Turk J Vet Anim Sci 28 (2004) 1087-1096 © TÜB‹TAK 1087 Pirüvat Kinaz›n Normal ve Tümör Meme Dokusundan Saflaflt›r›lmas› ve Karakterizasyonu* Seval YILMAZ, Sema TEM‹ZER OZAN F›rat Üniversitesi, Veteriner Fakültesi, Biyokimya Anabilim Dal›, Elaz›¤ - TÜRK‹YE ‹brahim Hanifi ÖZERCAN F›rat Üniversitesi, T›p Fakültesi, Patoloji Anabilim Dal›, Elaz›¤ - TÜRK‹YE Gelifl Tarihi: 19.08.2002 Özet: ‹nsan normal ve tümör meme doku pirüvat kinaz› saflaflt›r›larak moleküler a¤›rl›¤› ve kinetik özellikleri araflt›r›lm›fl, normal meme dokusu ile tümör meme dokusu pirüvat kinaz aktivite düzeyleri karfl›laflt›r›lm›flt›r. DEAE Sefadeks A-50 kromatografi ile meme dokusunda pirüvat kinaz›n iki farkl› formunun varl›¤› gösterilmifltir. SDS-PAGE ile alt birimlerinin moleküler a¤›rl›¤› normal dokuda I. pikte 30.000 Da, II. pikte 63.000 Da, tümör dokusunda ise I. pikte 16.500 Da, II. pikte 60.000 Da olarak saptanm›flt›r. Pirüvat kinaz aktivitesi tümör meme dokusunda normal meme dokusuna göre 5,2 kat fazla bulunmufltur. Pirüvat kinaz enzimi normal meme dokusunda I. pik 1591 kat, II. pik 636,4 kat, tümör dokusunda I. pik 219 kat, II. pik ise 318 kat saflaflt›r›labilmifltir. Reaksiyon h›z e¤risi normal ve tümör meme dokusu pirüvat kinaz›n›n I. pikinde hiperbolikti ve FDP taraf›ndan aktive edilmemifltir. II. pikte reaksiyon h›z e¤risi tümör meme dokusunda hiperbolik, normal meme dokusunda ise sigmoidald›r ve FDP taraf›ndan aktive edilmifltir. Hill katsay›s› PEP için enzimde en az iki ba¤lanma bölgesinin varl›¤›n› göstermifltir. ‹nsan meme dokusundan izole edilen pirüvat kinaz izoenzimleri di¤er doku pirüvat kinaz izoenzimleri ile karfl›laflt›r›ld›¤›nda bunlar›n M 1 ve M 2 izoenzim oldu¤u ve normal meme dokusundaki M 2 izoenzimin tümör meme dokusunda K izoenzime dönüflebilece¤i düflünülebilir. Anahtar Sözcükler: ‹nsan meme tümörü, pirüvat kinaz, saflaflt›rma, kinetik özellikler Purification and Characterization of Pyruvate Kinase from Normal and Tumor Breast Tissues Abstract: The molecular weight and kinetic properties of pyruvate kinase purified from human normal and tumor breast tissues were studied and the activity levels of pyruvate kinase from normal and tumor breast tissues were compared. The presence of 2 forms of pyruvate kinase in human breast tissue was demonstrated by DEAE Sephadex A-50 chromatography. The molecular weight of subunits estimated by SDS-PAGE in forms I. and II. in normal breast tissue and in forms I. and II. in tumor breast tissue were 30,000 and 63,000 Da and 16,500 and 60,000 Da, respectively. It was found that the pyruvate kinase activity in tumor tissue was 5.2 times higher than that in normal tissue. Peaks I and II of pyruvate kinase were able to be purified about 1591-fold and 636.4-fold in normal breast tissue, and 219-fold and 318-fold in tumor breast tissue, respectively. The reaction rate curve was hyperbolic in the first peaks of both normal and tumor breast tissue pyruvate kinase and was not activated by fructose-1, 6-diphosphate (FDP). Reaction rate curves in the second peaks of tumor breast tissue and normal breast tissue pyruvate kinase were hyperbolic and sigmoidal, respectively, and were activated by FDP. The Hill coefficient showed that there were at least 2 binding regions in the enzyme for PEP. When compared with other tissue pyruvate kinase isozymes, it seems likely that the isoenzymes of pyruvate kinase isolated from human breast tissue were M 1 and M 2 isozymes and that the M 2 isozyme of pyruvate kinase from normal breast tissue changed into K isoenzyme in tumor breast tissue. Key Words: Human breast tumour, pyruvate kinase, purification, kinetic properties Araflt›rma Makalesi * Bu çal›flma TÜB‹TAK (VHAG-1442) ve FÜNAF (327) taraf›ndan desteklenmifltir.