CC BY
16
Yusupova Umida Rakhmanovna, Senior teacher, National University of Uzbekistan Botirov Erkin Khojiakbarovich, Head of Chemistry department, professor, Surgut State University Almatov Karim Tajibayevich, doctor, of biological sciences, professor, E-mail: [email protected]
INFLUENCE OF HAPLOGENIN - 7 - GLYCOSIDE ON RESPIRATION AND OXIDATIVE PHOSPHORYLATION OF RAT LIVER MITOCHONDRIA
Abstract: It has been established that haplogenin - 7 - glycoside increases mitochondrial respiration of rat liver. Thus the ADP/O quotient increases slightly by glutamate, and by succinate on the contrary, decreases imperceptibly.
Keywords: flavonoids, haplogenin, oxidative phosphorylation, rat liver, mitochondria, glutamate, oxygen.
Introduction
It is known that in mitochondria occur processes as a result which energy accumulates in cells and these organelles possess all main functions of independent organism such as reduction, ion transportation and heredity [1]. It allows considering that mitochondria are rather complete substances of a living matter preserving their basic properties. If this so, a response of isolated mitochondria - their metabolic stations -should correspond to physiological laws of influence of living organisms on external actions. Differently, it is possible to believe that there is a correlation between metabolic station of isolated mitochondria and such stations, as excitation and braking. These terms often use for the description of system reactions of the whole organism. We use them in that sense in what they are applied in physiology to a designation of the basic laws of influence of a living tissue on external actions.
It's established that flavonoids possess anti-inflammatory, antiatherogenic, antivirus, anticytotoxic, membrane - stabilization [2, 3], anti-cancer, cytoprotective [4-6], neurocytoprotective [7-10] immune-modulation [11, 12] properties. However the questions, concerning influences haplogenin-7-glycoside on structure and functions of mitochondrial membranes still aren't studied.
Research an effect of haplogenin-7-glycoside on respiration and oxidative phosphorylation is of interest not only from a position of clarification of physiological and biochemical mechanisms the regulation of an organism activity, but also for an establishment pathogenetic importance of these parameters at various stressful influences and diseases.
Materials and Methods
Mitochondria were isolated from cells of rat liver according to a method [13]. Velocity of mitochondrial respiration at a various metabolic states was registered polarographically with the help of the rotating platinum electrode. Reactions were started with addition of a mitochondrial suspension into polarographic cell. An incubation medium has the following composition: sucrose - 0.25 M, KH2P04-5 mM, tris-HC1 - buffer - 10 mM (pH 7.4). As oxidizing substrata were used 10 mM of succinate (pH 7.4) and 10 mM glutamate (pH 7.4). Mitochondrial respiration and oxidative phosphorylation were analyzed at the consecutive addition 200 ^M of haplogenin-7-glycoside and ADP, 5.105 M of 2.4 - di-nitrophenol (DNP). Herewith the following velocity of respiration chemicals were determined: V2 - state 2 before the addition ofADP, V3 - active phosphorylation state, V4 - state 4 after an exhaustion of ADP in the polarographic cell; ADP/O ratio and respiration control quotient were calculated by a method of Chance and
Williams (V3: V4) [14]. Velocity of the substrata oxidation at a various metabolic states was expressed in nanogram atom oxygen min/mg of mitochondrial protein. Protein was defined by a method of Lowry et al. [15].
Experiments were carrying out in absence of flavo-noids and with addition haplogenin-7-glycoside into a polarographic cell in vitro. Haplogenin-7-glycoside was used in a manner of glycerin solution. The specified fla-vonoids were put into polarographic cell in final concen-
tration 20, 40, 60 mcgr/mg mitochondrial protein and studied the features of change of mitocondrial functional state. Haplogenin-7-glycoside has been kindly given by officials-professors Khushbaktova Z. A. and Syrov VV. of Institute of Plant substances' chemistry.
Results and discussion
Influence of haplogenin-7-glycoside on glutamate oxidation and oxidative phosphorylation of mitochondria was resulted in the (Table 1).
Table 1.- Influence Of Haplogenin-7-Glycoside On Glutamate Oxidation And Oxidative Phosphorylation Of Liver Mitochondria (M ± m; n = 5-6).
Readings Respiration rate, nanogram atom oxygen/min mg of protein
iaplogenin-7-glycoside, mcg/mg of protein
0 20 40 60
V2 18.0 ± 1.2 18.6 ± 1.4 19.0 ± 1.2 20.5 ± 1.6
% 100 103.3 105.5 113.9
V, 54.5 ± 1.8 57.4 ± 2.2 60.3 ± 1.9* 73.0 ± 1.8***
% 100 105,3 110,6 133,9
V, 17.6 ± 1.4 17.8 ± 1.4 17.9 ± 1.3 20.8 ± 1.7
% 100 101.1 101.7 118.2
V 68.8 ± 1.8 75.5 ± 2.2 80.0 ± 2.4* 96.5 ± 2.4****
% 100 109,7 116.3 140.2
RCch (V0: V,) 3.09 ± 0.10 3.22 ± 0.10 3.37 ± 0.09* 3,51 ± 0,10**
% 100 104.2 109.0 113,6
ADP/O 2.37 ± 0.09 2.81 ± 0.13** 3.00 ± 0.14*** 2.85 ± 0.10**
% 100 118.4 126.4 120.3
A note: Here and in the table 2 the quotient authenticity
Haplogenin-7-glycoside, in low concentrations (20 ^g/mg of protein) wasn't influence on glutamate oxidation and a respiration control value on Chance, however rose ADP/O quotient up to 18.4%. It's known that if a respiration control value on Chance reflects the degree of relationship of transformation processes and energy accumulation by mitochondria with energetic processes in the cell, that ADP/O value characterizes the functional organization of mechanisms, defining an ADP phosphorylation process in mitochondrial membrane and their relationship with activity of a terminal respiratory chain. The more a value of ADP/O, the less oxygen spent to phosphorylation, that accordingly higher mitochondrial coefficient of efficiency from energy storage point of view for further intracellular metabolic processes. The increase of haplogenin-7-glycoside concentration entered into the polarographic cell twice phosphorilative oxida-
s marked:*, *P < 0.05; **P < 0.002; ***P < 0.01;****P < 0.001.
tions of glutamate (V3) raises on 10.6% from control level. As a result, the value of the respiratory control on Chance and ADP/O quotient raised on 9.0 and 26.4%, respectively, from control level. The further increase of haplogenin-7-glycoside concentration (60 mkg/mg of protein) raises glutamate oxidation in various metabolic conditions of mitochondria.
Thus, a mitochondrial respiration in V2, V3 and V4 states increases on 13.9; 33.9 and 18.2% respectively, in comparison with control. Increase of respiration in phosphorylation condition leads to increase of value of the respiratory control on Chance and ADP/O quotient on 13.6 and 20.3%. It means that haplogenin-7-glycoside is a respiratory activator and especially, ATP synthesizing function of mitochondria at oxidation ofNAD - dependent substrates.
Influence of haplogenin-7-glycoside on succinate oxidation and oxidative phosphorylation of liver mitochondria is summarized in (Table 2).
In low concentration (20 mkg/mg of protein) haplogenin-7-glycoside slightly (on 12.9%) increases succinate phosphorilative oxidation. At the same time respiration of mitochondria in V2 and V4 metabolic states and value of respiration control on Chance hasn't change, however ADP/O quotient decreases to 12.1%. The increase of haplogenin-7-glyoside concentration, entered into a polarographic cell in two times leads to increase phosphorilative oxidation of succinate (V3) to 24.9% from control level, and respiration of mitochondria at the calm state (V4) - to 24.9%. Thus, value of the respiratory control on Chance does not change,
Table 2.- Influence of haplogenin-7-oxidative phosphoriltion of liver
Depending on dose, haplogenin-7-glyoside raises dinitropenolstimulative oxidation of substrates. So, if at the entering of haplogenin-7-glycoside into the polarographic cell in a dose of protein of 20 mkg/mg mitochondria, glutamate and succinate oxidation raise on 9.7 and 10.6% from control level, at the entering of protein of 40 mkg/mg - 16.3 and 37.8%, and 60 mkg/mg of protein -40.2 and 53.1%. Thus, haplogenin-7-glyoside increases transport of electrons from substrata to molecular oxygen along respiratory chain of mitochondria, and it considerably occurs on succinate oxidation pathway.
Analyzing the received results, it is possible to conclude that haplogenin-7-glycoside considerably raises mitochondrial respiration of rat liver. Thus, the ADP/O quotient with glutamate considerably raises, on the contrary, decreases with succinate.
however the ADP/O quotient raises on 16.0%. The further increase of haplogenin-7-glycoside concentration (60 mkg/mg of protein) leads to increase of suc-cinane oxidation in various metabolic states of mitochondria. Thus respiration of mitochondria in V2, V3 ana V4 states increases in comparison with control on 16.7; 39.8 and 34.7%, respectively. It means that hap-logenin-7-glyoside is the activator of the respiratory functions of mitochondria at oxidation of succinate oxidation pathway. Thus, the value of the respiratory control on Chance does not change, however ADP/O quotient decreases on 17.6%.
-glycoside on succinate oxidation and
mitochondria (M ± m; nb = 8-12)
At the analysis of oxidizing capability of mitochondria, the attention has been paid to characteristics of their conditions corresponding to a certain tissue activity. Now it became obvious that at functioning of mitochondria in vivo in quietness the main bulk of mitochondria are in a state 4 on Chance. This condition is characterized by good supply of mitochondria with oxygen and substrata. However, respiratory activity is suppressed because it is integrated to phosphorylation processes, and in a based tissue, the basic exchange fund of adenilnucleotides, using for endocellular transport of energy, appears in the form of ATP. Absence of corresponding acceptors of phosphate is the basic brake of cellular respiration. Increase of cell activity leads to energy expenses and ATP hydrolysis. Occurrence of phosphate acceptors in the form of ADP leads to activation of respiratory activity of mitochondria, and
Readings Rate of respiration, nanogram atom oxygen/min mg of protein
Haplogenin-7-glycoside, mcg/mg of protein
0 20 40 60
40.0 ± 2.4 40.9 ± 2.7 42.4 ± 3.2 46.7 ± 4.0
% 100 102.2 106.0 116.7
V3 112.0 ± 3.6 126.5 ± 4.4* 144.4 ± 5.7*** 156.6 ± 6.5****
% 100 112.9 128,9 139.8
V4 32.5 ± 2.5 34.8 ± 3.0 40.6 ± 3.5* 43.8 ± 3.7**
% 100 107.0 124.9 134.7
V 130.6 ± 5.6 144.5 ± 6.0 180.0 ± 6.4*** 200.0 ± 7.7****
% 100 110.6 137.8 153.1
RCch (V,: V4) 3.44 ± 0.13 3.63 ± 0.12 3.55 ± 0.12 3.57 ± 0.09
% 100 105.5 103.2 103.8
ADP/O 1.82 ± 0.09 1.71 ± 0.08 1.53 ± 0.11* 1.50 ± 0.08**
% 100 87.9 84.0 82.4
it will proceed until cell will spend energy of macroergic phosphoric connections and deliver ofATP to mitochondria. At presence of haplogenin-7-glycoside mitochondria pass from one metabolic state in higher metabolic state. In our opinion, high intensity of metabolism can be supported animals, received haplogenin-7-glycoside at the expense of a mitochondrial activation system of various tissues and, most likely, inner organs. Increase of mitochondrial respiration by haplogenin-7-glycoside can be connected with increase of translocase activity. It has shown [16] that, exchange of adenine nucleotides (ATP4-/ADP3-) between mitochondrial matrix and cyto-sole performed by special transport system - translocase, determines total speed of respiration. With use of fluorescence probe has been shown coexistence in a membrane not only not-mobile transmitting agents (a fixated portal pore) but also mobile ones, carrying out rotate and lateral diffusion in a membrane surface [17]. The most essential line of translocase is electrogenity. It means that in energized mitochondria transport of nucleotides is performed always in one direction: ADP from cytosole into mitochondria, ATP - from mitochondria into cytosole, and KM moreover, in 100 times higher for exogenous ATP than for exogenous ADP; relation ATP/ADP of cytosole: mitochondrial ATP/ADP is in direct linear dependence on the sizes of a membrane potential [16]. Translocase-adenine nucleotide, working synchronously with ATF-sintase and oxidizing enzymes system [18. 19] be under the control inner-mitochondrial pool of adenine nucleotides and linearly depends on the sizes of this pool [20].
In presence of haplogenin-7-glycoside increasing of ADP/O quotient by succinate and decreasing by glutamate in liver mitochondria is connected with an "anatropic electron transfer" phenomenon (restored NAD). In 1957 it has been found the phenomenon which has entered into bioenergetics under the name "oxidative phosphorylation convertibility", or, "ana-tropic transport of electrons" [26]. Succinate, added to mitochondrial suspension, invoked fast reduction of mitochondrial pyridine nucleotides. After addition of ADP pyridine nucleotides acidified and only after full phosphorylation ADP, they became reduced once again. The phenomenon of anatropic transport of electrons has found a rational explanation in frameworks of Mitchell's chemiosmotic theory [27]. It is known that from three proton pumps of a respiratory chain (respi-
ratory complexes 1, 3, 4) two (respiratory complexes 1 and 3) function reversible [28].
In vitro, for example can be created following conditions: at succinate oxidation, when the stream of protons, pumped out two proton pumps of a respiratory
chain (3 and 4), will be pump inside by the proton pump I. Anatropic transport ofprotons correlated with anatropic transport ofthe electrons moving against gradient ofredox potential ofisopotential groups of respiratory transmitting agents at the energy expense of an electrochemical gradient of protons on a mitochondrial membrane. Thus, in this case, succinate acts both as the donor ofprotons, pumped out by the pumps 3 and 4, and it is inversely to protons pumped in by the pump I. As a result, it can be registered by optical methods restoration of NAD+. It is obvious that anatropic transport of electrons can carry out only by "energized" mitochondria, i.e. possessing an electrochemical gradient of protons on a membrane.
Thus haplogenin-7-glycoside enhancing anatropic transport process of electrons raises ATP synthesis in mitochondria. Physiologically, that process is very expediently. It is known [29] that on each NADH molecule, oxidized by oxygen, ten protons carry through a membrane. Interrelation H+/O, equals 10. It responses to value of P/O for NAD-dependent substrates, succinate and ascorbate 10: 3 = 3, 3, 6: 3 = 2 and 4: 3 = 1, 3 respectively. It is known that extra membrane synthesis ATP from ADP and phosphate demands transport of three protons into mitochondrial matrix.
It is considered that transport of two protons is necessary for synthesis of one molecule of intra-mitochon-drial ATP, while transport of one more proton provides by antiport energy
(ADP3 ) t + (HPO4 ) t / (ATP4 ) . .
v 7 outer v 2 4 7 outer v 7 inner
Commutation of succinate oxidation to NAD-dependent substrates at presence of haplogenin-7-gluco-side represents the mechanism providing the possibility of completion of damage in high-energy compounds depot in a tissue which has arisen after excitation.
Conclusion
That commutation has an important power consequence. Considering that succinate oxidation rate is higher than NAD-dependent substrates, and transport intensity of high - energy compounds by respiration chain is higher at its burning (oxidation). This mechanism reacts as a spring, automatically backtracking
abovementioned system and is created by irritation. shown that at succinate use observed more complete
According to the modern biochemical reports, for re- cycle of mitochondrial changes with connection of ion
alization of protein and lipid synthesis it is necessary, transport, than at use of other substrates. It is possible
especially high power potential of mitochondria. Am- to think that this commutation represents the buffer
ber acid, as it is known, has no competitors in build- system, giving the chance to keep on the high levels of
ing of a high level of high-energy bonds and reduced native state.
pyridine nucleotide [30]. Therefore, it should possess Differently, at any normal physiological activity that
specific function of plasticity maintenance. It has been biochemical mechanism should be involved first of all.
References:
1. Zorov D. B., Isaev N. K., Plotnikov E. Yu et al., Mitochondria as multi-faced Janus. Review // Biochemistry,-2007.- Vol. 72.- Issue 10.- P. 1371-1384.
2. Rice-Evans C. A. Parker L. (Eds.) Flavonoids in Health and Diseases. Marcel Dekker, - New York,- 1997.
3. Paladini A. C., Marder M. Viola H. et al. Flavonoids and the central nervous system: from forgotten factors to potent anxiolytic compounds // J. Pharmacol.,- 1999.- V. 51.- P. 519-526.
4. Gramaglia A., Loi G., Mongioj V., et al. Increased survival in metastatic patients treated with stereotactic radiotherapy, omega three fatty acids and bioflavonoids // Anticancer Res.,- 1999.- V. 19.- P. 5583-5586.
5. Hirvoner T., Pietinen P., Virtanen M. et al. Intake of flavonols and flavonones and the risk of coronary heart disease in male smokers // Epidemiology,- 2000.- V. 12.- P. 62-67.
6. Bagchi D., Bagchi M., Stohs I. et al. Cellular protection with proanthocyanidins derived from grape seeds // Ann. N. Y. Acad. Sci.,- 2002.- V. 957.- P. 260-270.
7. Wang H., and Joseph J. A. Structure - activity relationships of quer-cetin in antagonizing hydrogen peroxide -induced calcium dysregulation in PC12 cells // Free Radic. Biol. Med.,- 1999.- V. 27.- P. 683-694.
8. Dore S., Bastianetto S., Kar S. et al. Protective and rescuing abilities of IGF-1 and some putative free radical scavengers against beta-amyloid toxicity in neurons // Ann. NY Acad. Sci.,- 1999.- V. 890.- P. 356-364.
9. Bastianetto S. Ramassamy C., Dore S. et al. The ginkgo biloba extract (Erb 761) protects hyppocampal neurons against cell death induced by betaamyloid // Eur. J. Neurosci,- 2000.- V. 12.- P. 1882-1890.
10. Middleton E., Kandaswami C., Theoharides T. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart diseases and cancer // Pharmacol. Rev.,- 2000.- V. 52.- P. 673-751.
11. Yuan J. and Yancuer B. Apoptosis in the nervous system // Nature,- 2000.- V. 407.- P. 802-809.
12. Chekman I. S. Flavonoids: clinic and pharmacologic aspects // Herbal therapy in Ukraine,- 2000.- V. 2.- P. 3-5.
13. Schneider W. C., Hogeboom G. N. Cytochemical studies of mammalian tissues the isolation of cell components by differential centrifugation. Cancer. Res.- 1951.- V. 19.- P. 1-22.
14. Chance B., Williams G. R. Respiratory enzyme in oxidative phosporylation. 1V. Respiratory chain. J. Biol. Chem.,- 1955.- No. 2.- P. 429-444.
15. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem.,- 1951.- V. 193.- No. 1.- P. 265-274.
16. Klingenberg M. The ADP/ATP translocation in mitochondria, a membrane potential controlled transport. Membrane Biol.- 1980.- V. 56.- P. 97-105.
17. Muller M., Krebs J. R., Cherry R. J., Kawato S. Rotational diffusion of the ADP/ARP translocator in the inner membrane of mitochondria and in proteoliposomes. J. Biol. Chem.,- 1984.- V. 259.- P. 3037-3043.
18. Gellerich F. N., Bohnensack R., Kunz W. Control of mitochondrial respiration. The contribution of the adenine nucleotide translocation depends on the ATP- and ADP consuming enzymes. Biochem. and biophys. acta.-1983.- V. 722. P. 381-391.
19. Krasinskaya I. P., Marshansky V. Ya., Dragunova S. F., Jaguzhinsky L. S. Synchronization ofrespiratory chain enzymes function and ATP - synthase of energized mitochondria. Biochemistry.-1984.- Vol. 49.- Issue 1.- P. 87-92.
20. Klingenberg M., Held H. W. The ADP/ATP translocation in mitochondria and its role in intracellular compart-mentation. Metabolic Compartmentation / Ed. H. Sies. Londonetc. Ac. Pr.,- 1982.- P. 101-122.
21. Khadjieva B. R. Comparative efficacy of different medicinal drugs of silimarin in the course therapy of alcoholic liver diseases. PhD., dissertation.- Moscow. Russia.- 2009.- 133 p.
22. Dorkina E. G. Hepatoprotective properties of flavonoids (pharmacodynamics perspectives of the clinic study). PhD dissertation. Volgograd. Russia. - 2010.- 296 p.
23. El-Beshbishy H. A. Hepatoprotective effect of Green Tea (Camellia sinesis) extract against tamoxifen-induced liver injury in rats // J. Biochem. Mol. Biol.- 2005.- Vol. 38.- No. 5.- P. 563-570.
24. Woodman O. L. Meeker W. F., Boujaoude M. Vasorelaxant and antioxidant activity of flavonols and flavones: structure-activity relationship // J Cardiovasc Pharmacol.- 2005.- Vol. 46.- No. 3.- P. 302-309.
25. H. van der Woude et al. Consequences of quercetin methylation for its covalent glutathione and DNA adducts formation // Chem. Biol. Interact.- 2006.- Vol. 160.- No. 3.- P. 193-203.
26. Chance B., Hollinger G. Succinate-linked pyridine nucleotide reduction in mitochondria // Federat. Proc.-1957.- V. 16.- Pt. 1.- 163 p.
27. Mitchell P., Moyle J. Chemiosmotic hypothesis of oxidative phosphorylation // Nature.- London,- 1967.- V. 213 (5072).- P. 137-139.
28. Wikstrom M., Krab K. Respiration-linked H+ translocation in mitochondria: stochiometry and mechanism // Current topics in bioenergetics.- 1980.- V. 10.- P. 52-100.
29. Skulachev V. P. Energy accumulation in the cell.- Moscow: Science.- 1969.
30. Kondrashova M. N. Metabolic states of mitochondria and main physiologic states of living tissues // Properties and functions of macromolecules and macromolecular systems.- Moscow: Science,- 1969.- P. 135-160.
