STATE OF OXIDATION-REDUCTION AND STATE OF BINDING IN CYTOSOLIC NADH-SYSTEM AS DISCLOSED BY EQUILIBRATION WITH EXTRACELLULAR LACTATE PYRUVATE IN HEMOGLOBIN-FREE PERFUSED RAT-LIVER
STATE OF OXIDATION-REDUCTION AND STATE OF BINDING IN CYTOSOLIC NADH-SYSTEM AS DISCLOSED BY EQUILIBRATION WITH EXTRACELLULAR LACTATE PYRUVATE IN HEMOGLOBIN-FREE PERFUSED RAT-LIVER
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DOI：
10.1111/j.1432-1033.1972.tb01840.x
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发表时间：
1972-01-01
期刊：
影响因子：
--
通讯作者：
KLEIN, F
中科院分区：
文献类型：
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作者：
BUCHER, T;BRAUSER, B;KLEIN, F
1细胞外[乳酸]/[丙酮酸]比率用作平衡细胞溶质NADH/NAD+系统的手段。这是通过在开放系统中逐步改变灌注液中的[乳酸]/[丙酮酸]来实现的。使用单波长或双波长方法同时记录NADH特定波长处的荧光和吸光度。借助于仪器计算机获得荧光激发谱和吸收差谱。通过输注内标校准读数，从而估计NADH特异性荧光量子产率的增强。2基于质量作用定律的数学处理允许外推至完全还原和完全氧化的胞质结合NADH。此外，获得了结合的NADH/NAD+和乳酸/丙酮酸的表观平衡常数（0.022至0.071），对应于结合的NADH的中点电位为-250至-265 mV。因此，与胞质结合位点相关的解离常数KNAD+/KNADH的比率为200-700。细胞溶质NADH结合能力估计约为80 nmol/g肝湿重。在灌流液中[乳酸]/[丙酮酸]为10的常氧条件下，细胞溶质结合NADH/游离NADH的含量比为100。3通过测量与乳酸输入耦合的净H+转运以及测定细胞内依赖性指示代谢物对α-甘油磷酸/二羟丙酮磷酸，表明系统中存在近平衡条件。讨论了有关乳酸/丙酮酸组织含量作为细胞溶质NADH/NAD+潜力指标的有效性的问题。
1The extracellular ratio [lactate]/[pyruvate] is used as a means to poise the cytosolic NADH/NAD+system. This is achieved by stepwise changes of [lactate]/[pyruvate] in the perfusate in an open system. Fluorescence and absorbance at NADH‐specific wavelengths is recorded simultaneously with single or dual wavelength methods. Fluorescence excitation and absorbance difference spectra are obtained with the help of an instrument computer. The readout is calibrated by infusion of internal standards, permitting an estimation of the enhancement of quantum yield of NADH‐specific fluorescence.2Mathematical treatment on the basis of the mass action law permits extrapolation to fully reduced and fully oxidized cytosolic bound NADH. In addition, the apparent equilibrium constant for bound NADH/NAD+and lactate/pyruvate is obtained (0.022 to 0.071), corresponding to a midpoint potential of bound NADH of – 250 to – 265 mV. Accordingly, the ratio of dissociation constants,KNAD+/KNADH, relevant for the cytosolic binding sites, is 200–700. Cytosolic NADH‐binding capacity is estimated to be around 80 nmol/g liver wet weight. Under normoxic conditions at [lactate]/[pyruvate] of 10 in the perfusate, the ratio of contents of cytosolic bound NADH/free NADH is 100.3The existence of near‐equilibrium conditions in the system is suggested by measurement of net H+transport coupled to lactate import, as well as by determination of an intracellular dependent indicator metabolite couple, α‐glycerophosphate/dihydroxyacetone phosphate. Problems regarding the validity of tissue contents of lactate/pyruvate as indicator for the cytosolic NADH/NAD+potential are discussed.