CONDICION DE LA VIA CON MANTENIMIENTO

threshold value (~50|iM) above which respiratory control of heart mitochondria is not a

function of free ADP concentration. What is not clear are the mechanisms that restrain

the ADP at this threshold and why free ADP does not rise in mild or short-lived

ischaemia. Even more difficult to explain is what limits the fall of ATP concentrations for

minutes when energy production is severely limited by ischaemia while a "bufier"

substrate like PCr disappears within tens of seconds of the onset of ischaemia (Bailey

and Seymour, 1981); or how the ATP pool is apparently replenished periodically in long­

term ischaemia as n.m.r. studies of frog skeletal muscle and heart seem to show (Keidel

et al, 1984). Similarly, in perfused rat livers, Murphy et al (1988) observed n.m.r.

resonances for ATP declining faster than extracted ATP and proposed that this missing

nucleotides represented an n.m.r.-invisible mitochondrial pool. However, there is

controversy regarding the n.m.r. visibility of mitochondrial ATP. In isolated

mitochondria (Hutson et al, 1989) and in perfused heart (Humphrey and Garlick, 1989)

all the ATP is reported to be n.m.r.-visible. Additional information is required for the

One potentially vital clue is the discovery in rat heart of an entirely new derivative of

ATP which exchanges rapidly with ATP so that they reach specific radioactivity

equilibrium within 10 min. (Mowbray et al, 1984b). In the isolated non-working

Langendorff perfused rat heart, Mowbray et al (1975) observed significant variations in

the tissue contents of cAMP and cGMP and indeed in the ratios of these cyclic

nucleotides during 60 min. perfusion. In view of the important intracellular regulatory

role of these cyclic nucleotides (e.g., heart contractility), the observed fluctuations stand

in contrast with the apparent steady-state of the heart preparation for up to 2 hrs. in the

Langendorff perfusion system, as judged by oxygen consumption, glucose uptake,

glycogen turnover, lactate turnover, alanine output (Mowbray and Ottaway, 1973a and

1973b), and protein synthesis rate (Mowbray and Last, 1974; Mowbray et al, 1975), beat

rate and contraction amplitude (Bates et al, 1978).

These conflicting results can be explained if, as has been suggested, cellular feedback-

regulation systems give rise to stable oscillations on which metabolic steady states rely

(Hess et al, 1969). Moreover, oscillations in the concentrations of adenine nucleotides

and IMP, apparently related to glycolytic oscillations, have been reported in particle-free

skeletal muscle extracts (Tomheim and Lowenstein, 1975).

Subsequently, Bates et al (1978) made a striking observation that the total extractable

adenine nucleotide pool was not constant but showed large very regular rapid variations

in size such that within 10 min. the tissue content could change by 1.3|xmol/g. wet wt.

and even show a net increase of 0.75|imol/g. wet wt. over the amount present

immediately after washout perfusion and before recirculation of perfusion medium. The

ATP/ADP ratio, cyclic AMP content, GTP content and the GTP/GDP ratio in the tissue

The magnitude of the observed changes, particularly in the ATP content, raised the

question as to why such variations had not been previously observed. It has been

generally found that the concentrations of the adenine nucleotides in the perfused heart

remain constant despite their rapid turnover (Neely et al, 1972; Nayler et al, 1976). A

possible explanation for this discrepancy can be attributed to the extraction technique

used by most workers. Freeze-clamped hearts are generally extracted with perchloric

acid or trichloroacetic acid (TCA) at 0°C or higher temperatures. These procedures have

been known to result in the redistribution of the phosphate content of the adenine

nucleotides (Seraydarian et al, 1961; Minard and Davis, 1962; Lowry et al, 1964) notably

because adenylate kinase and creatine kinase are not denatured under these conditions.

Opie et al (1971) reported significant changes in the adenine nucleotide content of

perfused rat hearts when extracted with cold perchloric acid / acetone / EDTA mixture

but not when perchloric acid was used alone. The use of a chelating agent in cold

organic solvent/aqueous mixtures has been shown to prevent phosphate exchange in

tissue extracts (Seraydarian et al, 1962; Lowry et al, 1964).

The net increases and decreases in the adenine nucleotides (see above) could not be

accounted for as extraction artifacts or as systematic variations between perfusions

(Bates et al, 1978) and no evidence was found of exchange of substantial quantities of

adenine nucleotides into other mono or di-nucleotides, nucleosides or bases. Nor was

the perfusion medium acting as a store although it did acquire (irreversibly) significant

quantities of xanthine, hypoxanthine and uric acid and (reversibly) small amounts of

adenosine and inosine. Further more, estimates of de novo synthesis rate (see Section l.D)

and purine salvage rate (see Section l.E) combined put these at a rate two orders of

magnitude lower than that required for the observed changes. Alternative cellular

changes in purine concentrations could also be eliminated since the amounts of

nucleotide produced were much greater than the total available from these sources (see

Bates et al, 1978).

Hence, it was suggested that some hitherto unknown compound capable of rapid

exchange with the soluble nucleotides must exist (Bates et al, 1978). Selective labelling of

the heart nucleotides using 0.25|iM[8-^'^C]-adenosine showed that 10-15% of

radioactivity was rapidly incorporated into a TCA/methanol insoluble species which

resisted repeated acid extraction but could be readily solubilised by dilute alkali

(Hutchinson et al, 1981; Mowbray et al, 1984b). On further extraction of the

TCA/methanol precipitate in 1% SDS buffer and phenol, 70% of the radioactivity

partitioned with the nucleic acid-rich fraction. This amount of radioactive material was

subjected to DEAE-cellulose chromatography in 7M Urea/lOmM sodium acetate buffer,

pH4.5 under sterile conditions and a major radioactive peak at specific radioactivity

equilibrium (-80,000d.p.m./^imol) with soluble heart ATP was found. This species had

an apparent Mr of about 3,000 as judged by gel-filtration chromatography on Sephadex

G-50 and its phosphate:purine ratio was 4.2:1 (Mowbray et al, 1984b). This material was

stable in sterile conditions in the presence of urea at pH4.5; however following desalting

it was found to be very labile.

Selective digestion of the purified compound allied to a variety of thin layer

chromatography and staining procedures has shown that it is composed of adenine,

ribose, glyceric acid and phosphate in the ratio 1:1:1:4. A n.m.r. analysis of the

compound revealed four distinct phosphate environments present in equal proportions:

two corresponded to the a and P phosphate of ATP, while the third was near the y, and

Figure 1.3

Structure ofoligophosphoglyceroyl-adenosine triphosphate (OPG-ATP).

H b HO' I O O Il II C “ 0 + P ”“ 0 “ P - ^ 0 — P —-Q OH OHI O O Il II c “ c “OF P ~“0 ” p “ O “ p “ O H O OH OHI OHI HO HO

compound produced enzymically assayable ATP. Enzymic digestion with snake venom

endonuclease supported the 3' phosphodiester link. Fast atom bombardment mass

spectrometry of the compound produced a number of species in the high-field positive-

ion spectrum. The largest corresponding to a pentamer. Based on these findings,

Hutchinson et al (1986a) proposed the structure for this derivative of the form p3

glyceroyl-l[-ppp5'-adenosyl-3'p3-glyceroyl-l]n-ppp5'adenosine (see Figure 1.3) and the

name of oligo-phosphoglyceroyl-adenosine triphosphate (OPG-ATP) has been

suggested.

Heyworth et al (1987) have subsequently shown that about 10% of incorporated

insoluble radioactivity is in a high molecular weight compound with a phosphate:purine

ratio of 0.8, which comparison of [^'^C]-uridylate and [^^C]-adenylate incorporate

suggests is a rapidly synthesised species of RNA that represents about 4% of total heart

RNA. Using techniques similar to those employed with hearts, the presence of OPG-

ATP has also been demonstrated in rat kidney (Hutchinson et al, 1986b). The discovery

and structural characteristics of OPG-ATP has been reviewed (Lawson and Mowbray,

1986).

1.F.1 Objectives o f this Study

The existence of nearly Ip-mol/g. wet wt. of OPG-ATP in perfused rat heart suggests that

this nucleotide has a major role to play in ATP homeostasis and that its inter-conversion

with free nucleotide is an important component of the mechanism by which ATP

concentrations are maintained during stress to provide both an energy currency and the

unidirectional drive for metabolism. In trying to elucidate the role of OPG-ATP in

OPG-ATP to free nucleotide.

b. Identify the enzymic product of OPG-ATP degradation.

c. Attempt to devise a rapid and sensitive assay for OPG-ATP in tissue extracts by

coupling the degradative enzyme(s) to enzymic determination of the released

nucleotide.

2. MATERIALS AND METHODS

2.A M aterials