苹果酸脱氢酶（MDH）来源于栖热菌

苹果酸脱氢酶是一种二聚体，其中每个亚基含有一个NAD结合结构域和一个活性所需的底物结合羧基末端结构域。 苹果酸脱氢酶是一种普遍存在的酶，其在真核细胞中有两种亚型。 苹果酸脱氢酶是一种细胞质同工酶，是三羧酸循环中的重要催化剂。 应用 该酶可用于临床诊断中L-苹果酸和谷氨酸草酰乙酸转氨酶(GOT)的测定。 Malic Dehydrogenase is a ubiquitous enzyme, which exists in two isoforms in eukaryotic cells. Malic dehydrogenase exists as a dimer with each subunit containing an NAD-binding domain and a substrate-binding carboxy-terminal domain required for activity. Malic dehydrogenase is a cytoplasmic isozyme and an important catalyst in the tricarboxylic acid cycle. Reagents A. 0.1 M Tris-HCl buffer (pH7.8) B. 0.01 M Phosphate buffer (KH2PO4-NaOH, pH 7.0) C. Triton X-100 solution (50 mg/ml) D. 0.01 M Phosphate buffer containing 0.1% Triton X-100 (KH2PO4-NaOH, pH 7.0) Dilute 20 ml of Triton X-100 solution (C) with approx. 800 ml of 0.01M Phosphate buffer (B). Fill up to 1,000 ml with 0.01M Phosphate buffer (B). E. NADH soluton Weigh 9 mg of NADH and dissolve in 0.1M Tris-HCl bufer (A). Fill up to 50 ml with 0.1M Tris-HCl Buffer (A). (Can be used for 5 days if kept refrigerated) F. Substrate solution Weigh 11 mg of oxaloacetic acid and dissolve in 0.1M Tris-HCl buffer (A). Fill up to 50 ml with 0.1M Tris-HCl buffer (A) (Make a fresh solution for each use.) G. Enzyme solution Weigh out Malate Dehydrogenase and dissolve in chilled 0.01M Phosphate Bufer containing 0.1% Triton X-100 (D). Enzyme solution should be prepared so that the value of AOD/minute becomes in the range of 0.025 ± 0.010. Procedure Pipette 2.0 ml of NADH solution (E) and 0.90 ml of Substrate solution (F) respectively into a quartz cell (d=10 mm) and keep at 25 + 0.5'℃ for 5 minutes. Then, pipete 0.10 ml of Enzyme solution (G) into the quartz cell and mix well immediately. Keep the reaction mixture at 25 ±0.5'C. Exaclly at 2 minutes and 5 minutes after the addition of Enzyme solution (G), measure the absorbances of the reaction mixture at 340 nm(A2 and A5). As a blank, pipette 0.01M Phosphate buffer (D) into another quartz cel (d=10 mm) instead of the Enzyme solution (G) and follow the same procedure described above (Ab2 and Ab5). Calculation Malate dehydrogenase activity (u/mg)=[(A2-A5)-(Ab2-Ab5)]/3*(1/6.22)*(n/0.1) Application This enzyme is used for the enzymatic determination of L-malate and gluamate oxalo-acetate transaminase(GOT)in clinical diagnosis.

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