FOSFATAZA ALCALINA din ficat

Clasa: Hidrolaze EC 3.1.3.1. CARN [9001-78-9] Nume sistematic: Fosfat monoester hidrolaza Reactie catalizata: Monoester fosforic +H2OAlcool+fosfat Surse biologice: Fosfataza alcalina este o enzima care se gaseste in cantitati mari in ficat si oase si care se elimina prin bila. Mai poate fi intalnita si in placenta si intestine. Caracteristici biochimice: Masa moleculara:494000(1), 306000(1), 200000(2) Compozitie: este alcatuita in principal din trei forme izoenzimatice (hepatobiliara, osoasa, intestinala), la care se adauga in timpul sarcinii o forma tranzitorie (forma placentara). Coeficientul de extinctie: E1%278= 7.2 Punct izoelectric: 6,2 Ph optim: 10,3-12 Constanta Michaelis: 0.1 M Tris, pH 8.0 la 25C: 4-fosfat nitrofenil, 3.6 M (rata relativa = 1.0); pirofosfat, 16 M (rata = 0.9); AMP, 18 M (rata = 1.2) ATP, 16 M (rata = 0.9); fosfoenolpiruvat 5.5 M (rata= 0.8). Inhibitori: Glicerolfosfat(2) Mercaptoetanol(2) Fenilalanina(2) Fenantrolina(1,10) Activatori: Albumina Histidina Colesterol Atenolol Anioni Specificitate: Fosfataza alcalina reprezinta o enzima pe baza de zinc cu rol in scindarea gruparii fosfat terminale a esterilor organici. Functioneaza optim in mediu alcalin. FAL hidrolizeaza esterii fosfati ai alcoolilor primari si secundari,alcooli ciclici alifatici,fenoli si amine.De asemenea mai hidrolizeaza pirofosfatul anorganic (la un Ph optim aproximativ egal cu 8), fosfatul 5-terminal ai DNA sau RNA. Enzima nu hidrolizeaza fosfodiesterii (R-O-PO2-O-R'; R, R':grupurile alchile)

Stabilitate: Enzima este stabila la temperatura de -20C, pierzand 18% din activitate dupa 2 ani. Mecanismul de reactie:

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Utilizari: Testul pentru determinarea nivelului de fosfataza alcalina se efectueaz ca parte a examenului general pentru disfuncionalitile ficatului. Niveluri foarte ridicate ale fosfatazei alcaline pot indica medicului c exista un blocaj la nivelul conductelor bilei. Adesea nivelul FAL este ridicat la persoanele care sufer de cancer ce s-a rspndit n ficat. Protocol de dozare: * Deoarece este o metoda directa ,se va lucra direct in cuva.Nu avem nevoie de proba martor. 1,9 ml Pnpp 0,1 ml EPT.

*Se masoara la 405 nm din 20 in 20 de secunde pana valoarea extinctiei ramane aproximativ constanta (45 de minute) deoarece se va consuma substratul. *Cu datele obtinute se traseaza graficul in Excel.
Dozarea Fosfatazei Alcaline

3.05 3 2.95 Extinctia (405 nm) 2.9 2.85 2.8 2.75 2.7 2.65 2.6 2.55 0 100 200 Timp (sec) 300 400
Series1

Unitatea de activitate enzimatica: Unitarea bodansky este cantitatea de fosfat alcalin care elibereaza 1 mg de fosfat anorganic in decurs de o ora de incubare in mediu tampon,continand ca substrat sodiu-beta-glicerofosfat. [U.I.]FOSFATAZA(ALCALINA) / l. SER = (EP/ES) 27,55 Unitatea Internationala [U.I.] exprima cantitatea de fosfor eliberata corespunzator la:1 mol P / minut / litru SER EP-extinctiile probei ES-extinctiile standardului

Calcul: E=cl Pnp405=18503

Referinte bibliografice: (1) Wright, P.J.; Leathwood, P.D.; Plummer, D.T., Enzymes in rat urine: alkaline phosphatase, Enzymologia,42 (1972) 317-327 (2) Spencer, T.; Macrae, S.L., Some properties of the alkaline phosphatase of HeLa cells, Enzymologia,42 (1972) 329-341 (3)Schweingruber, M.E. , Acid and alkaline phosphatases in yeast, Adv. Protein Phosphatases, 4 (1978) 77-93 (4)Dealwis, C.G.; Brennan, C.; Christianson, K.; Mandecki, W.; Abad-Zapatero C., Crystallographic analysis of reversible metal binding observed in a mutant (Asp153-->Gly) of Escherichia coli alkaline phosphatase, Biochemistry, 31 (1995) 13967-13973 (5) Stinson, R.A.; Chan, J.R.A., Alkaline phosphatase and its function as a protein phosphatase, Adv. Protein Phosphatases,4 (1987) 127-151 (6) Herz, F., Alkaline phosphatase isozymes in cultured human cancer cells, Experientia,41 (1985) 1357-1361 (7) Dealwis, C.G.; Chen, L.; Brennan, C.; Mandecki, W.; Abad-Zapatero, C., 3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity, Protein Eng.,8 (1995) 865-871 (8) Wyckhoff, H.W.; Handschumacher, M.; Murthy, H.M.K.; Sowadski, J.M., The three dimensional structure of alkaline phosphatase from E. Coli, Adv. Enzymol. Relat. Areas Mol. Biol.,55 (1983) 453-480 (9) Coleman, J.E.; Gettins, P., Alkaline phosphatase, solution structure, and mechanism, Adv. Enzymol. Relat. Areas Mol. Biol.,55 (1983) 381-452 (10) Heimo, H.; Palmu, K.; Suominen, I., Human placental alkaline phosphatase: expression in Pichia pastoris, purification and characterization, Protein Expr. Purif.,12 (1998) 85-92 (11) Klumpp, S.; Schultz, J.E., Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization, Biochim. Biophys. Acta,1037 (1990) 233-239 (12) Wang, H.; Gilles-Baillien, M., Alkaline phosphatase and ATPases in brush border membranes of rat jejunum: distinct effects of divalent cations and of some inhibitors, Arch. Int. Physiol. Biochim. Biophys,100 (1992) 289-294 (13) Sarciron, M.E.; Hamoud, W.; Azzar, G.; Petavy, A.F., Alkaline phosphatase from Echinococcus multilocularis: purification and characterization, Comp. Biochem. Physiol. B,100 (1991) 253-258 (14) Reid, T.W.; Wilson, I.B., E. coli alkaline phosphatase, The Enzymes, 3rd Ed. (Boyer, P.D., ed.),l4 (1971) 373-415

(15) Fernley, H.N., Mammalian alkaline phosphatases, The Enzymes, 3rd Ed. (Boyer, P.D., ed.),4 (1971) 417-447

Stoica Laura Biochimie Anul II