Blanco C E, Sieck G C, Edgerton V R
Department of Anatomy, University of California, Los Angeles 90024.
Histochem J. 1988 Apr;20(4):230-43. doi: 10.1007/BF01747468.
A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., 'nothing dehydrogenase' activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and 'nothing dehydrogenase') when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.
开发了一种组织化学技术，用于定量测定肌肉横切片中的琥珀酸脱氢酶（SDH）活性，使用1-甲氧基吩嗪硫酸甲酯（mPMS）作为外源电子载体，并使用叠氮化物作为细胞色素氧化酶的抑制剂。确定了SDH反应孵育培养基的最佳组成。将该组织化学方法与使用吩嗪硫酸甲酯（PMS）代替mPMS以及使用氰化物代替叠氮化物的方法进行了比较。用mPMS和叠氮化物替代导致硝基蓝四唑（NBT；反应指示剂）的非特异性还原，即“无脱氢酶”活性大幅降低。在反应介质中使用mPMS和叠氮化物时，在酶促反应期间NBT甲臜的产生至少9分钟呈线性。相比之下，使用PMS和氰化物时，在反应（SDH和“无脱氢酶”）的初始阶段NBT的还原是非线性的。同时使用mPMS和叠氮化物还消除了使用PMS和氰化物时出现的NBT单甲臜的产生。该方法被证明符合为组织化学反应定量制定的各种标准。
