facebook
twitter
vk
instagram
linkedin
google+
tumblr
akademia
youtube
skype
mendeley
Wiki

DYNAMICS OF ACTIVITY OF KEY METABOLIC ENZYMES HEME AND SOME CYTOCHROMES RATS IN POSTNATAL ONTOGENY

Автор Доклада: 
Kryzhnaya S. I.
Награда: 
DYNAMICS OF ACTIVITY OF KEY METABOLIC ENZYMES HEME AND SOME CYTOCHROMES RATS IN POSTNATAL ONTOGENY

UDK 577.151.63:57.017.6

DYNAMICS OF ACTIVITY OF KEY METABOLIC ENZYMES HEME AND SOME CYTOCHROMES RATS IN POSTNATAL ONTOGENY

Kryzhnaya Svetlana Ivanovna, candidate of Medical Sciences, Associate Professor of Pathophysiology
National Pharmaceutical University

Investigated the activity of key enzymes of heme metabolism and content of some hemoproteins in the liver of male rats 1,5, 5 and 24 months of age. It was established that the activity of δ-aminolevulinat synthase (EC 2.3.1.37) is reduced to 3 months of age and remains at the same level in the rat adulthood. The content of microsomal cytochrome P-450 activity and holoenzyme tryptophan 2.3 dioxygenase increased to 5 months of age. The total activity of tryptophan 2.3 dioxygenase above at 24 months as compared to those aged 1,5 month. Hemoxygenase activity and the content of microsomal cytochrome b5 due to age do not change.
Keywords: heme oxygenase, tryptophan dioxygenase 2.3, δ-Aminolevulinat synthase, cytochrome P450, B5

Исследовали активность ключевых ферментов метаболизма гема и содержание некоторых гемопротеидов в печени крыс-самцов 1,5, 5 и 24-месячного возраста. Установлено, что активность δ-аминолевулинат-синтазы (КФ 2.3.1.37) снижается к 5 месячному возрасту и остается на том же уровне у крыс зрелого возраста. Содержание микросомального цитохрома Р-450 и активность холофермента триптофан 2,3 диоксигеназы увеличиваются к 5 месячному возрасту. Общая активность триптофан 2,3 диоксигеназы выше у 24 месячных по сравнению с 1 месячными животными. Гемоксигеназная активность (КФ 1.14.99.3) и содержание микросомального цитохрома b5 с возрастом не изменяются. 
Ключевые слова: гемоксигеназа, триптофан 2,3 диоксигеназа, δ-аминолевулинат-синтаза, цитохром P450 and b5.
 

Activity of enzymes catalyzing reactions stages of biosynthesis and degradation of heme (δ-aminolevulinat synthase and heme oxygenase, respectively), is controlled by many factors, one of which is the content of heme in the cell. The level of activity of key metabolic enzymes of the heme determines its content in the liver and subsequent utilization for the synthesis of hemoproteins [1]. Based on these series of articles [7, 8] formed the view that the activity of δ-aminolevulinat synthase and hemoxygenase most closely associated with the content and activity of hemoproteins such as microsomal cytochrome P-450 and 2.3 tryptophan dioxygenase. However, unknown to the relationship between the activity of key enzymes of heme metabolism and the content or activity of hemoproteins in the process of individual development and aging. Currently available literature data on the activity of δ-aminolevulinat synthase and heme oxygenase are scarce and contradictory [9, 10], which makes the conclusion even on the direction of change of these enzymes in the postnatal development. Also contradictory data on age characteristics of content and activity of hemoproteins. The purpose of this study - the study of δ-aminolevulinat synthase and hemoxygenase activities, the content of microsomal cytochrome P450 and b5, and 2.3 tryptophan dioxygenase activity in rat liver during postnatal ontogenesis.

Materials and methods
Experiments were conducted in 30 nonlinear white male rats a mass of 60-280 g of 1,5, 5 and 24 months of age, which were kept in standard vivarium diet, constant temperature and humidity. Animals were decapitated, livers perfused with saline in situ. δ-Aminolevulinat-synthase activity was determined in liver homogenate was expressed as pmol δ-aminolevulinic acid in 1 h per 1 mg of protein. Activity of hemoxygenase was determined in the microsomal fraction [5]. Substrate served methhemalbumin (final concentration of hemin in the cell 0.033 mM, serum albumin 2.5 uM). The duration of incubation is 10 min at 37° C. The enzyme activity was expressed as pmol of bilirubin formed per 1 min order of 1 mg protein using molar extinction coefficient of 40,000 M-1 cm-1. Tryptophan 2.3 dioxygenase activity was determined in fractions post mitohondrial substrat. Total kinurenina formed and expressed in nanomoles kinurenina for 1 h at 1 mg protein, while the activity of holoenzyme tryptophan 2.3 dioxygenase was determined in the absence of overall activity in the presence in the incubation medium of exogenous hemin [2]. The degree of saturation of the heme dioxygenase tryptophan 2.3 was judged by the ratio between the activities of the holoenzyme to the total activity and expressed as%. The content of microsomal cytochrome established by differential spectrophotometry and expressed in pmol per 1 mg mikrosomal protein using a molar extinction coefficient for cytochrome b5, 164 ×10-3 M-1 cm-1, and for cytochrome P450 - 91 × 10-3 M-1cm-1. The total content of heme in liver microsomes [12] was expressed as pmol mg per g protein. Protein was determined by the method of Miller, using as a standard human serum albumin. Enzyme activity, content of cytochromes and gema was determined on the SF-40 and SF-26 spectrophotometer.

We used the following reagents: Pyridoxal-5-phosphate, glucose-6-phosphate dehydrogenase (Fegak, Germany), glucose-6-phosphate («SIGMA» USA), glycine, and albumin from human serum, tris, L-tryptophan, NADP («Reanal», Hungary) and other reagents of national production. At the end of experiment we carried out of statistical processing of data [3].

Results and discussion
The studies found that the δ-aminolevulinat-synthase activity in liver was expressed in rats predpubertat period and decreased by 32% to the mature age and remained at that level in the rat sexually mature age. These results agree with those of other studies, which showed similarity in size to reduce the activity of δ-aminolevulinat synthase in mature rats compared with young mature (5 months). A more significant decrease in activity in old rats, 12 monthly, compared with younger monthly 1.5 observed by other authors [5]. In the activity of liver microsomal heme oxygenase 1,5, 5 - and 24-month-old animals revealed no significant differences. Similar results are given in [6]. The level of microsomal cytochrome b5, in rat liver investigated age groups did not differ. The content of cytochrome P450 in liver microsomes is increased by 58% from 1,5 to 5 months of age and remains at the same level in old animals. A similar age dynamics of the concentration of cytochrome P450 in the rats liver is shown in article [8]. Total content of heme in microsomes varies with age is similar to changes in the level of microsomal cytochrome P450.

Table

Activity of key enzymes of heme metabolism and content of cytochrome in liver of rats of different ages (M±m; n=5±8)

Studied parameters

Age, months

1,5

5

12

δ-Aminolevulinat synthasepmol δ-aminolinovoyacids at 1 mg protein in 1 h

148±15

99±12*

103±7*

Hemeoxygenase pmol of bilirubin per 1 mg protein 1min

68±6

56±8

61±7

Р450, pmol mg protein b5, pmol per 1 mg of protein

461±43

713±33*

632±42*

Heme microsomes pmol per 1 mg of protein

379 ±21

382±22

388±19

Tryptophan 2.3 dioxygenase nanomol kinurenina to 1mg protein 1 h:

712±29

1096±53*

985±52*

The total activity

19,2±2,6

25,0±3,4

33,7±3,9*

Active holoenzyme

7,1±1,3

13,1±1,5*

12,4±1,1*

Percentage saturation of the enzyme heme

37,9±4,2

54,1±2,5*

40,5±3,2

Note: * - p <0.05 as compared with the 1.5 month old rats

The total activity of 2.3 tryptophan dioxygenase in rat liver is increased in old rats as compared to 1.5 periods. Holoenzyme activity in 5 month old rats by 91% higher than the 1.5 month and not statistically different from the old rats. Degree of saturation of the heme dioxygenase 2.3 tryptophan maximum at 5 months of age. Thus, the results indicate that in the liver of old rats there is no significant change in the intracellular concentration of heme, since the activity of key metabolic enzymes heme, content and activity of the hemoproteins studied in this group of animals did not differ from the young adult. Changes in activity of δ-aminolevulinat synthase, cytochrome P450 content, activity of holoenzyme tryptophan 2.3 dioxygenase observed before puberty between 1,5-5 months of postnatal development, the direction of changes in the activity of a key enzyme synthesis heme and hemoproteins studied the content has the opposite character. These seemingly contradictory data is understandable, considering the content of cytochrome P450 holoenzyme activity and percentage saturation of tryptophan dioxygenase 2.3 as indicators of heme regulatory pool of heme in the cell. As is known, the activity of key enzymes of heme metabolism is controlled by the concentration of the heme through the so-called regulatory pool of heme: activity δ-aminolevulinat synthase is regulated by the final product on the principle of reciprocal negative relationship, and heme oxygenase activity induced by an excess of heme [1, 2]. The author of [14] suggests that the regulatory heme is used in the synthesis of cytochrome P450, and consequently, the content of hemoproteins may serve as an indicator of the regulatory pool of heme. However, another group of researchers believes that the activity of tryptophan 2.3 dioxygenase and especially the percent saturation of the enzyme heme more than the content of cytochrome P450, reflects the state of the pool of regulatory heme in the liver. Thus, if we consider these two hemoproteins as indicators of the regulatory heme pool, according to information received, the low concentration of regulatory heme in the liver of young immature animals induces δ-aminolevulinat-synthase activity. By 5 months of age of free regulatory heme in the liver increases, this leads to reduced activity of δ-Aminolevulinat synthase. In old rats a similar relationship is observed between the activity of a key enzyme of the synthesis of gem, the content of cytochrome P450 activity and holoenzyme of tryptophan dioxygenase 2.3, but the degree of heme saturation of tryptophan 2.3dioxygenase.

Conclusions:

  • 1. Hence, in young animals indicator of the pool of regulatory pool in the liver may serve as the content of cytochrome P450 holoenzyme activity and percentage of heme saturation of tryptophan 2.3 dioxygenase, and in older animals - the content of cytochrome P450 activity and holoenzyme of tryptophan 2.3. dioxygenase
  • 2. Elevated levels of microsomal heme, cytochrome P450, and the holoenzyme of tryptophan 2.3 dioxygenase from 1.5 to 5 months of age against a background of reducing δ-Aminolevulinat-synthase activity.
  • 3. Age-related changes do not affect the rates of metabolism gem.


Literature:
1.Калиман П.А., Баранник Т.В. Метаболизм гема и оксидативный стресс // Укр.биохим.журн. – 2001. – Т.73, №1. – С.5-15.
2.Камышников B.C. Справочник по клинико-биохимической и лабораторной диагностике. - М., 2004. - 834 с.
3.Лапач С.Н. Статистические методы в медико-биологических исследованиях с использованием Еxel/ / С.Н.Лапач, А.В.Чубенко, П.Н.Бабич. – К.: «Морион», 2000. – 320с.
4.Лемешко В. В. Система микросомального окисления при развитии и старении организма // Биохимия.— 1980.— 45, №11.— С. 1964—1969.
5.Руководство по клинической лабораторной диагностике. Ч.3. Клиническая биохимия: Учеб. пособие / [М.А.Базарнова, З.П.Гетте, Л.И.Кальнова и др. ]; Под ред.проф. М.А.Базарновой, проф. В.Т.Морозовой.– 2-е изд., перераб. и доп.– К.: Вища шк.,2000.–319 с.
6.Abraham G. N., Levere R. D., Friedman M. L. Effect of age on rat liver heme and drug metabolism // Exp. Gerontol.— 1985 — 20, N 5.— P. 277—284.
7.Control of synthesis of hepatic Δ-aminolevulinic acid synthase and cytochrome Р450: relationship to hepatic porphyrias /I. A. Borthwick, G. Srivastava, A. A. Hobbs et al. //Cell. Regul. and Malignant Growth. Lipmann Tymp., May 24—28, 1984. Tokyo; Berlin e. a., 1985.— P. 144—151.
8.De Matteis F. Hepatic porphyria caused by 2-allyl-2 isopropyl acetamide, 3,5-di-ethooxycarbonyl-l,4-dihydrocollidme, griaetofulvin and' related compounds // Heme and Hemoproteins.—Berlin e. a., P. 129—155.
9.Fujii., Dale G.L., Beutler E. Glutatione-dependent protection against oxidative damage of the human red cell membrane // Blood. – 1998. – 34, №10. – P.1632-1644.
10.Kikuchi G., Yoshida T. Function and induction of the microsomal heme oxygenase // Моl. and Cell. Biochem.— 1983.— 53954, N 1.— P. 163—183.
11.Leusui S.F., Tomaro M.L. Heme oxygenase and oxidative stress. Evidence of involvement of bilirubin as physiological protector against oxidative damage //Ibid. – 1994. – 1223, №1. - P 9-14.
12.Maines М.D., Kappas A. Prematurelly evoked synthesis and induction of δ-aminolevulinate synthetase neonatal. Evidence for metal ion repression of enzyme formation// J. Biol. Chem.— 1978.— 253, N 7.— P.Y2321-2326.
13.Paterniti J. R., Lin C.-I. P., Beattie D. S. Regulation of heme metabolism during senescence: activity of severa heme-containing enzymes and heme oxygenase in the liver and kidney of aging rats.— Mech. Ageing, and Develop.—1980.— 12, N 1.— P. 81—91.
14.Peng J., Jonnes G.L., Watson K. Stress Proteins as Biomarkers of Oxidative Stress: Effects of Antioxidant Supplements // Free Radic. Biol. Med. – 2000. – 28, №11. – P. 1598-1606.

6.75
Your rating: None Average: 6.8 (4 votes)

I'm deeply grateful for the

I'm deeply grateful for the brilliant ideas and innovations which have been contributed into your research and announced in this paper.
PARTNERS
 
 
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
Would you like to know all the news about GISAP project and be up to date of all news from GISAP? Register for free news right now and you will be receiving them on your e-mail right away as soon as they are published on GISAP portal.