient with
calcium pyrophosphate deposition disease. Ordinate equals the
reciprocal counts per minute per 5 mg wet tissue per 2%-hour
incubation at 3TC, of I4C-AMP plus ''C-adenosine.
as substrate (24). These discrepancies may have resulted from strong pH dependence of the Km, as well
may be important
as the fact that endogenous Ca '+
for activity. Chelation of endogenous Ca+' by the
substrate might inhibit enzyme activity and produce
overestimation of the Km.
Table 7 shows the effect of adding other nucleotide triphosphates as substrates in lieu of ATP. The
percentage activity of CTP and UTP was roughly onethud to one-half, and GTP one-fifth of the level of
activity found for ATP under conditions studied here.
Similar results were found by Ryan et a1 in assays on
cartilage slices (5).
To our knowledge, these studies represent the
first characterization of an NTP pyrophosphohydrolase partially purified from normal or diseased articular cartilage, and indeed its first identification anywhere in humans. The pH range activities in the
presence of metal ions, organic inhibitors, and other
properties are consistent with the view that this enzyme is similar to that studied in other cartilages (6,7),
as well as in liver (8-12). The contention that these
enzyme activities are truly separated in these plasma
membrane fractions is strengthened by: 1) the finding
of the same results using two separate chromatographic techniques for measuring pyrophosphate, 2) the
quantitative correlation of these findings with AMP
and adenosine production using the (8-14C)-labeled
ATP, and 3) previous measurements of PPi production
from ATP by spectrophotometric methods (3,4).
The assayed levels of adenyl cyclase were low
compared with the levels found for NTP pyrophosphohydrolase. Roughly baseline enzymatic activity in the
presence of 1 mM Mgff or 1 mM Gaff is consistent
with a surface membrane-involved enzyme, since
such concentrations are present in extracellular fluid.
It was fortunate for purposes of studying this enzyme
that the levamisole series of inhibitors could block
Table 7.
Substrate specificity*
% activity
* The reaction mixtures were prepared as described in Patients and
Methods. ATP and the other nucleotides were added at 1 mmole,
with specific activity of 2 mCilmmole (n = 6 patients).
alkaline phosphatase selectively, with no effect on the
NTP pyrophosphohydrolase.
In a previous study, PPi elaboration by articular
cartilage was greatly suppressed by cysteine and dithiothreitol, despite the fact that alkaline phosphatase
activity was reduced in those studies (1). This seemed
engimatic, in that we had anticipated reduced catabolism of extracellular pyrophosphate ions by the incubation with these enzyme inhibitors. The results of
those studies could readily be explained now by simultaneous inhibition of the postulated NTP pyrophosphohydrolase ecto-enzymatic function, if there were a
nucleotide substrate available in the cartilage slices.
Possibly, the increase of NTP pyrophosphohydrolase
reflects a homeostatic change to accelerate calcium
efflux, resulting from cell injury during production of
osteoarthritic lesions. Whether the increased NTP
pyrophosphohydrolase is needed for a nucleotide salvaging function, as suggested by Ryan et a1 (5) and
previously alluded to in the introduction to this paper,
remains untested. Whether there is up-rcgulation of
calcium efflux due to cell leakages and influx also
requires further study.
Finally, if PPi wcre not sufficiently removed
from the system in CPDD because of deficient extracellular pyrophosphatase function relative to need, the
net result would be increased PPi bioavailability for
mineralization. Further study is needed on whether
actual functional disturbances in the intact chondrocytes of CPDD patients in vivo are reflected in the
findings on extracted enzymes. Obviously, disruption
of the membrane apparatus for ion transport by triton
extraction requires cautious interpretation of the enzyme activities reported here. Nevertheless, this significantly disturbed activity of component enzymes
could have significance for the pathogenesis of CPDD.
Torp-Pedersen et a1 found substantial activity
of NTP pyrophosphohydrolase in most organs, including kidney, small intestine, and salivary glands (23). It
is tempting to postulate that CPDD-osteoarthritic cartilages, as displayed in Table 2, have a high enzyme
activity level related to phenomena secondary to rapid
matrix synthesis or responses to injurious physical
forces. In particular, to sort out what isolated specific
conditions affect NTP pyrophosphohydrolase, and
what function this enzyme serves, will require study of
cells in culture.
The authors are deeply indebted to Dr. Jerry Enis,
Clinical Professor of Orthopedic Surgery, University of
Miami School of Medicine for the samples of osteoarthritic
and chondrocalcinotic cartilage. We wish to thank Mrs.
Geneva Jackson for her excellent secretarial assistance.
1. Howell DS, Muniz OE, Pita JC, Enis JE: Extrusion of
pyrophosphate into extracellular media by osteoarthritic
cartilage incubates. J Clin Invest 56: 1473-1480, 1975
Ryan LM, Cheung HS, McCarty DJ: Release of pyrophosphate by normal mammalian articular hyaline and
fibrocartilage in organ culture. Arthritis Rheum 24: 15221527, 1981
Tenenbaum J, Muniz OE, Schumacher HR, Good AE,
Howell DS: Comparison of phosphohydrolase activities
from articular cartilage in calcium pyrophosphate deposition disease and primary osteoarthritis. Arthritis
Rheum 24:492-500, 1981
Howell DS, Muniz OE, Morales S: S'nucleotidase and
pyrophosphate (PPi) generating activities in articular
cartilage extracts in calcium pyrophosphate deposition
disease (CPDD) and in primary osteoarthritis (OA),
Epidemiology of Osteoarthritis. Edited by JG Peyron.
Paris, Ciba-Geigy, 1980, pp 99-108
Ryan LM, Wortmann RL, Karas B, McCarty DJ: Nucleoside triphosphate pyrophosphohydrolase activity in
canine articular cartilage (abstract). Arthritis Rheum
(suppl) 25:S30, 1982
Cartier P, Picard J: La mineralization du cartilage ossifiable. 111. Le mecanisme de la reaction ATPasique du
cartilage. Bull SOCChem Biol 37: 1159-1 176, 1955
Hsu HHT: Purification and partial characterization of
ATP pyrophosphohydrolase from fetal bovine epiphysea1 cartilage. J Biol Chem 258:3463-3468, 1983
Lieberman I, Lansing AI, Lynch WE: Nucleoside triphosphate pyrophosphohydrolase of the plasma mernbrane of the liver cell. J Biol Chem 242:736-739, 1967
Ray TK, Tomasi V, Marinetti GV: Hormone action at
the membrane level. I. Properties of adenyl cyclase in
isolated plasma membranes of rat liver. Biochim
Biophys Acta 22:20-30, 1970
Franklin JE, Tramz EG: Metabolism of coenzyme A and
related nucleotides by liver plasma membranes. Biochim
Biophys Acta 230: 105-1 16, 1971
House PDR, Poulis P, Weidemann MJ: Isolation of a
plasma membrane subfraction from rat liver containing
an insulin sensitive cyclic-AMP phosphodiesterase.
Eur J Biochem 24:429-437, 1972
Flodgaard H, Torp-Pedersen C: A calcium ion-dependent adenosine triphosphate pyrophosphohydrolase in
plasma membrane from rat liver. Biochem J 1712317820, 1978
Schumacher HR, Giblisco P, Reginato A, Cherian U ,
Gordon G: Implications of crystal deposition in osteoarthritis, Clinical Patterns and Pathological Features
in Osteoarthritis. Edited by TD Cooke. J Rheumatol
(SUPPI9) 10~41-42, 1983
McCarty DJ Jr: Calcium pyrophosphate crystal deposition disease: pseudogout: articular chondrocalcinosis,
Arthritis and Allied Conditions. Ninth edition. Edited by
DJ McCarty. Philadelphia, Lea and Febinger, 1979, pp
1276-1 299
Kellgren JH, Lawrence JS: Radiological assessment of
osteoarthrosis. Ann Rheum Dis 16:494-502, 1957
Mankin HJ, Dorfman H, Lipiello L, Zarins A: Biochemical and metabolic abnormalities in articular cartilage
from osteoarthritic human hips. 11. Correlation of morphology with biochemical and metabolic data. J Bone
Joint Surg 53A:523-537, 1971
Dixon TF, Purdom M: Serum S'nucleotidase. J Clin
Pathol 7:341-343, 1954
Russell RGG, Bisaz S, Donath AJ, Morgan DB, Fleisch
H: Plasma pyrophosphate in bone disorders. J Clin
Invest 50:961-969, 1971
Fullerton PD, Finch LR: Use of polyethyleneiminecellulose thin layers in assay of pyrophosphate: ATP
exchange reactions. Anal Biochem 29:544-547, 1969
Lehninger AL, editor: Biochemistry: The Molecular
Basis of Cell Structure and Function. New York, Worth
Publishers, Inc., 1970
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ:
Protein measurement with the fohn phenol reagent. J
Biol Chem 193:265-275, 1951
Ceriotti G: A microchemical determination of deoxyribonucleic acid. J Biol Chem 198:297-303, 1952
Top-Pedersen C, Flodgaard H, Saermark T: Studies on
a Ca2+ dependent nucleoside triphosphate pyrophosphohydrolase in rat liver plasma membranes. Biochim
Biophys Acta 57194-104, 1979
Decker K, Bischoff E: Purification and properties of
nucleotide pyrophosphatase from rat liver plasma membranes. FEBS Lett 21:95-99, 1972
Без категории
Размер файла
672 Кб
characteristics, osteoarthritis, chondrocalcinosis, pyrophosphohydrolase, cartilagesome, human, biochemical, ntp
Пожаловаться на содержимое документа