A. Kemp

Specific photolabelling of beef-heart mitochondrial ATPase by 8-azido-ATP.

1. 8-Azido-ATP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1) 2. 8-Azido-ATP is hydrolysed slowly by F1 in the dark. Photolysis at 350 nm in the presence of F1 leads to inhibition of the ATPase activity. The presence of ATP during illumination prevents the inhibition. Illumination of F1 in the absence of 8-azido-ATP causes no inhibition. 3. Added Mg2+ is not necessary for the binding of the 8-azido-ATP to F1. 4. 8-Azido-ATP binds specifically to the beta subunits of F1. 5. The ATPase activity is completely inhibited when 2 mol of 8-azido-ATP are bound per mol F1.

Oxidative phosphorylation as a function of temperature

Abstract 1. The activation energy of succinate oxidation by rat-liver mitochondria changes at a temperature of about 17° in State 3 as well as in the uncoupled state. 2. Over the whole temperature range investigated (0–23°) the rate of phosphorylation of intramitochondrial ADP during succinate oxidation exceeds that of added ADP. 3. The activation energy of the ADP-ATP and P i -ATP exchange reactions and of the 2,4-dinitrophenol-induced ATPase also changes at about 17°. 4. The temperature coefficients of the State-3 oxidation and of the P i -ATP and ADP-ATP exchange reactions are similar and, at temperatures below 17°, are high in comparison with that of the phosphorylation of intramitochondrial ADP. 5. The translocation of ADP and ATP through the inner membrane is rate-limiting for the process of oxidative phosphorylation in rat-liver mitochondria.

Nucleotide-binding properties of native and cold-treated mitochondrial ATPase

1. The bound nucleotides of the beef-heart mitochondrial ATPase (F1) are lost during cold inactivation followed by (NH4)2SO4 precipitation. The release of tightly bound ATP parallels the loss of ATPase activity during this process. 2. During cold inactivation, the sedimentation coefficient (s20, w) of the ATPase first declines from 12.1 S to 9 S, then to 3.5 S. (NH4)2SO4 precipitation of the 9-S component also leads to dissociation into subunits with s20, w of 3.5 S. 3. The 9-S component still contains the bound nucleotides, which are removed when it dissociated into smaller subunits. 4. Reactivation of cold-inactivated ATPase by incubation at 30 degrees C is increased by the presence of 25% glycerol. ATP, however, does not have any clearcut effect on the degree of reactivation in the presence of glycerol. 5. ADP is an inhibitor of the reactivation, probably because it exchanges during reactivation for bound ATP giving rise to an inactive 12-S component. 6. The exchange of tightly bound nucleotides with added adenine nucleotides is more extensive and faster with cold-inactivated ATPase than with the native enzyme. During reactivation up to 1.6 moles of ATP and 1.0 mole ADP can exchange per mole enzyme. 7. Incubation with GTP, CTP or inorganic pyrophosphate induces an increased activity of the ATPase, which, however, soon declines in the presence of ATP. It also disappears on precipitation of GTP-treated enzyme with (NH4)2SO4.

Prolyl 4-hydroxylase activity in relation to the oxidation state of enzyme-bound iron. The role of ascorbate in peptidyl proline hydroxylation.

In agreement with others (Myllylä, R., Kuutti-Savolainen, E.-R. and Kivirikko, K.I. (1978) Biochem. Biophys. Res. Commun. 83, 441-448), it was found that, in the absence of ascorbate, prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) catalyses the hydroxylation of peptidyl proline, stoicheiometrically coupled to the oxidative decarboxylation of 2-oxoglutarate, at a high initial rate. Under these conditions the enzyme becomes inactivated by at least 90% within 1 min in the presence of 400 microM 2-oxoglutarate, in the presence or absence of the peptide substrate (Pro-Pro-Gly)10. The enzyme can be partly reactivated by ascorbate, but not by Fe2+. Addition of a stoicheiometric amount of iron to the enzyme gives rise to a small EPR signal at g = 4.3, which is typical of a high-spin d 5 ion in a rhombic environment. After subsequent incubation for 30 s at 37 degrees C in the presence of 2-oxoglutarate, the amplitude of the EPR signal at g = 4.3 increases 3-4-fold and corresponds to virtually all of the iron added. In addition, an EPR signal at g = 2.0 is formed under these conditions. The signal at g = 4.3 decreases after subsequent addition of ascorbate. It is concluded that in the presence of 2-oxoglutarate enzyme-bound Fe2+ is rapidly converted to Fe3+, leading to inactivation of the enzyme. Enzyme-bound Fe3+ can be reduced again by ascorbate, thus reactivating the enzyme, or, in the absence of 2-oxoglutarate, by Fe2+.

Comparison of ADP and ATP as substrates for the adenine nucleotide translocator in rat-liver mitochondria

Abstract 1. 1. The exchange of ATP with endogenous adenine nucleotides is biphasic in contrast to that of ADP and is dependent on the endogenous ATP/ADP ratio. 2. 2. It is shown that the rapid initial phase of the ATP exchange is an electro-neutral exchange of external ATP with internal ATP. 3. 3. Uncoupler stimulates both the ATP and the ADP exchange. 4. 4. Competition experiments show that the adenine nucleotide translocator has a high specificity for exogenous ADP compared to exogenous ATP. This affinity difference is more pronounced than that reported by Pfaff and Klingenberg ( Eur. J. Biochem. (1968) 6, 66–79). 5. 5. The competition between ATP and ADP for the translocator is dependent on the energy state of the mitochondria, the K i for ATP increasing from 1.5 μM in the presence of uncoupler to 200 μM, under high-energy conditions, whereas the K m for ADP is unaffected. 6. 6. The K m for ATP is also dependent on the energy state of the mitochondria, and is under both high- and low-energy conditions about the same as the K i for ATP in the ADP exchange. 7. 7. Since the energy state has no effect on the maximum velocity these effects on K m reflect a change of K D . The different K D values for ATP and ADP in the controlled state explain the asymmetric behaviour of the adenine nucleotide translocator. 8. 8. The influence of the energy state of the mitochondria on the K i and K m for ATP suggests the existence of different conformational states of the adenine nucleotide translocator.

Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction

In the absence of a peptidylproline substrate, the oxidative decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase (prolyl-glycyl-peptide,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) is stoicheiometrically coupled to the oxidation of ascorbate. The Km and Kd for O2 in this partial reaction are 1.5 mM, this value being one order of magnitude higher than the Km and Kd for O2 in the complete reaction in the presence of (Pro-Pro-Gly)5, indicating that in this case O2 can become enzyme-bound predominantly after the interaction of the peptide substrate with the enzyme. The Km values for 2-oxoglutarate in the partial and the complete reactions are the same. In the absence of both a peptide substrate and ascorbate 2 mol CO2 per mol enzyme are produced in the first 1-1.5 min, during which the enzyme becomes inactivated and, as shown earlier (De Jong , L., Albracht , S.P.J. and Kemp, A. (1982) Biochim. Biophys. Acta 704, 326-332) enzyme-bound Fe2+ becomes oxidized to Fe3+. The results are consistent with a mechanism in which a Fe2+O complex is the O-transferring intermediate involved in peptidylproline hydroxylation.

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.

The ATP-and ADP-binding sites in mitochondrial coupling factor F1 and their possible role in oxidative phosphorylation.

EIsevier/North-Holland Biomedical Press causes inactivation of the ATPase activity, the inactivation being proportional to the amount of %nitrenoATP (the photolysis product of 8azido-ATP) that is covalently bound to the Fr, complete inactivation being obtained when 2 molecules 8.nitreno-ATP are bound per molecule of Fr [9]. ATP and ADP (but not AMP) protect against the inactivation. The nitreno-ATP is bound to the P-subunit [9]. The ATPase is also completely inactivated when 2 molecules of 8-nitreno-ADP are covalently bound by irradiation of Fr with 8azido-ADP, either in the presence or absence of Mg 2+. ATP and ADP (but not AMP) protect against the inactivation. In the presence of Mg2+, the El-nitreno-ADP is covalently bound mainly to the o-subunit ; in the absence of Mg”, it is bound about equally to both the (Yand P-subunits. The amount of firmly bound ATP and ADP is not appreciably altered by irradiation in the presence of azidoATP [8]. When Fr is irradiated in the presence of azido-ATP in the absence of Mg2+, followed by irradiation in the presence of both azido-ADP and Mg2*, a total of 3.1 mol photoaffmity label was covalently bound to the Fr without appreciably affecting the amount of firmly (but not covalently) bound ATP and ADP (R.J.W., unpublished observations). After the first irradiation, the label was practically entirely in the P-subunit; the extra label after the second irradiation was predominantly in the o-subunit. Thus, the binding site for ATP in the absence of Mg2+ and that for ADP in its presence appear to be independent and to be located on the fland a-subunits, respectively.

The function of ascorbate with respect to prolyl 4-hydroxylase activity

1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.

Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP

1. In addition to the previously studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). 2. Photolysis at 350 nm of 8-azido-ADP in the presence of isolated F1 leads to inactivation of ATPase activity. Both ATP and ADP (but not AMP) protect against the inactivation. 3. In the absence of Mg2+, 8-azido-ADP binds almost equally to the alpha and beta subunits of F1, whereas in the presence of Mg2+ the alpha subunits are predominantly labelled. 4. The ATPase activity is completely inhibited when two molecules of 8-azido-ADP are bound per molecule F1. 5. 8-Azido-ATP and ATP are competitive substrates for F1, indicating that in the presence of Mg2+ 8-azido-ATP binds to the same site as ATP. 6. The amount of tightly bound nucleotides in F1 is not significantly changed upon incubation with 8-azido-ATP either in the light or the dark. 7. 8-Azido-ATP is also a suitadrial particles, photolabelling leading to inactivation of ATPase activity. 9. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabelling Mg-ATP particles with 8-azido-ATP. 10. In contrast to the uncoupled ATPase activity, where the two ATP-binding sites do not interact, cooperation between the two sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.