M. A. Orlova

Magnesium isotope effects in enzymatic phosphorylation.

Recent discovery of magnesium isotope effect in the rate of enzymatic synthesis of adenosine triphosphate (ATP) offers a new insight into the mechanochemistry of enzymes as the molecular machines. The activity of phosphorylating enzymes (ATP-synthase, phosphocreatine, and phosphoglycerate kinases) in which Mg(2+) ion has a magnetic isotopic nucleus 25Mg was found to be 2-3 times higher than that of enzymes in which Mg(2+) ion has spinless, nonmagnetic isotopic nuclei 24Mg or 26Mg. This isotope effect demonstrates unambiguously that the ATP synthesis is a spin-dependent ion-radical process. The reaction schemes, suggested to explain the effect, imply a reversible electron transfer from the terminal phosphate anion of ADP to Mg(2+) ion as a first step, generating ion-radical pair with singlet and triplet spin states. The yields of ATP along the singlet and triplet channels are controlled by hyperfine coupling of unpaired electron in 25Mg+ ion with magnetic nucleus 25Mg. There is no difference in the ATP yield for enzymes with 24Mg and 26Mg; it gives evidence that in this reaction magnetic isotope effect (MIE) operates rather than classical, mass-dependent one. Similar effects have been also found for the pyruvate kinase. Magnetic field dependence of enzymatic phosphorylation is in agreement with suggested ion-radical mechanism.

Spin biochemistry: magnetic 24Mg-25Mg-26Mg isotope effect in mitochondrial ADP phosphorylation.

The rates of adenosine triphosphate (ATP) production by isolated mitochondria and mitochondrial creatime kinase incubated in isotopically pure media containing, separately, 24Mg2+, 25Mg2+, and 26Mg2+ ions were shown to be strongly dependent on the magnesium nuclear spin and magnetic moment. The rate of adenosine 5′-diphosphate phosphorylation in mitochondria with magnetic nuclei25Mg is about twice higher than that with the spinless, nonmagnetic nuclei24.26Mg. When mitochondrial oxidative phosphorylation was selectively blocked by treatment with 1-methylnicotine amide, 25Mg2+ ions were shown to be nearly four times more active in mitochondrial ATP synthesis than 24,26Mg2+ ions. The rate of ATP production associated with creatine kinase is twice higher for 25Mg2+ than for 24.26Mg and does not depend on the blockade of oxidative phosphorylation. There is no difference between 24Mg2+ and 26Mg2+ effects in both oxidative and substrate phophorylation. These observations demonstrate that the enzymatic phosphorylation is a nuclear spin selective process controlled by magnetic isotope effect. The reaction mechanism proposed includes a participation of intermediate ion-radical pairs with Mg+ cation as a radical partner. Therefore, the key mitochondrial phosphotransferases work as a magnesium nuclear spin mediated molecular machines.

Fullerene-based Low Toxic Nanocationite Particles (Porphyrin Adducts of Cyclohexyl Fullerene-C60) to Treat Hypoxia-induced Mitochondrial Dysfunction in Mammalian Heart Muscle

BACKGROUND This is the first report on the targeted delivery of fullerene-based low toxic nanocationite particles (porphyrin adducts of cyclohexyl fullerene-C(60)) to treat hypoxia-induced mitochondrial dysfunction in mammalian heart muscle. METHODS The magnetic isotope effect generated by the release of paramagnetic (25)Mg(2+) from these nanoparticles selectively stimulates the ATP overproduction in the oxygen-depleted cell. RESULTS Because nanoparticles are membranotropic cationites, they will only release the overactivating paramagnetic cations in response to hypoxia-induced acidic shift. The resulting changes in the heart cell energy metabolism result in approximately 80% recovery of the affected myocardium in <24 h after a single injection (0.03-0.1 LD(50)). CONCLUSIONS Pharmacokinetics and pharmacodynamics of the nanoparticles suggest their suitability for safe and efficient administration in either single or multi-injection (acute or chronic) therapeutic schemes for the prevention and treatment of clinical conditions involving myocardial hypoxia.

Effect of 67Zn-nanoparticles on leukemic cells and normal lymphocytes.

Porphyrin-fulleren-based nanoparticles (NP), containing magnetic isotopes Mg, Zn and zinc of natural isotope composition (Zntotal-NP), have been tested on leukemic cells of patients with T-ALL, B-ALL, AML and lymphocytes of healthy donors. Reliable differences in action of magnetic and non-magnetic zinc isotopes for some types of cells were obtained. Magnetic magnesium isotopes and pure nanoparticles of porphyrinfulleren did not demonstrate any effects. Zn-NP induced high cytotoxicity in cells of acute B-lymphoblastic leukemia with LD50 almost three times lower, than those for healthy donors, and 4 times lower in comparison with Zntotal-NP. Also evaluation of apoptosis process in granulocytes of healthy donors in the case of the preparates were performed by method of flow cytometry.

Dependence of Mitochondrial ATP Synthesis on the Nuclear Magnetic Moment of Magnesium Ions

Isotopes continue to be a reliable tool in studies of molecular mechanisms of chemical and biochemical processes: first, as isotope labels and, second, as a source of isotope effects, which are markers of the mechanisms of transformation of molecules. The fractionation of isotopes in chemical and biochemical reactions induced by isotope effects is based on two fundamental properties of atomic nuclei: mass and magnetic moment. Classical isotope effect provides fractionation of isotope nuclei in accordance with their mass; magnetic isotope effect provides selection of isotopes in accordance with magnetic moments of their nuclei. The physical grounds and mechanisms of the two effects are absolutely different.

Perspectives of Fullerene Derivatives in PDT and Radiotherapy of Cancers.

Nanoparticles of fullerenes and their water-soluble derivatives have been firmly introduced into solution of medical problems. Although there are still debates about their toxicity and long-term consequences of their application in the clinic, the success of fullerenes application in some sections is undeniable, in particular, in photodynamic therapy (PDT) of cancer tumors. Besides there are interesting data on radiotherapy where fullerenes appear to be more transporters than drugs, but due to own cytoprotective properties, the fullerene adducts can also participate in the combined treatment. This review evaluates the status of these sections of fullerene chemistry in terms of development and recent trends.

Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli - Substrate specificity and stability under irradiation

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41→ His and Phel43→ Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phel43→ Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals.The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2′-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and o-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for o-phenylene diamine.

Porphyrin-fullerene nanoparticles for treatment of hypoxic cardiopathies

In this study, low-toxic fullerene-based nanocationite particles (adducts of porphyrin with cyclohexyl fullerene C60) designated for targeted delivery of the paramagnetic stable magnesium isotope to heart muscle is reported for the first time; these particles exhibit a sharp clinical effect of 80% recovery from tissue hypoxia in less than 24 h after a single injection (0.03–0.1 LD50). This therapy is based on a novel principle: 25Mg2+ released by nanoparticles due to the magnetic isotopic effect selectively stimulates the additional production of ATP in oxygen-depleted cells. These cationite “smart nanoparticles,” which possess a membranotropic effect, release hyperactivating paramagnetic cations only in response to a metabolic acidic shift. The final positive changes in the energy metabolism of heart muscle cells are capable of helping to prevent and/or treat local hypoxia of heart muscle, and therefore, protect heart muscle from serious damage in a wide variety of clinical cases of hypoxia, including cardiotoxic side effects of doxorubicin and 1-methylnicotineamide. Both the pharmacokinetics and pharmacodynamics of the proposed drug allow safe and efficient administration in single-and multi-injection (acute and chronic) therapeutic schemes.

Enzyme activation and inactivation induced by low doses of irradiation

Activation phenomenon has been observed with two sets of enzymes under the conditions of low dosage irradiation. Activation was registered for angiotensin-converting enzyme under in vitro γ-irradiation (0.662 MeV, pulse duration approx 10s) at dose levels of 1–3 Gy and under X-ray irradiation (approx 9 keV, pulseduration approx 10−9s) at dose levels of 2×10−5 Gy. An activation effect has also occurred for native and recombinant horseradish peroxidase and tobacco peroxidase under γ-irradiation. The phenomenon observed is rationalized in terms of a kinetic model suggesting the existence of at least one activated enzyme conformation induced by radiolysis. The activity oscillations registered in dense plasma focus experiments were rationalized using the same model with the corresponding kinetic equation converted into the form describing the decaying oscillations caused by exciting force. The model analysis is presented.

Effect of the 67Zn isotope on leukemic cells and normal lymphocytes

Porphyrinofullerene nanoparticles (NP) containing magnetic isotopes 25Mg and 67Zn (25Mg-NP and 67Zn-NP) and the natural isotopic composition of zinc (Zn) were tested on human leukemic cells of patients with acute leukemia and on lymphocytes of healthy donors. The fundamental differences in the cytotoxic effect of magnetic and nonmagnetic zinc isotopes on tumor cells were observed, as well as the complete absence of the influence of the magnetic magnesium isotope and pristine nanoparticles. The 67Zn-NP manifested high cytotoxicity towards cells of acute B-lymphoblast leukemia with LD50 almost three times lower than that of healthy donors and four times lower than that of the Zn-NP. Apoptosis was evaluated by cytofluorimetry for the drugs used.