CC BY
9UJ
Wschodnioeuropejskie Czasopismo Naukowe (East European Scientific Journal) #11, 2016
NAUKI ROLNICZE / CEAbCK0X03flMCTBEHHblE HAYKM
XANTHINE OXIDASE OF CAMEL MILK HARBORS NITRATE AND NITRITE REDUCING
ACTIVITIES
Dyussembayev K. A., Kulataeva M. S., Alikulov Z.
L.N. Gumilyov Eurasian National University, Astana, Kazakhstan
The aim of this study was to investigate nitrate- and nitrite reducing activity of camel milk. For the first time nitrate and nitrite reducing activity of camel milk xanthine oxidase was studied. It was found that heat treatment of the milk in the presence of the molybdate and cysteine resulted in dramatically increase of the enzyme activity in vitro. These results show that camel milk contains demolybdoenzyme populations of XO. Since plant proteins are potential inducers of xanthine oxidase biosynthesis, in the summer when the content of total protein is the highest in living plants, the level of the associated activities XO were also higher. It is supposed that milk XO biosynthesis is also very sensitive to the level of xenobiotic compounds of plants.
Key words: cysteine, molybdenum, nitrite reductase, nitrate reductase, molybdocofactor, tungsten, xanthine oxidase,
During the investigation of molybdenum cofactor from xanthine oxidase (XO) in 1980 we found that homogenic cove milk XO catalyzes the reduction of inorganic nitrate and nitrite. XO reduces nitrate to nitrite, but a product of nitrite reduction by the enzyme remained unidentified [1, 3-4]. Later English scientists using a chemiluminescent method demonstrated that the end product of nitrate and nitrite reduction by xanthine oxidase is nitric oxide (NO) [2, 2-3]. Therefore, now much attention has been focused on this enzyme because of its possible involvement in NO biosynthesis.
NO, a gas, is found to control a seemingly, limitless range of functions in animals. Nitric oxide is involved in many physiological and pathological processes including: vasodilation inhibition of platelet aggregation, neurotransmission and cytotoxic host defense mechanisms [3, 3-5]. It has been convincingly shown that NO synthase (NOS, EC 3.14.13.39) is involved in the intracellular production of NO through the enzymatic conversion of L-arginine to L-citrulline in the presence of O2 and NADPH [4, 2-4].
This well known L-arginine:NO pathway has been generally regarded as the mechanism of NO biosynthesis within mammalian cells. However, later observations, in which reperfused myocardium generates NO in a manner dependent on nitrite but independent of NOs inhibitors, suggested that alternative mechanisms for NO synthesis other than the L-arginine:NO pathway may operate within ischaemic tissues [5, 4-6]. It is conceivable that the efficiency of NO generation by NOs will be greatly reduced once the oxygen supply to the tissue is limited, because both oxygen and L-arginine are indispensable substrates for NO synthesis by NOS [6, 3-4].
Nitrate reductase and nitrite reductase, the two metalloenzymes (containing molybdenum and iron atoms) involved in the assimilatory and dissimilatory pathways in bacteria and plants, catalyze the reduction of nitrate to nitrite and nitrite to NO, respectively. However, to date, mammalian nitrate and nitrite reductases have not been identified.
Levels of XO are high in mammalian liver although it is present in similar concentrations in lactating mammary tissue and is concentrated 1000-fold in milk fat globule micelles. In addition to molybdenum and FAD, it contains two iron-sulfur redox centers and has a wide substrate specificity, typically hydroxilating purines and concomitantly reducing either NAD+ (dehydrogenase form of the enzyme, EC 1.1.1.204) or molecular
oxygen (primarily the oxidase form (EC 1.1.3.22). The latter reaction generates the reactive oxygen species, superoxide anion and hydrogen peroxide [7, 2-4].
In contrast to the large body of knowledge regarding XO in animal body cells, the physiological role of the enzyme in milk has been obscure. Another point which arises from other works is that bovine milk xanthine oxidase, which is readily available commercially, is not typical of mammalian XO with regard to its substrates' specificity. A number of physiological functions have been proposed, but even the involvement of XO in purine metabolism may not be essential in mammals. Moreover, the actual function of the enzyme in animal milk is not currently known. Much less well known is XO's capacity of an animal's milk, with the exception of cove (camel, in our research), to reduce nitrate and nitrite. Therefore, the aim of this study was to examine the capacity of camel milk for reduction of nitrate and nitrite.
The reaction mixture for determination of enzyme activity consisted of 100 ^l fresh camel milk, 100 ^l of 0.1 M KNO3 (or NaNO2), 50 ^l of 10 ^M methyl- or benzyl viologen reduced by ditionite (sodium hydrosulfite, Na2S2O4) and 300 ^l of 0.1 M K-Na phosphate buffer (pH 6). NADH was not effective reductant for all associated activities of camel milk XO. One assay was used to determine nitrate reducing (NaR) as well as nitrite reducing (NiR) activities of milk XO: (1) nitrite formation from nitrate by NaR, and (2) nitrite utilization by NiR. At pH 2-2.5 nitric acid reacted with sulfanylamide forms diazonic compound. The latter reacted with N-(1-naphtyl)-ethylenediamine dichloride forming red colorazodye.The absorbance of the resulting color solution measured at 548 nm wavelength of the spectrophotometer. The mutant nit-1 of the fungi Neurospora crassa was grown and its crude extract was used for detection of molybdenum cofactor of XO. The cofactor activity was determined by highly sensitive method developed earlier by our team [8, 2-3].
Only fresh camel milk showed XO and associated activities, i.e. its nitrate and nitrite reducing activities. Deep freezing (at -87oC) of fresh milk makes possible its storage for up to six month without any changes its XO and associated activities. In contrast, its storage at -20oC resulted in gradually decrease of these activities after two months. Second freezing of once thawed camel milk led to totally lost of all activities of the enzyme.
First experiments showed that fresh camel milk possesses detectable nitrate and nitrite reducing activities. Optimal
114
© Dyussembayev K. A., Kulataeva M. S., Alikulov Z., 2016
uu
Wschodnioeuropejskie Czasopismo Naukowe (East European Scientific Journal) #11, 2016 IIIB§9JMj
condition for determination of enzyme catalytic activities was incubation of the reaction mixture at 35-37oC for 10 min. These activities had a pH optimum of between 6.0 and 6.0 whilst pH optimum for XO was 8.0. However, it was not clear whether these activities regard to milk XO. The milk lost both activities after boiling for 5 min but was relatively stable at 60 to 80oC. Heat treatment of the milk at 80-100oC in the presence of sodium molybdate and cysteine sharply increased nitrate- and nitrite reducing activities of XO. Heat treatment without thiol reagents led to the disappearance of these activities. Further experiments showed that heat treatment at 80-85oC temperature for 5 min, was optimal for the increase of associated activities of XO. Optimal final concentrations of molybdate and cysteine necessary for heat treatment were 10 ^M and 1.0 ^M, respectively. Effect of cysteine in the activation of all activities of camel milk XO was approximately three times higher than glutathione. Other artificial thiols such as mercaptoethanol, dithiothreitol and unithiol inhibited nitrate and nitrite reducing activities of milk XO.
Heat treatment of camel milk in the presence of sodium tungstate resulted in total inhibition of all activities of XO. It is well known that in the absence of molybdenum its chemical analog tungsten easily replaces the molybdenum in the active center of molybdoenzymes. However, tungsten-enzymes become inactive due to its inability to transport the electrons from a donor to an receiver during the catalytic reaction of molybdoenzyme. It is well known that allopurinol, a specific inhibitor of XO, inhibits enzyme activity by acting at its molybdenum site and prevents electron donation to Mo. Percentage inhibition of 10 ^M of allopurinol was 97%. These results show that nitrate and nitrite are clearly meant to interact with Mo center of XO of camel milk. These results convincingly show that in camel milk prevailing amount of XO molecules do not contain molybdenum atoms, i.e. the milk contain demolybdo-population of native XO. The existence of the demolybdoforms of XO in animal milk is widespread [9, 4-5].
Quantitative transfer of the molybdenum cofactor from xanthine oxidase of camel milk to the deficient enzyme of the nit-1 mutant of Neurospora crassa carried according to Hawkes and Bray confirmed the existence of demolybdoforms of camel milk XO. This method was carried out in our modification [10, 5-6]. Molybdenum cofactor activity of camel milk XO in the absence of exogenous molybdenum in the reaction mixture was only 2-3% of that of molybdenum presence.
One of possible ways of restoration the activity of such a demolybdo-XO is its release from milk fat globule micelles (MFGM) by heat treatment. Additionally, heat treatment leads in partial denaturation of XO molecules and incorporation of exogenously added molybdenum occurs. It is generally known that XO belongs to thermostable enzymes - it does not loss activity at 75-80oC temperature in several minutes. Thus, heating the milk at such temperature in the presence of phospholipids may result in disruption of MFGM and consequently cause the release of XO molecules from them. On the other hand, under high temperature XO molecules denature partially and
availability of enzyme active center for oxygen increased. In this case molybdenum-free forms of XO molecules quickly deactivated as a result of oxidation of sulfhydrile (-SH) groups of molybdocfactor in active center. The main active center of the molybdoenzyme xanthine oxidase is a molybdenum cofactor buried in a cavity. In all molybdoenzymes, Mo atoms bond with these sH-groups of the cofactor. Therefore, to protect them against oxidation the presence of strong reductants is necessary and it should be natural and harmless for health. Only natural antioxidants may play a role in reductions, such as cysteine and glutathione. Thus, heating results in disruption of MFGM by phospholipids and the release of XO molecules. Partial denaturation of enzyme molecules by heat treatment increases the availability of exogenous molybdenum and antioxidants for cofactor molecule in the active center. Antioxidants (cysteine and glutathione) protect SH-groups of the cofactor against oxidation and molybdenum atom easily binds with the sulfhydrile groups of the cofactor. After renaturation molybdenum-free molecules of the enzyme become active.
Finally, we found that dietary factors are also implicated in the regulation of XO. It was known before that species differences in the levels of xanthine oxidase are more pronounced with herbivores containing the highest levels of the enzyme [11, 4-6]. We investigated the seasonal changes in activities of XO of camel milk, i.e. their dependence on forage quality. We found that XO and its nitrate and nitrite reducing activities reached their maximum in the summer season, i.e. in May and June (2014). Very low activity of XO and negligible nitrate and nitrite reducing activities observed in autumn-winter season, i.e. from the end of September to the middle of May (2014-2015). Determination of molybocofactor activity confirmed such changes in the activities associated with XO [12, 2-4]. In fresh camel milk collected in winter season the activity of the cofactor determined by using nit-1 mutant of Neurospora crassa was negligible, but reaching the maximum in the summer season. Some results suggested that a depletion of dietary protein causes a10-fold drop in XO activity due to decreased synthesis of the enzyme in rat liver. Protein extracts more readily from soft fresh leaves than from those that are dry [13, 2-3]. Thus, our results express doubt that milk XO is an essential protein in milk fat droplet secretion from the lactating mammary gland [14, 3-4].
In human milk such a changes in nitrate- and nitrite reducing activity of was not observed. Furthermore, we believe that animal XO, milk XO in particular, is inducible also by xenobiotics of plant origin. It is known that plants have hundreds of different low molecular compounds which might be a potential xenobiotics for animal XO and their amount is high in the summer season, i.e. green fresh plants have more xenobiotics than dried ones in the winter season.
The presence of XO in camel milk remains a subject to be clarified. To our knowledge, this is the demonstration of the presence of demolybdo-population of XO in camel milk. For understanding physiological roles of XO in animal milk, development of new approaches that can provide information on the effects of NO on infants health will have special importance.
References:
1. Alikulov, Z.A., L'vov N.P., Kretovich, V.L. (1980) Nitrate and nitrite reductase activity of milk xanthine oxidase. Biokhimiia 45:1714-1718.
2. Millar T.M., Stevens C.R., Benjamin N., Eisenthal R., Harrison R., Blake D.R. 1998. Xanthine oxidoreductase catalyzes the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Letters. 427: 225-228.
NAUKI ROLNICZE 115
ии
ШДЖЭИ Wschodnioeuropejskie Czasopismo Naukowe (East European Scientific Journal) #11, 2016
3. Zhang Z., Nauthon D., Winyard P.G., Benjamin N. 1998. Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity. Bioch.Biophys.Res.Comm. 249:767-772.
4. Bryan N.S., Bian K., Murad F. 2009. Discovery of the nitric oxide signaling pathway and targets for drug development. Frontiers in Bioscience. 1-18.
5. Hemmens B and Mayer B. 1988. Enzymology of nitric oxide synthases. In: Nitric oxide protocols.Humana Press Totowa. Pp 1-32.
6. Beedham C. Molybdenum hydroxylases. In: "Enzyme systems that metabolize drug and xenobiotics" Ed. Costas Ioannidis. 2001. John Wiley & Sons Ltd. 146-188.
7. Savidov N. Alikulov Z. Lips H. Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts. Plant Science, 1998, v. 133 pp 33-45.
8. Atmani D, Benboubetra M, Harrison R. Goat's milk xanthine oxidoreductase is grossly deficient in molybdenum. J.Dairy Res.
2004. 71:7-13.
9. Godber BLJ, Schwarz G, Mendel RR, Lowe DJ, Bray RC, Eisenthal R, Harrison R. Molecular characterization of human xanthine oxidase: The enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulfur centers. Bioch. J.Immediate Public.
2005. 1-29. Manuscript BJ20041984.
10. Hawkes T.R. and Bray R.C. 1984. Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase. Biochem J. 219(2): 481-493.
11. Rowe P.B. and Wyngaarden J.B. 1966. The Mechanism of Dietary Alterations in Rat Hepatic Xanthine Qxidase Levels. J.Biol. Chem. Vol. 241, No. 23, PP. 5571-5576.
12. Cherry, D.M.; Amy, N.K. 1987. Effect of dietary protein and iron on the fractional turnover rate of rat liver xanthine oxidase. Journal of Nutrition; v. 117(12); p. 2054-2060.
13. PIRIE, N.W. 1975. The effect of processing conditions on the quality of leaf protein. In Protein nutritional quality of foods and feeds, ed. by M. Friedman, p. 341-354. New York, Marcel Dekker.
14. Vorbach, C.; Scriven, A.; Capecchi, M. R. 2002. The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland. Genes Dev. 16:3223 - 3235.
ВЫРАЩИВАНИЕ КАРТОФЕЛЯ В УСЛОВИЯХ ЮГА УКРАИНЫ
ЛАВРИНЕНКО ЮРИЙ АЛЕКСАНДРОВИЧ
доктор сельскохозяйственных наук, профессор, член-корреспондент НААН Украины, Институт орошаемого земледелия
НААН Украины БАЛАШОВА ГАЛИНА СТАНИСЛАВОВНА доктор сельскохозяйственных наук, Институт орошаемого земледелия НААН Украины
ЮЗЮК СЕРГЕЙ НИКОЛАЕВИЧ младший научный сотрудник, Институт орошаемого земледелия НААН Украины
Исследован технологический процесс выращивания картофеля при капельном орошении в условиях Южной Степи Украины. С учетом закономерностей водного, питательного режимов почвы. А также показатели роста, развития растений и формирования урожая картофеля весенней посадки в зависимости от элементов технологии полива и способов внесения удобрений. Схема опыта предусматривала изучение и увлажнение разных расчётных слоев почвы и способов внесения удобрений. Исследования проводились в соответствии с общепринятыми методиками.
Ключевые слова: картофель, капельное орошение, расчетный слой почвы, способы внесения удобрений, урожайность, биологическая спелость, водопотребление.
GROWING POTATOES IN THE SOUTH OF UKRAINE
LAVRINENKO Y. A.
Doctor of Agricultural Sciences, Professor, Corresponding Member of NAAS of Ukraine, Institute of irrigated agriculture NAAS of
Ukraine BALASHOVA G. S.
Doctor of Agricultural Sciences, Institute of irrigated agriculture NAAS of Ukraine
YUZYUK S. N.
Junior Researcher, Institute of irrigated agriculture NAAS of Ukraine
The process of growing potatoes under drip irrigation in the conditions of South Steppe of Ukraine. Given the patterns of the water, soil nutrient regimes. As well as indicators of growth, plant development and the formation of potato planting spring crops, depending on the elements of irrigation techniques and methods of fertilizer application. The experimental setup includes the study of different computational and hydration layers of the soil and methods of fertilizer applica-tion. The studies were conducted in accordance with generally accepted procedures.
Keywords: potato, drip irrigation, the settlement layer of soil, fertilizer application methods, yield, biological ripeness, water consumption.
116
© Лавриненко Ю. А., Балашова Г. С., Юзюк С. Н., 2016
