2.3 NORMATIVA DE LIQUIDACIONES PARA EL SECTOR FINANCIERO
2.3.2 DE LA LIQUIDACIÓN
2.3.2.2 Liquidación forzosa
2.3.2.2.2 Del liquidador
TCA/methanol derivative, OPG-ATP, first identified by Hutchinson et al (1986a). Nearly
all of the soluble radioactivity was confined to ATP, ADP, AMP, GTP and the monomer
PG-ATP (see Table 3.1 and Table 3.2). The amount of insoluble derivative in these hearts
is estimated to be 0.62|imol/g. wet wt. adenylate equivalent (see Section 3.B) which after
phenol extraction has been purified by DEAE-cellulose chromatography under sterile
conditions to give the major radioactive species with a phosphate:purine ratio of 3.9:1.
Although Fitt et al (1987) have confirmed this rapid incorporation of substantial amount
of ^'^C-adenosine in TCA/methanol insoluble material in perfused hearts, they report
that the product, which accounts for -70% of the insoluble radioactivity, resembles RNA
or polyA. Based on the specific radioactivity of tissue adenylate, their data imply
incorporation of more than 0.4^mol adenine/g. tissue. The total RNA in a Ig. heart is
estimated to contain l.l|im o l of adenine and maximally 40nmol polyA (Heyworth et al,
1987). Thus, a turnover of more than 30% of total tissue RNA w ould be required in a 20
min. perfusion period if the product observed by Fitt et al (1987) were really RNA.
Incorporation of radioactivity into the insoluble derivative was much less when hearts
were perfused with 10pM[8-^^C]-adenine or 2.5|iM-hypoxanthine, even in the presence
of O.SmM ribose, compared to that with adenosine perfusion. These differences can be
attributed to the disproportionate labelling of adenine nucleotides in the endothelial cells
and myocytes (see Section l.E) and to the rate limiting enzymes, notably APRT, HGPRT
In heart, OPG-ATP appears to be present in both cell types (Lawson and Mowbray, 1986)
in direct proportion to the quantities of free adenine nucleotides in the whole tissue.
Using techniques similar to those employed with hearts, the presence of OPG-ATP in rat
liver has been demonstrated (see Section 3.F). Liver contains 5-10 times more OPG-ATP
per g. than heart and cell fractionation experiments showed that virtually all of tissue
OPG-ATP was confined to the mitochondrial fraction; over 90% of insoluble radioactivity
was found to co-distribute with succinate dehydrogenase activity (Section 4.C; Patel et al,
1991a).
The labile mixed anhydride link in OPG-ATP (see Figure 1.3) is remarkably stabilised in
neutral solution by Mg^"^ ions above 2mM (Section 3.E). Addition of a chelating agent
(e.g., EDTA) results in over 80% breakdown of the oligomer into two major products, one
proposed to be the monomer ppp5'A3'p3Gri (PG-ATP) and the other corresponding to a
chemically synthesised phosphodiester A3'p3Gri. Alkaline hydrolysis of OPG-ATP also
results in the above compounds. The mechanism for stabilisation of OPG-ATP by Mg^"*"
or even Mn^"^ above 2mM (Mowbray et al, 1987) is not clear but could be similar to the
metal-ion containing "head-tail" stacks of ATP (Corfu and Sigel, 1991) which involve
inter-molecular ionic interactions as well as hydrogen bonding between ATP molecules
(see Figure 3.13).
By using an assay based on the ability of OPG-ATP to precipitate in cold 50% aqueous
ethanol, a specific OPG-ATP 3' phosphodiesterase activity has been partially purified
and characterised from rat liver mitochondria (Section 4.B) which selectively hydrolyses
the 3' ester link in OPG-ATP (see Figure 4.11). This activity has been purified over 40-
fold from liver mitochondria by (NH4)2S0 4 precipitation and f.p.l.c. (Table 4.7). The
and not associated with lysosomal or endoplasmic reticular subcellular compartments
(see Section 4.C). Although the activity is latent in intact mitochondria, it is not
associated with intact phosphorylating inner-membrane vesicles (mitoplast), but is
released during the preparation of mitoplast implying an inter-membrane space location
(Table 4.8).
The enzyme, as purified, contains tightly bound OPG-ATP which can be released,
without inactivation of the enzyme, by mild alkaline treatment (see Section 4.B). This
suggests that the oligomer is also located in the mitochondrial inter-membrane space.
The finding that OPG-ATP is mitochondrial together with its association with Mg^"^ ions
and the above enzyme may account for its n.m.r. invisibility and explains why the
existence of such a large pool of a highly phosphorylated compound has not been
observed by others.
The apparent for OPG-ATP is -35pM which is consistent with its content in tissues:
greater than ImM in heart and kidney and at least 5mM in liver (see Section 4.B).
However, the relatively low V^ax of 6nmol / min. / mg protein (~30nmol/min./g. wet
tissue) is in contrast with the rapid turnover of OPG-ATP with soluble ATP
(~60runol/min./g. wet tissue) observed in heart and kidney (Mowbray et al, 1984b;
Hutchinson et al, 1986b) and the less complete equilibration but comparable rate of
exchange found in liver (Section 3.F.2; Patel et al, 1991a). This, allied to the poor recovery
of total activity from f.p.l.c. suggests the loss of some regulatory factor during
purification. Another possible factor for the low V^ax may be the presence of a high
concentration (lOmM) of Mg^^ ions in the assay mixture which by altering substrate
conformation could conceivably be inhibitory to the enzyme.
The product of the enzyme is proposed to be the monomer 3-phosphoglyceroyl^-
findings that it migrates on t.l.c. in borate buffer indicating that the 2' and 3' hydroxyls of
the ribose are free and also that the Pi:purine ratio was found to be 3.8:1 (see Section 4.D).
On anion-exchange h.p.l.c., this compound elutes between AMP and ADP as does a low
molecular weight-related compound B found in tissue extracts (Section 3.D). However,
these two compounds differ in their migration pattern on 2-D t.l.c. The enzymic product
co-chromatographs with ATP in the borate (pH7) system whereas the extracted
compound (B) does not migrate in this system and is proposed to be of the form
ppp5'A3'p3Gri. All soluble extracts from heart and liver contain a purine compound
that co-chromatographs on h.p.l.c. with PG-ATP.
The identity of the enzymic product PG-ATP was further supported by the finding that
digestion with snake venom phosphodiesterase 1 (which requires free 2' and 3'-OH on
ribose) yields ADP (see Figure 4.12). Based on these results, a sensitive luminometric
assay was attempted (Section 4.E) for OPG-ATP by linking the 3' phosphodiesterase
product via snake venom phosphodiesterase to produce ADP and then to ATP with PEP
and PK; the ATP produced being detected using luciferin-luciferase. Several problems
were encountered which are discussed in Section 4.E, and optimal conditions remain to
be established if this is to prove a useful assay.
5.B The P o ssib le Function o f O PG -A T P
The function of OPG-ATP in the heart remains conjectural. The findings that OPG-ATP
is mitochondrial and is bound to the inter-membrane space 3'-phosphodiesterase, an
enzyme résistent to dénaturation by TCA at 0°C (see Section 4.C), like adenylate kinase
and creatine kinase, suggests that OPG-ATP has a major role in adenine nucleotide
pyrophosphate bonds or both. The presence of similar types of nucleotides, e.g., Ap^A
(diadenosine 5', 5"'-P^-P^-tetraphosphate) and Gp^G (diguanosine tetraphosphate) have
been reported (Anderson, 1989). Accumulation of Ap^A in prokayrotic and eukaryotic
organisms in response to oxidative stress or heat shock have been found (Lee et al, 1983a,
1983b; Brevet et al, 1985). Gp4G has been observed to provide purines during
embryogenesis in a range of aquatic species (Warner and Finamore, 1965). Consistent
with such a storage role are the observations that the total acid-insoluble radioactivity in
^'^C-Ado perfused hearts increases following short-term ischaemia (Hutchinson et al,
1981; Fitt et al, 1985). The ability to replenish the 10-20% loss of ATP from myocytes (to
endothelial cells) as nucleosides and bases following short-term ischaemia could be an
important property of the healthy heart. OPG-ATP seems unlikely to be a high energy
source for contractile work, at best providing 5 s. of energy. Nevertheless, it could act as
a source to replenish rapidly small amounts of Ado released as vasodilatory agent for
vascular smooth muscle in heart (Berne, 1980).
At variance with a potential storage role for OPG-ATP is the rapid attainment of specific
radioactivity equilibrium between OPG-ATP and soluble ATP (Mowbray et al, 1984b),
implicating a more dynamic role for this compound. Such a function might be a kind of
buffer for the ATP/ADP ratio and this may explain why free [ADP] does not rise above a
low threshold value of approx. 50|iM even at high workloads (From et al, 1986; Balaban
et al, 1986). A mechanism for the preservation of a high ATP/ADP ratio is essential to
maintain the unidirectional flux in metabolic pathways and to provide an energy
currency for cell viability. The observed synchronous oscillations in soluble adenine
nucleotide content of Langendorff perfused heart (Mowbray et al, 1984a) may be the
result of such regulatory mechanisms, bereft of sympathetic innervation and blood borne
phosphorylation without allowing the free [ADP] to rise appreciably. The factors which
initiate the synchronous oscillations in heart are not yet clear. One possible factor
involved in the rapid (10 min. interval) sequestration and mobilisation of adenine
nucleotides may be a brief period of ischaemia which the heart experiences on removal
from the animal into the cooling medium (2°C) for cleaning prior to cannulation. Such a
possible mechanism is supported by two lines of evidence. Firstly, Hutchinson et al
(1981) reported a two-fold increase in TCA/methanol insoluble radioactivity following a
2 min. period of ischaemia which is restored to control values on reperfusion. Secondly,
n.m.r. study of the Langendorff perfused hearts (Bailey and Seymour, 1981) showed
that following a 6 min. period of global ischaemia, the PCr content which disappeared
during ischaemia rapidly returned to its pre-ischaemic level, whereas the contents of
ATP showed pronounced variation over the subsequent 30 min. of perfusion. These
variations are comparable to those reported by Mowbray et al (1984a).
Consistent with such a buffering function is a ^^P n.m.r. study of the perfused rat heart
in which the soluble nucleotides and Pi were measured at various times during the
cardiac cycle (Fossel et al, 1980). Systematic and inverse changes in PCr and ATP with Pi
were observed during each contraction-relaxation period (214ms). However, estimates
of the amount of O2 which would be required using oxidative phosphorylation to
rephosphorylate the resultant ADP with Pi were more than 4 times the observed O2
consumption. This implies that free nucleotide is replenished from an n.m.r. invisible
high phosphate compound such as OPG-ATP. The oligomeric nature of OPG-ATP and
its mitochondrial location as well as its binding to divalent metal ions and the enzyme
3'-phosphodiesterase, could contribute to this n.m.r. invisibility.
Humphrey et al, 1991), liver (Desmoulin et al, 1987; Iles et al, 1985) and kidney (Stubbs et
al, 1984) somewhat less than 50% of the extractable Pi pool is ever n.m.r. visible. Thus,
OPG-ATP may play some role in the control of [Pi] and its function in glycogenolysis and
glycolysis at key phosphorolytic steps.