Određivanje proteinaza trichomonas-a tenax-a : doktorska disertacija - Šegović, Sanja; Buntak-Kobler, Dunja (Zagreb : S. Šegović, 1997)
Sažetak: U radu smo demonstrirali karakteristike proteolitičkih sustava protzoona Trichomonas tenax, uzgojenih iz zubnog plaka i korijenskih kanala zubi s gangrenoznom pulpom ili periapeksnim procesom. Uzorci su nasađeni na modificirani Diamondov tekući hranjivi medij i očitavani svjetlosnom mikroskopijom svježeg preparata svakih 24 sata do petog dana nakon nasađivanja. Izolati su smrznuti na -20 C do pripreme staničnih lizata. Lizati ispranih stanica su pripremljeni dodavanjem 0,25% (v/v) Triton x-100 suspenziji parazita u 0,25 M sukrozi.Lizati su odmah korišteni ili pohranjivani na -20 C.
Nativnom elektroforezom na poliakrlamidom gelu utvrđene su četiri proteinske trake kod obiju vrsti izolata oralnog trihonomonasa, a elektroforezom na poliakrilamidnom gelu uz natrijev dodecil sulfat i uz dodatak želatine utvrđena je za obje vrste izolata multipla proteolitička aktivnost najviše izražena u području proteina molekulske težine 35 kDa i kod inkubiranja gela u pH 4,6 i pH 5,6. Komparativna proteiolotička aktivnost lizata ispitana je na supstratima; azokazeinu i peptidima nitroanilida. U staničnim lizatima oba izolata oralnog trihonomonasa opažene su 3 protolitičke aktivnosti: AZCaza, aktivnost na azokazeinu ovisno o ditiotreitolu; te dvije različite aktivnosti na peptidima nitroanilidskih derivata (jedna optimalna kod pH 7,0 i stimulirana ditiotreitolom; i druga optimalna kod pH 5,0 i neovisna o ditiotreitolu).
AZCaza aktivnost je bila očigledna u širokom pH rasponu. S obzirom na komparativnu proteolitičku aktivnost prema navedenim supstratima, izolati protozoona Trichomonas tenax pokazuju podjednaku aktivnost i pripadaju cistein proteinazama. Iako žive u različitim mikrosredinama usne šupljine, oba izolata protozoona Trichonomonas tenax pokazuju sličnu proteolitičku aktivnost.
Summary: In this study a number of common characteristics in the proteolytic systems of Trichonomonas tenax culturing from dental plaque and from the teeth with necrotic pulp or with periapical lesion have been demonstrated. Samples were cultivated in modified liquid Diamond's axenic medium and light microscopy of native smears were taken in intervals of 24 h until the fifth day from sampling. Izolates were frozen at -20 C until preparation of cell Iysates was completed. Lysates of washed parasites were obtained by the addition of 0,25% (v/v) triton x-100 to suspensions of parasites in 0,25 M sucrose. The Iysates were used immediatly or stored at -20 C. Native electrophoresis on polyacrylamide gel determined 4 protein bands for each species of Trichonomonas tenax.
Electrophoresis on SDS-polyacrylamide gel containing gelatin determined multiple proteolytic activity for each species of Trichonomonas tenax the most detectible at M 35000 and pH optimum at 4,6 and 5,6. Comparative proteolytic of two different izolates of Trichonomonas tanex were examined towards the following substrats: azocasein and peptide nitroanilide derivates. At least three proteolytic activities were detected in each lysate. There were AZCaza, a dithiothreitol dependent activity on azocasein; and two distinct activities towards the peptide nitroanilide derivates - one was optimally at pH 7,0 and stimulated by dithiothreitol; the other had no dithiothreitol requirment and was highly active at pH 5,0. AZCaza were active over a broad pH range.
Overall, with respect to these three activities, oral Trichonomonas tenax from dental plaque and from root canales were quite similar and belong to cysteine proteinases. Trichonomonas tenax from dental plaque and from root canals live in different microenvironments, but have similar proteilytic activities.
Mentor: Dunja Kobler-Buntak
Sveučilište u Zagrebu, Stomatiološki fakultet, Zagreb, 1997
Bibliografija: str. 76-86
Summary
Keywords: Oralni trihomonas -- Proteolitička aktivnost; Oralna higijena -- Trichomonas tenax