min only at which point significant breakdown could be detected Where different

substrate concentrations were used, 15 min. time courses at the lowest and highest

concentrations were used to check that the initial rate was proportional to [S]. Figure 4.3

shows a Lineweaver-Burk plot of the rate of solubilisation of ^^C-OPG-ATP in aqueous

50% cold ethanol over a range of substrate concentrations. The apparent for

adenosine equivalents of OPG-ATP can be estimated at about 35pM and the V^ax at

6nm ol/m in./m g of protein. Comparable values were obtained with dialysed 60% satd.

(NH4)2S0 4 precipitates from soluble mitochondrial extract. The is consistent with

estimates of purine equivalents of OPG-ATP (>lmM) in heart (Section 3.B and Mowbray

et al, 1984b) and in kidney (Hutchinson et al, 1986b), and at least 5mM in liver (Section

3.F and Patel et al, 1991a). However, the V^ax (for OPG-ATP) of 6nmol/ min. / mg protein

would be equivalent to 30nm ol/m in./g. wet tissue for heart (20mg total mitochondrial

protein/g. wet wt., 0.25mg enzyme protein/mg mitochondria; 5mg enzyme protein/g.

wet wt.) which is lower than the value necessary for turnover of OPG-ATP (0.6p,mol/g.

wet heart) to attain specific radioactivity equilibrium with soluble ATP within 10 min.

found in heart and kidney (Mowbray et al, 1984b and Hutchinson et al, 1986a) and the

less complete equilibration, but comparable rate of exchange seen in perfused livers

(Patel et al, 1991a). This, along with the relatively poor recovery of total activity from

f.p.l.c., suggests that some activating or regulatory factor has been lost during

purification. Another contributory factor to low V^ax could be the presence of

Figure 4.2

Time-course of OPG-ATP Solubilisation

100-1 80-

I

§

» .

Approxim ately 2Ünmol (adenosine equivalents) of ^XZ-OPG-ATP in lOmM-MgCl^ /Im M -ad en osin e /25m M -H ep es buffer, pH7.4 were incubated at 37°C for upto 15 m in w ith 0.4mg protein, open circles (O) - protein from 60% (NH^)^ SO^ mitochondrial fraction and closed circles (# ) - protein from M ono Q f.p.l.c. fractions. The reactions w ere terminated by the addition of an equal volum e of cold aqueous ethanol and the sam ples left at -20°C for 2hr. before they were filtered and assayed for radioactivity (see section 2.K.I.).

Figure 4.3

Double-Reciprocal plot of the initial velocities measured at 2min

bJD

I

I

Between 7 and 40nm ol (adenosine equivalents) of [^^C]- OPG-ATP in lOmM- M g C iy Im M -adenosine/25m M -H epes buffer, pH7.4 were incubated at 37°C for 2min w ith 0.4mg of en zym e protein from M ono Q fractions. The reaction w as stopped by the addition of 50% cold aqueous ethanol and the sam ples left at -20°C for 2 hrs, before they were millipore filtered and assayed for radioactivity, (see section 2.K.I.).

high salt fractions (see Figure 4.1) confirmed this. The OPG-ATP activity and protein

were both absent in these salt fractions, but they absorbed strongly at 259nm indicating

the presence of purine containing material. The U.V. absorption spectrum (220-300nm)

of this peak matched that of the monomer PC-ATP (see Figure 3.5). However, not all the

bound substrate could be separated from the enzyme by chromatography on a Mono Q

column because a substantial blank value was observed when a sensitive luminometric

enzyme assay for OPG-ATP linked to the above activity was attempted (see Section 4.D).

This obstacle was tackled by making use of the property that OPG-ATP is stabilised by

Mg^"^ ions but can be readily broken down under alkaline conditions in the presence of

EDTA (see Chapter 3). The 60% (NH4)2S0 4 protein was dissolved in 25mM

Hepes/5m M EDTA buffer, pH7.4 and the made alkaline, pH8.5, by the addition of 4M

KOH. The mixture was left at room temperature for up to 30 min. before re-adjusting

the pH to 7.4 with IM HCl. The protein solution was dialysed, at 4°C, initially against a

small volume (2ml) of 25mM Hepes buffer, pH7.4, and then against two changes of

500ml of the same buffer over a 6 hr. period. Analysis of the initial dialysis buffer

(dialysate) showed the presence of substantial quantities of material absorbing light at

259nm which is presumed to be broken down purine compound crossing the dialysis

membrane. Ths U.V. absorption spectrum of this material was similar to those of the

high salt Mono Q peak (see above) and the monomer PG-ATP (see Figure 3.5). Anion-

exchange h.p.l.c. of the dialysate also resulted in a peak eluting before ADP with

retention time as for the monomer PG-ATP (see Figure 3.6). These results suggest that

the oligomer is bound to the mitochondrial cleavage enzyme which releases monomeric

units of PG-ATP and appears to be a regulated enzyme like glycogen phosphorylase

Table 4.6

Effict of alkali treatment on mitochondrial OPG-ATP cleavage activity.

Fraction Total Protein mg Specific Activity Activity nmol/min. (mU) mU/mg

Total Activity mU Sonicated mitochondria 157 ±13 1.7 ±0.07 7.2 ±0.7 1139 ±161 Soluble extract 68 ±3 1.4 ± 0.2 3.3 ± 0.4 218 ± 17 60% (NH4)2S0 4 44± 2 1.4 ± 0.2 3.3 ±0,4 145 ± 20 Alkali treated (NH4)2S0 4 44 ±2 3.0 ± 0.04 13.0 ±0.9 559 ±11

Mitochondrial preparation was from 5 rat livers. Fractionation and alkali treatment was as detailed in Section 2.J.I. Each fraction was assayed for OPG-ATP cleavage activity by incubation of ~30nmol ^^C-OPG-ATP in 25mM Hepes/lOmM

MgCl2/lm M adenosine buffer, pH7.4, with 200|ig protein at 37°C for 2 min. (the

linear range of substrate solubilisation). The reactions were terminated by the addition of an equal volume of cold ethanol and the samples left at -20°C for 2 hr. before filtration and assay of radioactivity (see Section 2.K.1). Values are presented as mean ± S.E.M. from 3 mitochondrial preparations.

rate of added substrate solubilisation and a 3.5 fold increase in the specific activity and

total activity compared to the activity of the untreated 60% (NH4)2S0 4 protein (see Table

4.6). Since the cleavage activity was measured in terms of the amount of radioactive

substrate solubilised, presence of cold endogenous substrate would have resulted in the

lower activity values obtained with the earlier untreated enzyme preparations (see

Tables 4.4 and 4.5).

Purification of a protein solution treated as above (to alkali) on an f.p.l.c. Mono Q H 5/5

column resulted in a 45 fold increase in the specific activity of this cleavage enzyme

(Table 4.7). The yield, however, was much lower than before (see Table 4.5). Most of the

activity eluted between 0.07-0.1M-NaCl (see Figure 4.4). 10% of the total activity loaded

was eluted before the start of the salt gradient. The active gradient fractions from 5 such

column runs were pooled and concentrated to approximately 2m g/m l by ultrafiltration.

In order to make an approximate estimate of the molecular size of the active enzyme, a

sample (100|il) from Mono Q (see above) was subjected to gel filtration chromatography

in 50mM Tris-Cl buffer, pH7.4, containing O.llM NaCl, on a Superose 12 HR 10/30

(Pharmacia) f.p.l.c. column calibrated with a range of molecular weight standards (see