ISRAEL JOURNAL OF
VETERINARY MEDICINE
MULTIPLE ISO-FORMS OF CAPRINE
ADENOSINE DEAMINASE
L. F. S. Rodrigues1 G. H. Freire2 and M. R. Vale2
1 - Faculty of Agricultural Sciences of Pará, Brazil
2 - Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of
Ceará, Brazil
abstract
Adenosine has been established as an important inihibitor of the
immune system. Its tissue levels can be modulated by adenosine deaminase
(ADA). ADA activity is used as a marker for infectious diseases. Nothing was
known about ADA in caprine tissues. This paper describes the detection of
ADA in caprine serum, liver, heart and duodenum. In serum the levels of ADA
(19.5 + 6.0 U/L) are similar to those in human serum. The duodenum shows
about 25 times more ADA activity (108 + 2.4nmols NH3/mg protein/minute)
than liver (3.9 + 0.7 nmols) and heart (3.7 + 0.3 nmols). Molecular filtration of
a fresh duodenal homogenate produced 2 ADA peaks: peak 1 representing a
high molecular weight and peak 2, an intermediate molecular weight.
Molecular filtration of a previously frozen homogenate produced an additional
peak (peak 3) with a low molecular weight beyond peaks 1 and 2. The
rechromatography of lyophylized material from the original peak 1 produced
only peak 3. Lyophylized material from peak 2 or peak 3 did not produce
different peaks after rechromatography. It is proposed that the isoenzyme
profile of caprine ADA activity is similar to the human.
INTRODUCTION
It is well established that the nucleoside adenosine (Ad) is an important extracellular
signal. It possesses receptors in many tissues including adipose, nervous, liver,
smooth and cardiac muscles, as well as in the cells of the immune system. A number
of papers report the inhibitory actions of adenosine on the immune system (1, 2, 3, 4).
Adenosine levels are mainly modulated by two enzymes: adenosine kinase and
adenosine deaminase (ADA, E.C.3.5.4.4).
In human ADA is expressed as two isoenzymes: ADA1 and ADA2.
ADA1 is synthesized in all tissues and is found either free or associated with
CD26 (5, 6). When ADA is absent for genetic reasons severe combined
immunodeficiency (SCID) develops as a consequence of high levels of
adenosine, 2’-deoxiadenosine and dATP formation. The latter phenomenon
has been implicated in the blockade of lymphocyte proliferation due to its
inhibitory effect on DNA replication (7). ADA2, exclusively synthesized in
monocytes and macrophages, has been used as a marker of infectious
diseases such as tuberculosis (8, 9), AIDS (10) and typhoid fever (6).
Nothing is known about ADA from caprine tissues or the role of
adenosine on the immune system of the goat. It should be of interest to study
ADA in caprine tissues to determine if ADA could be used as marker of
infectious processes like Caprine Arthritis-Encephalitis (CAE), in which the
monocytic-macrophagic system is involved (11, 12).
This paper presents the results of the purification of ADA isoenzymes
and some of their kinetic properties.
MATERIAL AND METHODS
Animals and collection of biological material
Healthy male crossbred goats aged 18 to 40 months old weighing 40 to
60Kg, and slaughtered in the city slaughter-house (Fortaleza-Ceará, Brazil),
were used.
Before slaughter, blood (10ml) was collected from the jugular vein and
kept on ice until processing in the laboratory. After clotting the serum was
separated, centrifuged (3000xg, 10 minutes) and used for ADA assay.
Following slaughter, the abdomen was open and portions of duodenum (5
cm), heart (15g) and liver (15g) were removed. The tissues were quickly
washed with cold saline and immediately taken to the laboratory on ice. The
tissues were again washed, this time with phosphate buffer, 50mM, pH 7.0.
Homogenization was processed in the same buffer in a proportion of 1 g of
tissue to 4ml of buffer in a Potter homogenizer (1500rpm, 5 strokes). The
homogenates were then centrifuged at 26,000xg for 30 minutes in a
refrigerated centrifuge (5ºC). The supernatants were divided in aliquots and
frozen. A fresh aliquot (not frozen) was used for ADA assay, analysed for its
protein content and chromatographed as described below. When using
thawed aliquots they were centrifuged before loading on the column to
eliminate denatured proteins.
ADA assay
The enzyme assay is based on the determination of ammonia formed
in the deamination of adenosine by ADA by the method of Giusti (13) with
slight modifications. In brief, to a final volume of 220 μl was added 200μl
adenosine (21mM) in phosphate buffer, 50mM, pH7.2 and 20μl of the enzyme
source (serum or supernatant of tissue homogenates). The tubes were then
incubated in a water bath at 37o C for 1 hour. The reaction was terminated
with 600μl phenol/potassium nitroprussiate (106mM/101.7mM). In control
samples the enzyme source was added after this procedure. After addition of
600μl 11mM sodium hypochloride in 125mM NaOH the samples were
reincubated at 37o for 30 minutes. When analysing the activity of fractions
from chromatographic column 100μl adenosine (21mM) and 100μl of fractions
were added for the first incubation period. Control tubes were also run without
adenosine to discard the ammonium from other sources such as deamination
of proteins or aminoacids. Ammonium sulphate (75μM) was used as ammonium
standard. For the determination of ADA optimum pH in the fractions from the
chromatographic column the same buffer was used (sodium phosphate buffer, 50mM)
to solubilize adenosine by varying the pH from 6.0 to 8.0. The other procedures were
the same as described above.
Protein assay
The protein concentration of the homogenates was assayed by the
method of Lowry (14).
Molecular filtration
A Sephadex G200 column (30x3cm) was equilibrated and eluted with
phosphate buffer 50mM, pH 7.2, and a flow rate of 0.5ml/minute was
maintained. One mililiter of duodenum supernatant was applied to the column.
Fractions of 2ml were collected and analysed for ADA activity and for their
absorbance at 280nm. Fractions from the 3 peaks obtained were pooled and
concentrated by liophylization. These new samples were then frozen and
thawed. After centrifugation they were applied separatelly to the same
column, eluted and analysed following the same procedures described above.
RESULTS
ADA activity of 19.5+6.0 U/L (n=27) was found in the goat serum. In
duodenum, liver and heart homogenates the ADA levels were 108.8+ 2.4,
3.9+0.7 and 3.7+0.3 nmols of ammonium formed/mg of protein/minute,
respectively. The ADA specific activity in duodenum was about 25 times
higher than in liver and heart homogenates.
Figure
1:
Chromatographic profile of ADA activity and absorbance at 280nm in a fresh
homogenate of caprine duodenum in Sephadex G200. The column was
equilibrated and eluted with phosphate buffer (50mM, pH7.2) at a flow rate of
0.5ml/min. Fractions of 2ml were collected.
Figure 2 :
Chromatographic profile of ADA activity of a frozen and thawed homogenate of caprine
duodenum in Sephadex G200. The column was equilibrated and eluted with phosphate buffer
(50mM, pH7.2) at a flow rate of 0.5ml/min. Fractions of 2ml were collected.
Figure 3 :
Graph A
Chromatographic
profile of ADA
activity in the
Sephadex G200
column of
lyophylyzed
fractions of peak 1
from figure 1. The
column was
equilibrated and
eluted with
phosphate buffer
(50mM, pH7.2) at
a flow rate of
0.5ml/min.
Fractions of 2ml
were collected.
Graph B
Chromatographic
profile of ADA
activity in the
Sephadex G200
column of
lyophylyzed
fractions of peak 2
from figure 1. The
column was
equilibrated and
eluted with
phosphate buffer
(50mM, pH7.2) at
a flow rate of of
0.5ml/min.
Fractions of 2ml
were collected.
The chromatographic profile of protein (absorbance in 280nm) and of
ADA activities from duodenum homogenate are given in figure 1. It shows a
small peak of ADA activity (peak 1) about fraction 15 and a larger one (peak
2) about fraction 40. It can also be observed that the first protein peak
coincides with peak 1 of ADA activity. Peak 1 presents reduced ADA activity
and an optimum pH varying from 6.8 to 7.0. Peak 2 indicates a low protein
concentration and shows the largest ADA specific activity eluted from the
column with the samples showing an optimum pH=7.2.
The chromatographic profile was altered when the applied sample was
not fresh. Figure 2 shows that after freezing the activity of peak 2 decreased
to one third of the original values as observed in figure 1. In addition, the
frozen and thawed sample produced an additional peak of ADA activity (peak
3) about fraction 68. This new peak showed an optimum pH varying from 7.0
to 7.2.
The rechromatography of fractions from peak 1 (figure 3A) or from
peak 3 (data not shown) produced only one peak of ADA activity which
appeared in the same position as the original peak 3 from figure 2. The
rechromatography of the fractions from peak 2 also produced only one peak
of ADA activity observed about the same position of the original peak 2
(figure 3B).
DISCUSSION
ADA activity was found in caprine serum in levels comparable to those
found in healthy human beings (13). Since no reference was found in the
literature about ADA in goats the present results cannot be evaluated
objectively.
The largest enzymic activity was found in the duodenum which
expresses at least 25 times more ADA activity than liver and heart
homogenates. Similar results were found for cattle (15).
The chromatographic profile of ADA activity from the fresh duodenal
homogenate revealed 2 peaks showing in the first a large isoform and in the
second a smaller isoform. When the chromatography was performed with a
frozen sample a third peak was eluted presenting an even smaller molecular
weight. In addition, when the lyophylized fractions from peaks 1 and 3 were
rechromatographed, the chromatographic profile exhibited only one activity
which peaked about the original peak 3. This result probably indicates that the
large molecule from peak 1 underwent modification after freezing with loss of
molecular weight, that is, a large molecule with ADA activity had lost part of its
structure and became smaller.
In contrast, ADA protein from peak 2, did not undergo any apparent
changes in its molecular weight after freezing. However, this isoform
presented a large fall in its activity, indicating that freezing had somehow
denatured it.
As already mentioned, in humans ADA activity is expressed by at least 2
isoforms: a small form (ADA1) which can be found free or bound to a large
protein (CD26) (5, 6) and an intermediary form (ADA2) which exhibits
different properties (16). The present results indicate that the goat similarly
expresses ADA. It seems that freezing unbinds the two proteins, and under
very low temperatures the big molecule formed by small ADA and a binding
protein, eluted in peak 1, are no longer together and they separate to release
the free small molecule, expressed in peak 3.
Further work is required to establish whether the role of ADA in the
goat immune system is similar to that in human immune system.
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