Serum prolidazın optimal deney koşulları

Prolidaz (EC 3.4.13.9) C terminalinde prolin ve hikroksiprolin içeren dipeptidlerin hidrolizini kataliz ederek prolinin geri kazanılmasında önemli bir işlev üstlenmektedir. Bu çalışmada insan serum prolidazının optimal deney koşulları saptandı. Bazı metal iyonlarının (Mn+2, Fe+2, Zn+2, Hg+2, Mg+2, Cu+2, Sr+2) ve maddelerin (etilendiamintetrasetikasid [EDTA], lodoasedamid [IAA], p-kloromerkuribenzoate [p-CMBA] and glutatyon) enzim aktivitesi üzerine etkisi araştırıldı. Serum prolidaz için optimal deney koşulları 37 0C'de 2,5 mmol/l Mn+2 ve 40 mmol/l (pH: 8) tampon içinde 2 saat preinkübasyon ile sağlandı. Ayrıca optimal aktivite için final substrat konsantrasyonu (gly-pro) 30 mmol/l, ve Km değerleri gly-pro ve ala-pro substratları için sırasıyla 13 ve 14 mmol/l olarak saptandı. Serum prolidaz aktivitesinin Mn+2 iyonu ile 23 kez arttığı saptandı. Mn+2 dışında kullanılan diğer metal iyonlarının aktiviteyi artırmadığı gözlendi. EDTA, IAA ve p-CMBA'in ise enzim aktivitesini inhibe ettiği tespit edildi.

Optimal conditions for the human serum prolidase assay

Prolidase is an iminodipeptedase that plays and important in the recycling of proline in collagen synthesis. In this study, the optimal assay conditions of prolidase obtained from human serum were determined. The effects of some metal ions (Mn+2, Fe+2, Zn+2, Hg+2, Mg+2, Cu+2, Sr+2) and substances (etildiamintetraceticacid [EDTA], lodoacedamid [IAA], p-chloromercuribenzoate [p-CMBA] and glutathione) were also investigated. Optimum conditions of serum prolidase were found as follows; preincubations at 37 0C, for two hours with at 2,5 mmol/l Mn+2 concentration and buffer concentration 40 mmol/l (pH:8). Serum prolidase activity was increased 23 times with Mn+2 ion. The final substrate concentration (gly-pro) for serum prolidase was 30 mmol/l. The Km values were found as 13 mmol/l, 14 mmol/l for gly-pro and ala-pro respectively. There is no activator affect on human serum prolidase activity of investigated metal ions except Mn+2. The enzyme activity was inhibited by EDTA, IAA, and p-CMBA.

Kaynakça

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