Frederiks W M, Marx F, Van Noorden C J
Laboratory of Histology and Cell Biology, University of Amsterdam, The Netherlands.
Histochem J. 1988 Nov;20(11):624-8. doi: 10.1007/BF01324081.
Creatine kinase (EC 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP. The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues. In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.
使用含聚乙烯醇的孵育介质和辅助酶在大鼠心肌中证实了肌酸激酶（EC 2.7.3.2）活性。通过使用终点测量和动力学测量的显微密度测定法定量该活性。在不存在磷酸肌酸和ADP的情况下进行对照反应。对于终点测量和动力学测量，在等吸收波长（585nm）下还原型硝基蓝四唑的吸光度对孵育时间的线性回归线都非常显著。从终点测量获得的活性低约40%。这是由于在反应结束时除去孵育介质时，组织切片中生成的甲臜反应产物损失所致。对于心肌和骨骼肌，肌酸激酶活性（测试减去对照反应）与切片厚度之间的关系均不是线性的；然而，对照反应显示两种组织的活性与切片厚度均呈线性关系。辅助酶向切片中的渗透受限可能是造成这种不成比例的原因。因此，在解释不同组织获得的定量数据时应谨慎。总之，多步酶反应可用于定量组织化学目的，前提是要考虑到反应性与切片厚度不成比例。
