CALIFICACIÓN PARA EL CRITERIO DE ACCESIBILIDAD

estimated from the absorbance at 259nm (molar absorption coefficient of 15.4 x 10^

M“^.cm~^ for ATP at pH7) were between 9-15|imol/g. dry acid insoluble material

obtained from livers of 3-4g. wet weight.

This amount of material was subjected to anion-exchange chromatography by the same

method as that used for heart (see Section 2.C.5). Figure 3.15.A shows that the liver

extract too produced a major peak eluting at around 0.3M NaCl which contained

virtually all of the radioactivity applied to the column (compare with Figure 3.3). About

30% of the material absorbing at 259 was not retained. The material from the major peak

was recovered after precipitation with 50% cold aqueous ethanol for approximately 10

days. The Pi:Purine molar ratio of the purified material from two livers gave values of

3.3:1 and 3.8:1, strongly supporting the suggestion that it is OPG-ATP. Although it is not

Figure 3.15

DEAE-cellulose Chromatography of the Nucleotide-rich fraction of the Acid/Alcohol Insoluble Material B 2.0 o\ ID (N 1.5; l.Qi 0.5 0.5 0.1 100 150 200 0.1 0 200 50 100 150 300 200 100

1

I

(A) From perfused liver : The com bined aqueous layers from the buffered p h en ol/ch loroform /isoam ylalcoh ol extraction of 0.8g of w ash ed and dried (see section 2.C.2) acid /a lco h o l insoluble material were dissolved in 0.5ml of Im M EDTA/TM U rea/lO m M sodium acetate buffer (pH4.5) and w ash ed on to a 20x1.4cm colum n (30ml) o f DEAE-cellulose w ith 30ml of buffer at a flo w rate of 2 7 m l/h . A bout 30% of the material absorbing at 259nm w as not retained. The colum n w as developed w ith a linear gradient of 0.1-0.5M NaC l in the sam e buffer and SOOpl fractions were collected and assayed for radioactivity (shaded area).

(B) From Isolated Mitochondria: 2ml sam ples from m itochondrial preparations (approxim atley 20mg p rotein /m l) were m ixed w ith an equal volum e o f cold T C A /m ethanol/M gC l^ and the w ashed, dried insoluble material (see section 2.E.) stored at -70°C. Sam ples from three different preparations w ere com bined, extracted w ith buffered phenol (see section 2.C.3.) and the nucleotide-rich aqueous fractions applied to the colum n just as in A.

Figure 3.16

Mr estimation of purified liver OPG-ATP

lOOOOi

I

I

I

OPG-ATP sam ples (purified according to section 2.C.5) from tw o perfused rat livers were applied to a sterile sephadex G-15 colum n (23.5x1.3cm) calibrated w ith authentic AMP, ATP and Ap^A in 7M -urea/ lOOmM-sodium acetate/lO m M MgClj buffer, pH4.5, and developed in the sam e buffer. ApsA represents

P , P -bis (adenosyl) pentaphosphate. The void volum e of the colum n w as determ ined w ith Blue Dextran. Kav w as calculated as for figure 3.7.

OPG-ATP in liver may be substantially greater than in these organs.

The specific radioactivity of ^'^C-OPG-ATP calculated from these fractions ranged from

900-l,800d.p.m./^imol adenylate equivalent. This is around 10 times lower than the

estimate for the specific radioactivities of the soluble adenine nucleotides (Section 3.F.1)

and contrasts sharply with the observations with both perfused hearts (Mowbray et al,

1984b and this study) and kidney (Hutchinson et al, 1986b) that OPG-ATP reaches

specific activity equilibration with soluble nucleotides in 10 min. or so.

That the proportion of total radioactivity rapidly incorporated into the acid-insoluble

fraction was certainly no lower in liver (Table 3.9) suggests that the rate of exchange

between soluble nucleotides and OPG-ATP was as rapid in liver as in heart. The

evidence that there is substantially larger amount of OPG-ATP in liver implies that it is

this larger pool size which does not allow specific radioactivity equilibrium in the same

short perfusion regime as in heart and kidney.

The apparent Mr of material purified from two perfused livers was estimated to be 1340

and 1470 on a calibrated sephadex G-15 column (Figure 3.16). Fast atom bombardment

mass spectrometry of heart OPG-ATP detected about 80% of the material with a mass

number of 1329 and the Mr of a unit of OPG-ATP was deduced to be 656 (Hutchinson et

al, 1986a). The liver samples additionally contained lOmM MgCl2. Thus, if liver OPG-

ATP is also complexed to Mg^^ as suggested in Section 3.E, then these two samples

ppp5'A3'p3Gri --- > ppp5'A3'p3Gri lppp5'A3'p3Gri I ^ unit Mr 656 Mg-Dimer Mr 1350 Grilppp5'A3'p3Grilppp5'A3'p3Gri I ^ I_ _ _ _ _ ^

Mg^^

Mg^'^

where Dimer = 655 x 2 + 1 oxygen: Mg^"*" adds 22 (with loss of 2H), A represents

adenosine and Gri represents glyceric acid.

3.F.3 OPG-ATP in Liver M itochondria

Rat liver mitochondria were prepared and extracted with TCA/methanol/MgCl2 as in

method Section 2.E. The acid-alcohol precipitates from three different preparations were

combined and phenol extracted exactly as had been the insoluble fraction from freeze-

clamped perfused livers. Only mitochondrial preparations that gave a respiratory

control ratio (RCR) of greater than 3.5 in the presence of succinate and ADP (i.e., coupled

mitochondria when measured using an oxygen electrode) were used for extraction. The

quantities of purine nucleotide present in the aqueous extracts were between 3-5|imol/g.

dry acid-insoluble material (estimated as in Section 3.F.2). These phenol extracts (from

approximately llOmg mitochondrial protein) were chromatographed on the same

DEAE-column as the perfused liver extracts. As shown in Figure 3.15.B, a substantial

peak elutes in the position expected for OPG-ATP. This material was harvested by

precipitation in the cold with 50% ethanol (recovery was 65%) and shown by assay with

a specific OPG-ATP 3' phosphodiesterase (see Chapter 4) to be OPG-ATP. The U.V.

Table 3.10

OPG-ATP from perfused rat liver and isolated mitochondria.

Sample OPG-ATP ^mol/g. dry wt.

Perfused liver 11.7 ± 1.1

(n = 4)_________________________________

Mitochondria 4.1 ± 0.3

(n = 4)

Rat livers weighing 3-4g. wet tissue were perfused and extracted as described in Section 2.D. Isolation of liver mitochondria and extraction of OPG-ATP was according to method 2E. Values are expressed as means ± S.E.M. For mitochondria, n = 4 indicates the number of mitochondrial preparations, each from 5-10 rat livers weighing 7-8g. wet tissue.

The yield of mitochondria used for extractions was equivalent to 30-40% of total

mitochondria in a 7-8g. liver. The protein content of liver is 129mg/g. wet wt. and

mitochondria have 30% total liver protein (see Reid, E., 1961). That the amount of OPG-

ATP purified from mitochondria is about 33% of that purified from whole liver (see

Table 3.10) implies, therefore, that all of the liver OPG-ATP may be present in the

mitochondrial fraction assuming the extraction procedures gave comparable yields in

both cases. This was tested more directly in a labelling experiment in which instead of

freeze-clamping the liver after perfusion with 0.25pM [8-^^C] adenosine it was used to

prepare subcellular fractions. The cell debris/nuclei, the mitochondrial and the post-

mitochondrial fractions were each then examined for TCA/methanol precipitable

radioactivity. Succinate dehydrogenase was assayed as a mitochondrial marker (see