Izolovanje i delimična karakterizacija kisele fosfataze ekstrakta polena Artemisia vulgaris

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE(under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and α-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM), molybdate (Ki = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide. Kisela fosfataza ekstrakta polena visokog korova (Artemisia vulgaris) je prečišćena 48 puta kombinacijom jonoizmenjivačke i gel-hromatografije. Molekulska težina enzima je 76 kDa i 73 kDa, određena gel-filtracijom na matriksu Sephadex G-100 sf i SDS PAG elektroforezom (pri redukujućim i neredukujućim uslovima), respektivno. Pri izoelektrofokusiranju, enzim se sastoji iz dve vrlo bliske trake pI vrednosti oko 4,2. Optimalno pH za aktivnost enzima je 5,4. PrividnoKmza hidrolizu p-nitrofenil-fosfata je procenjeno da je 0,16 mM. Prečišćeni enzim ima široku specifičnost hidrolizuje p-nitrofenil-fosfat i α-naftil-fosfat. Pirofosfat i O-fosfo-L-tirozin su procenjeni kao najbolji od potencijalnih in vivo supstrata ovog enzima. Enzim je inhibiran kompetitivno fosfatom (Ki=1,25 mM), molibdatom (Ki=0,055 mM) i pirofosfatom (Ki=6,7 mM) a nekompetitivno fluoridom (Ki= 9,8mM). Joni metala, kao što su Hg2+, Cu2+ i Zn2+ iskazuju inhibitorni efekat na enzim, dok je efekat ne-jonskih detergenata, kao što su Tween 20 i Triton X-100 blago aktivirajuć i. Nema promene u aktivnosti enzima u prisustvu tartarata, citrata, EDTA 1,10-fenantrolina i modifikatora sulfhidrilnih grupa kao što su p-hloromerkuribenzoat i N-etilmaleimid.