Chivdzhiyan, M.

Kaprelyan, G.

Metodiev, K.

Rusev, V.

English

Varna Medical University Press: Journals

STUDY OF INHIBITORY E F F E C T OF GLUTATHIONЕ-REDUCED, L-CYSTEINE AND ZINC IONS ON NEURAMINIDASE N2 A C T I V I T Y IN INFLUENZA VIRUS T Y P E A M. Chividzhyan, V . Rusev, K. Metodiev, G. Kaprelyan Key-words: influenza virus type A - neuraminidase N2 - glutathione-reduced — L-cysteine - zinc ions The increased interest in studying the inhibitors of ortomixoviruses and some paramixoviru-ses is due to the fact that these inhibitors can be applied as chemotherapeutic drugs by means of suppression of the enzyme neuraminidase, too. Sulfhydril preparations inhibit neuraminidase activity of influenza viruses A(Japan) 305/72 (H2N2) and PR8 (HON1) at concentrations of lO^M (9). There are literature data that some structurally different compount such as phenyl-glioxane, phenyl-oxaminic acid, ft-phenyl-JC-me reap to-acrylic acid, etc. exert also an inhibitory action (2). Edmond et al. (1966) proved that derivatives of both phenyl-glioxane and of oxaminic acid inhibited neuraminidase activity of influenza vims A(England) 1/61 (H2N2). N-phenyloxaminic acid is proved to be the most effective inhibitor of the whole group of compounds. It inhibits the enzyme activity by 50 per cent at concentration between 2 and 5.10-4M. In order to create specific neuraminidase inhibitors some investigators synthetize compounds which are deri­vatives of N-acetylneuraminic and of desoxyneuraminic acids specifically suppressing enzyme activity (2). 2-desoxy-2,3-dihydro-N-trifluoroacetyl neuraminic acid (FANA) possesses the strongest ac­tivity. It inhibits competitively enzyme activity of strains A/Me 1/35 (HON1) with K m = 7.9 at concentration of inhibitor of 10* 'M (2). The aim of the present work is to study the influence of some'inhibitors on neuraminidase N2 activity. Material and Methods Investigations were carried out of standard influenza virus type A strains with neuraminida­se, serotype 2: A(Singapore 1/57 (H2N2); A(Hong Kong) 1/68(H3N2); A(Victoria) 35/72 (H3-N2); A(Texas) 1/77 (H3N2); as well as of some influenza virus strains isolated in our country: A(Sofia) 1/57 (H2N2); A(Sofia) 142/69 (H3N2) (both provided kindly by Dr. Kotzeva from tliе Research Institute of Infectious and Parasitic Diseases, Sofia); A(Varna) 123/76 (H3N2), and A(Vama) 31/7/84 (H3N2) isolated in the Department of Microbiology and Virology of the Higher Institute of Medicine, Varna. The strains studied were selected with a view to neurami­nidase incorporated in them. Virus strains having passed through 2 passages on developing chick embryos with infectious titre 6.5 - 7.6 lg EID50/0.2 cm 3 were used in our study. The working inhibitor concentration was 0.1M t.c. Preliniinarily, the following concentra­tions were selected: from 0.110*3M till 0.1 M. At the last concentration enzyme inhibition was maxinial. л N + 1 X S В о s ев 3 Е г-•о r S 0 0 5 ON т*- г о C N CO О 00* •* о • о О* "J-00* Г* h* v n vO rsl •o d 0.01 0.17 0.01 0.20 0.01 0.17 0.02 0.10 + 1 •и + 1 +| + 1 -н +i «о с SO г* Г* 00 о t̂-ON С Л V ) г о ЧУ" »-« о •о сК © r S O N г -* о с о г о VO 3 3 vo m O N ON' «о о* «о r s • О с VO r f о 4* О d d +1 r s r s о r s о 4> rn V O О d -H о d d 4 I d +1 О Op о »н Оч »o O N О d + 1 8 d о +! О о d -H 2 d O N O N CO ОЛ m CO Ол « О oo r o т г О «-Ч О d d d + 1 -H +i GO CO r o «/"> r o •o rs" « o v n •я с О О О Г -d d d ^' -ft +« ON .—t ON —« — J VO —« CO g g § r S ГЧ I со «o rs О О О VO o d d d +1+1+1 +1 S ON VO VO r o r s * « n rs* г ч r s r s m О О О d d d +t+i +i О О О **. **. **". «О VO* r s r i r s w> ^ го o o o o d d d rs" +i +i +»+i r s r s Ov О vo Ю vo >—1 £ <1 3 3 r-SoS d o d o * s o" 3 5 о d Г Л Р 2 o Z O ^ Z o Z ^ Z S ^ Z o p Z « Z 1 z 98 M. Chividzhyan, V . Rusev, К. Metodiev. The rest enzyme activity after inhibitory action was determined after Aminoff s method (1961). The results were calculated in mkg protein and then statistically processed by the me­thods of variation analysis for p < 0.05 (1 ,3) . Results and Discussion Our data are summarized on table 1 and fig. 1. One can see that glutathione inhibits relati­vely less standard strains^as compared with local ones isolated by us excepting the strains A(Sin-gapore) 1/57 (H2N2) and A(Sofia) 1/57 (H2N2) where inhibition is equally manifested (60.15 per cent and 59.98 per cent, respectively). 100 50 Glutathione-reduced L -cysteine Zinc ions 7?/ у C3 standard strains EZ2 local strains Fig. 1. Charges of neuraminidase N2 activity influenced by glutath jone-ге duce d, L-cysteine and zinc ions Legend: [ j standard strains i—i focal strains There are analogous results when L-cysteine action is concerned. Local strains isolated by us are inhibited to a greater extent as compared with the standard ones: A(Singapore) 1/57 (H2 N2) - 40.43 per cent; A(Sofia) 1/57 (H2N2) - 50.05 per cent; A(Hong Kong) 1/68 (H3N2) -42.62 per cent; A(Sofia) 142/69 (H3N2) - 50.85 per cent; A(Victoria) 35/72 (H3N2) - 44.96 per cent: A(Varna) 3 73/76 (H3N2) - 47.56 per cent; AOexas) 1/77 (H3N2) - 39.96 per cent, and A(Varna) 31/84 (I13N2) - 45.64 per cent. Zincs ions exert the same effect on strains A(Victoria) 35/72 (H3N2) (25.51 per cent inhi­bition) and A(Varna) 123/76 (H3N2) (34.87 per cent). Standard grains A(Singapore) 1/57 (H2N2)S A(Hong Kong) 1/68 (H3N2) and A(Texas) 1/77 (H3N2) are inhibited to a greaterex­tent in comparison with local ones isolated by us under zinc ion influence by 1.5 per cent, 7.62 per cent, and 721 per cent, respectively. it is evident that glutathione is die best inhibitor among the three ones at these concentra­tions selected by us. Most probably, it is due to its structure inhibiting competitively this en-Study of inhibitory effect of. 99 zyme. Our investigations argue for an approximately equivalent inhibitory action of L-cysteine which is possibly due to its active sulfhydril group - SH-group. There are rather great variations when zinc ions are concerned. This probably confirms the statement that neuraminidase type 2 (N2) is heterogenous and divided into subtypes (4 ,5) . We can conclude that at concentration of 0.1M glutathione, L-cysteine, and zinc ions inhibit neuraminidase N2. Glutathione and L-cysteine do it to a greater extent when local strains are concerned in comparison with standard ones. Zinc ions inhibit neuraminidase N2 of standard strains to a greater extent than that of local ones excepting A(Varna) 123/76 (H3N2). R E F E R E N C E S 1. А ф и ф и, А., С. Э й з е н. Статистический анализ. Подход с использованием ЭВМ. Москва, Мир, 1982. - 2. К и л ь б у р н, Э. Д. Вирусы гриппа и грипп. Москва, Медицина, 1978. - 3. Л ь в о в с к и й , Е. Н. Статистические методы построения эмпирических формул. Москва, Вышая школа, 1982, 160-168. - 4, Л ю б о в ц е в а, О. В., М. А. Я х н о, Л. Я. 3 а к с т е л ь с к а я. Вопр. вирусол., 1987, № 3, 283-285. - 5. Н и к о л о в а, 3. , Р. К о ц е в а, Г. К а п р е л я н, и др . В: Конгр. микробиол. , В. Вар­на, 12-14. X. 1981 г. София, 1982, т. 1, 343-347. - 6. С м о р о д и н ц е в, А. А., Д. Г о л у б е в, Т. Я. Л ю з а н и н а , Г. И. К а р п у х и н . Вопр. вирусол., 1981, № 4, 499-504. - 7. A m i n о f f, D. Biochem. J., 81, 1961, 384-392. - 8. Е d m о n d, J. D. , R. G. J о h n s о n, D. К i d d, et al. Brit. J . Pharmacol, 27, 1966, No 2, 415-426. - 9. R a f е 1 s о n, M. Е., M. S с h n е i r , D. W. W i l s о n, Arch. Biochem., 193, 1963, No 3 ,424-430. • ' ИЗУЧЕНИЕ ИНГИБИТОРНОГР ДЕЙСТВИЯ РЕДУЦИРОВАННОГО ГЛУТАТИОНА, L-ЦИСТЕИНА И ИОНОВ ЦИНКА НА АКТИВНОСТЬ НЕВРАМИНИДАЗЫ N2 ПРИ ВИРУСЕ ГРИППА ТИПА А М. Чивиджян, В. Русев, К. Методиев, Г. Капрелян Р Е З Ю М Е ч В настоящей работе авторы ставят перед собой цель изучения влияния некоторь^ ингибиторов (редуцированного глутатиона, I-цистеина и ионов цинка) на активность невраминидазы второго серотипа N2. Исследования проведены на эталонных штаммах вирусов гриппа и местных штаммах, изолиро­ванных в Софии и Варне. В выбранной авторами концентрации - 0,1 М, редуцированный глутатион, L-цистеин и ионы цин­ка действуют как ингибиторы невриминидазы Nz. Редуцированный глутатион и L-цистеин ингиби-руют в более высокой степени энзим из изолированных местных штаммов по сравнению с энзимом из эталонных штаммов. Под действием ионов цинка невраминидаза N2 эталонных штаммов инги-бируется в большей степени по сравнению с невраминидазой N2 местных штаммов, за исключением штамма А /Варна/123/76. 

STUDY OF INHIBITORY EFFECT OF GLUTATHIONÐ•-REDUCED, L-CYSTEINE AND ZINC IONS ON NEURAMINIDASE N2 ACTIVITY IN INFLUENZA VIRUS TYPE A

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STUDY OF INHIBITORY EFFECT OF GLUTATHIONÐ•-REDUCED, L-CYSTEINE AND ZINC IONS ON NEURAMINIDASE N2 ACTIVITY IN INFLUENZA VIRUS TYPE A

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