Citokemijsko spremljanje polifenol oksidaz v netresku : diplomska naloga = Cytochemical identification of polyphenol oxidases in houseleek : graduation thesis
Slamič, Vesna
Rastline netreska smo 9 ur inkubirali s hlapi 10 % etanolne raztopine metil jasmonata (MeJa) in istočasno s 95 % raztopino etanola. Po končani indukciji smo liste zamrznili s tekočim dušikom in jih ...shranili pri -20 ŚC. Zamrznjene liste induciranih rastlin smo uporabili za ekstrakcijo in delno čiščenje polifenol oksidaze. PPO smo ekstrahirali s pomočjo neionskega detergenta Tritona X-114 po metodi temperaturno inducirane dvofazne ločbe po Sanchez-Ferreru. Povečano aktivnost PPO smo spremljali spektrofotometrično pri 400 nm po dodatku substrata 4-metil katehola in nasičenju reakcijske zmesi s kisikom, brez in po dodatku katalaze. PPO smo tudi predinkubirali s tripsinom. Na osnovi rezultatov lahko zaključimo, da je metil jasmonat induciral, tripsin pa aktiviral PPO. Na bistveno povečanje aktivnosti vplivata kombinacija dodatka tripsina in daljše zamrzovanje supernatanta 6 (S 6). Aktivnost encima smo spremljali tudi na mestu reakcije (in situ) s citokemijsko metodo po Vaughn-u. Koščke svežih listov induciranih rastlin smo inkubirali s substratom DOPA. Z dodatkom osmijevega tetraoksida je nastalo osmijevo črno oziroma elektronsko gost produkt, ki smo ga v kloroplastih opazovali z elektronskim mikroskopom. Opazili smo, da je tako v velikih kot v starih listih reakcija na PPO negativna in da je celotna specifična aktivnost pripadla peroksidazi.
Type of material - undergraduate thesis
; adult, serious
Publication and manufacture - Ljubljana : [BF, Oddelek za živilstvo] : [V. Slamič], 1999
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