Abstract: The rate of enzymatic ATP synthesis is shown to depend on the zinc isotopes. The ATP producing activities of creatine kinase and pyruvate kinase in which Zn²⁺ ions have magnetic nuclei ⁶⁷Zn are found to be 2-6 times higher than that of enzymes in which Zn²⁺ ions have nonmagnetic nuclei ⁶⁴Zn. The isolated rat heart muscle mitochondria exhibit a similar effect. An expression of this magnetic isotope effect in enzymatic ATP synthesis processed in the presence of Zn²⁺ ions is in a favor to the ion-radical mechanism of ATP production occurred within a high zinc concentration range. Both fundamental aspect and a possible pharmacological significance of the phenomenon described are under discussion.

INTRODUCTION

The rate of enzymatic ATP synthesis, a Mg involving process was recently shown to strongly depend on the magnesium isotopes. Activity of ATP synthase and ATP producing kinases in which Mg²⁺ ion has magnetic nucleus ²⁵Mg was found to be 2-3 times higher than that of enzymes in which Mg²⁺ ion has nonmagnetic nuclei ²⁴Mg or ²⁶Mg (Buchachenko et al., 2005a-c; Buchachenko, 2009).

The effect was shown to be a function of the concentration of Mg²⁺ ions at low concentration there is no isotope effect, i.e., classical generally accepted nucleophylic mechanism of the ATP synthesis dominates. If concentration of Mg²⁺ ions exceeds intracellular one by a few tens a huge isotope effect appears which gives evidence that the new, spin-dependent ion-radical mechanism of ATP synthesis is switched on (Buchachenko et al., 2008). Providing additional and considerable enzymatic source of ATP.

Similar effect was also observed for the calcium ions: the activity of creatine kinase with catalytic sites, loaded with ⁴³Ca²⁺ ions having magnetic nuclei ⁴³Ca was found to be 2.0±0.3 times higher than that of enzyme in which Ca²⁺ ions have even, nonmagnetic nuclei ⁴⁰Ca, ⁴²Ca or ⁴⁴Ca. (Kuznetsov et al., 2010).

Since ATP syntheses, catalyzed by magnesium and calcium are very similar in concentration dependences and isotope effects one can suppose that ion-radical mechanism is a universal phenomenon and may be detected for other metals as catalysts (Zn for instance).

MATERIALS AND METHODS

To verify this idea which would be useful to stimulate ATP production and prevent biomedical pathologies related to deficiency of ATP in the living organisms, we prepared two series of samples of Creatine Kinase (CK), Pyruvate Kinase (PK) and mitochondria. In one series the enzymes were loaded with Zn²⁺ ions of natural isotope composition in the other one the enzymes were loaded with Zn²⁺ ions strongly (by 78.4%) enriched with magnetic isotope ⁶⁷Zn (nuclear spin 5/2, magnetic moment+0.8 mB). Then both series were tested for their enzymatic activities in the identical conditions.

CK, E.C.2.7.3.2 was isolated from the Vipera xanthia lyophilized venom and purified according to (Kuznetsov et al., 2004). Rabbit reticulocyte PK, E.C.2.6.9.17 was purchased in the ammonium sulfate precipitated form from Worthington, Inc., Durham. The substrates, [³²P] phosphocreatine, 26.6-29.2 Ci mmoL^-1 and [³²P] phosphoenolpyruvate, 33.6-37.4 Ci mmoL^-1 were manufactured by the Amersham Radiochemical Centre, UK. The enzyme activity A was conventionally evaluated as the amounts of radioactive ³²P decays per minute found in the HPLC-separated nascent [³²P] ATP pool produced by 1.0 mg of pure enzyme during the 40 min incubation time in the Mg²⁺/Ca²⁺ free samples. This time was shown to be enough for the ATP yield to reach a limiting value. For controls, the metal-free samples incubation at +37°C as well as ice-cold incubation tests were carried out (Buchachenko et al., 2005a; Kuznetsov et al., 1986) the yield of ATP in these experiments was shown to be small in comparison with that in the presence of Zn²⁺ ions.

The rat heart muscle mitochondria were isolated according to Randall with their following 60 min long incubation as described by Buchachenko et al. (2005b) to conduct then the oxygen consumption measurements (Rezayat et al., 2009) and the total ATP yield estimations (Buchachenko et al., 2005c). The optimum-balanced mitochondria incubation mixtures (Buchachenko et al., 2005a, b; Rezayat et al., 2009) were employed as they are as well as in their metal-lacking and Zn-supplemented modified forms, same like in experiments with pure enzyme specified above. For protein and DNA quantitative estimations, conventional colorimetric procedures were applied (Bradford, 1976).

Two samples of ZnCl₂ were prepared using a routine acidic treatment of the two sorts of zinc oxides: ZnO with natural isotope composition (⁶⁴Zn, 48.6%; ⁶⁶Zn, 27.9%; ⁶⁷Zn, 4.1%; ⁶⁸Zn, 18.8%; ⁷⁰Zn, 0.6%) and ⁶⁷ZnO enriched with magnetic nuclei (78.4% of ⁶⁷Zn versus 4.1% of that in natural ZnO), respectively. Enzymatic activity of the free metal CK, PK and mitochondria (the contents of Ca²⁺ and Mg²⁺ in the latter were negligibly small, 30-35 and 16-18 μg mg^-1 of DNA, respectively) was measured as a function of the ZnCl₂ concentration.

RESULTS AND DISCUSSION

The yield of ATP produced by CK as a function of ZnCl₂ concentration is shown in Fig. 1. It exhibits the following remarkable features:

Fig. 1:	The yield of ATP produced by CK as a function of 64ZnCl2 (1) and 67ZnCl2 (2) concentration

Both samples of CK contain zinc isotopes in different shares. In the samples with natural ZnCl₂ the share of magnetic nuclei ⁶⁷Zn is 4.1%; the total share of nonmagnetic isotopes is 95.9% (further we will conventionally denote the set of nonmagnetic isotopes as ⁶⁴Zn). In the samples loaded with enriched ⁶⁷ZnCl₂ the shares of ⁶⁷Zn and ⁶⁴Zn are 78.4 and 21.6%, respectively. Now the activity A of the both samples of CK may be presented as a sum of additive contributions coming from catalytic sites carrying ⁶⁷Zn and ⁶⁴Zn:

(1)

(2)

Here A (⁶⁷Zn) and A (⁶⁴Zn) characterize the true values of enzymatic activity of catalytic sites with ⁶⁷Zn²⁺ and ⁶⁴Zn²⁺ ions, respectively. Substituting into the (Eq. 1, 2) A₁ = 7000 (Fig. 1, curve 1) and A₂ = 30200 (Fig. 1, curve 2) for the ZnCl₂ concentration 5 mM, it is easy to derive A (⁶⁷Zn) 37000 and A (⁶⁴Zn) = 5753. Their ratio A (⁶⁷Zn)/A (⁶⁴Zn) = 6.4±0.3 is the magnitude of isotope effect in enzymatic ATP synthesis by CK. It demonstrates that the CK catalytic sites with ⁶⁷Zn²⁺ ions produce ATP by 6 times more efficiently than those with ⁶⁴Zn²⁺ ions.

In order to compare both nuclear spin dependences of the ATP synthesis directed by CK from Vipera xanthia venom and that of the mitochondrial CK promoted one, some additional experiments were carried out using the above specified identical standard conditions.

Fig. 2:	The yield of ATP produced by PK as a function of 64ZnCl2 (1) and 67ZnCl2 (2) concentration

As a result, no marked difference found whatsoever. Both CK species tested promotes the very same isotope-related specific activity response whatever metal concentration range studied (0.5-50.0 mM). There is no magnetic isotope effect revealed in the isolated mitochondria oxygen consumption at all it means that isotope effect arises in substrate ATP synthesis itself and has no relation to the NAD-engaging oxidative mitochondrial processes.

ATP synthesis by PK exhibits generally similar but not identical behavior (Fig. 2). The differences are quite evident (Fig. 1 and 2) they assumed to refer to those in molecular mechanics of these two enzymes, CK and PK. At the concentration of ZnCl₂ 5 mM there is no isotope effect for PK (unlike of CK for which it is 6.4±0.3) however at 50 mM of ZnCl₂ it reaches 2.2±0.3 (calculated according to Eq. 1 and 2).

Observation of magnetic isotope effect in ATP synthesis catalyzed by Zn²⁺ ions evidently demonstrates that the ion-radical mechanism of enzymatic ATP synthesis is a universal phenomenon. It includes electron transfer from Zn (ADP)^3- complex to the hydrated Zn (H₂O)²⁺_n complex as a primary reaction of ATP synthesis. The reaction generates ion-radical pair composed of Zn (H₂O)_n⁺ ion and ADP anion-radical coordinated to Zn²⁺ ion. The addition of the anion-radical to the substrate P = O bond results in ATP formation. Populations of the singlet and triplet states and singlet-triplet spin conversion in the pair are controlled by hyperfine coupling of unpaired electrons with magnetic ⁶⁷Zn and ³¹P nuclei. Due to this interaction the yield of ATP is a function of nuclear magnetic moment as discussed in detail for magnesium induced ATP synthesis (Buchachenko et al., 2010). For the same reason ATP yield depends on the magnetic field (Buchachenko and Kuznetsov, 2008).

Two results need to be commented. First, the three enzymes, CK from the Vipera xanthia venom, mitochondrial CK and PK exhibit specific isotope effects. Both CK demonstrate ion-radical mechanism of ATP synthesis in the same range of ZnCl₂ concentration, however, isotope effects for these two enzymes are slightly different. These differences may be attributed to the differences in molecular structure and dynamics of protein domains in catalytic sites.

Second, large isotope effects are not unexpected and suspicious because they are induced by spin dynamics rather than chemical reactions themselves. Their magnitudes are controlled by the rates of singlet-triplet spin conversion in the ion-radical pairs and depend on the electron-nuclear (hyperfine) coupling of unpaired electrons with magnetic nuclei. In principle, there is no limit on the magnitude of magnetic isotope effect, (Buchachenko, 2009) in contrast to classical, mass-dependent one which is known to be limited on the ratio of nuclear masses.

CONCLUSION

Apart from its obvious fundamental significance, the phenomenon described possesses some clear pharmacological potential. Thus, the low toxic cation exchanging nanoparticles formed on a basis of the fullerene-C60 porphyrinic adducts were found to be the reliable carriers for magnetic bivalent metal isotopes suitable for a targeted delivery of the latter’s ions in vivo followed then by a marked local hyper-activation of the pre-suppressed ATP synthesis (Rezayat et al., 2009; Amirshahi et al., 2008a).

This alone made a remarkable contribution to either prevention or treatment of several hypoxia related syndromes including the ones associated with some myocardial and lymphoid tissue energy metabolism disorders (Buchachenko, 2009; Kuznetsov et al., 2010; Amirshahi et al., 2008b). As per the phenomena described in a present study, they are no doubt worthy the further nanopharmacological testing in a way offered and pre-programmed recently (Buchachenko, 2009; Kuznetsov et al., 2010; Rezayat et al., 2009; Amirshahi et al., 2008a, b).

ACKNOWLEDGEMENTS

This research was financially supported by Russian Ministry of Science and Education (Grant NS-2010.3) and Russian Foundation for Basic Research (Grant 08-03-00141). The European INTAS-06/09 Magnetic Field Biological Effects Research Grant is also acknowledged.