Becker D M, Knipprath W G, Perlman S L, Nissenson C, Kark R A
Clin Chim Acta. 1982 May 6;121(1):1-9. doi: 10.1016/0009-8981(82)90204-2.
Widely different method have been used to assay lipoamide dehydrogenase in tissues from patients with neurological diseases. We have re-examined conditions of assay in homogenized human platelets in the light of results of optimal and inhibitory conditions others have found for the purified pig and rat liver enzymes. Optimal conditions in homogenized platelets for the forward, physiological direction were pH 8.0, 2-4 mmol/l dihydrolipoamide and 1.6-2 mmol/l NAD+ and for the reverse reaction, pH 7.3, 1.2-2 mmol/l lipoamide and 0.125-0.2 mmol/l NADH. Km values by the Lineweaver-Burke method were approximately 420 mumol/l dihydrolipoamide, 180 mumol/l NAD+, 600 mumol/l lipoamide and 27 mumol/l NADH. The optimal conditions and Km values are similar to those reported for the purified pig and rat enzymes. Assays by the present methods should therefore reflect the activity of lipoamide dehydrogenase and not the effects of substrate or cofactor inhibition nor the effects of other, interfering enzyme activities.
人们已采用大不相同的方法来测定神经疾病患者组织中的硫辛酰胺脱氢酶。鉴于其他人所发现的纯化猪和大鼠肝脏酶的最适条件及抑制条件的结果，我们重新审视了人血小板匀浆中的测定条件。血小板匀浆中正向生理反应方向的最适条件为pH 8.0、2 - 4 mmol/L二氢硫辛酰胺和1.6 - 2 mmol/L NAD⁺，逆向反应的最适条件为pH 7.3、1.2 - 2 mmol/L硫辛酰胺和0.125 - 0.2 mmol/L NADH。用林-贝氏法测得的Km值分别约为420 μmol/L二氢硫辛酰胺、180 μmol/L NAD⁺、600 μmol/L硫辛酰胺和27 μmol/L NADH。这些最适条件和Km值与已报道的纯化猪和大鼠酶的结果相似。因此，采用本方法进行测定应能反映硫辛酰胺脱氢酶的活性，而不是底物或辅因子抑制的影响，也不是其他干扰酶活性的影响。
