Purification and kinetic properties of human liver pyruvate kinase

İnsan karaciğer pirüvat kinazı saflaştırılarak kinetik özellikleri araştırılmıştır. İnsan karaciğer pirüvat kinazı amonyum sülfat ile çöktürme, dializ ve Sefadex G-200 kromatografiyi içeren 3 safha ile saflaştırılmıştır. L tip pirüvat kinaz enzimi insan karaciğer dokusunda %13.2 ürünle yaklaşık 26.3 kat saflaştırılabilmiştir. Enzimin spesifik aktivitesi 2.6 U/mg protein bulunmuştur. İnsan karaciğer pirüvat kinazı için optimal pH 7.4 olarak saptanmıştır. Ca+2 iyonu enzimi inhibe, K+, Mg+2, Mn+2 ve Na+ iyonları ise aktive etmiştir. Pirüvat kinaz FDP, ADP ile düzenlenmiş, alanın ile inhibe edilmiştir. PEP ve ADP için Km değerleri sırası ile 1.2 ve 0.3 mM bulunmuştur. 2,3-DPG ve ATP konsantrasyon artışına bağlı olarak başlangıçta enzimi aktive daha sonra inhibe etmiştir.

Anahtar Kelimeler:

pirüvat kinaz, karaciğer, insan hastalıkları, kinetik, saflaştırma

İnsan karaciğer pirüvat kinazının saflaştırılması ve kinetik özellikleri

The kinetic properties of pyruvate kinase purified from human liver were investigated. Human liver pyruvate kinase was purified by three-step process involving ammonium sulphate precipitation, dialysis and Sephadex G-200 chromatography. L type of pyruvate kinase enzyme was able to be purified about 26.3-fold with 13.2% yield in human liver. Specific activity of the enzyme was found to be 2.6 U/mg protein. The optimal pH for human liver pyruvate kinase was found to be 7.4. K+, Mg2+, Mn2+ and Na+ ions activated enzyme whereas Ca2+ ions inhibited it. The enzyme was regulated by FDP, ADP and pyruvate kinase activity was inhibited by alanine. The Km values for PEP and ADP were 1.2 and 0.3 mM respectively. 2,3-DPG and ATP were found to initially activate and subsequently inhibit the enzyme, as the concentration of 2,3-DPG and ATP increased.

Keywords:

pyruvate kinase, liver, human diseases, kinetics, purification,

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