Frederiks W M, Marx F, Van Noorden C J
Laboratory of Histology and Cell Biology, Academic Medical Centre, Amsterdam, The Netherlands.
Histochem J. 1987 Oct-Nov;19(10-11):529-32. doi: 10.1007/BF01687359.
Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread on object glasses before mounting the sections to be incubated. In this way, the auxiliary enzymes were interposed between glass slide and section thus preventing loss of formazan generated within the sections. Creatine kinase activity was found to be localized in finely dispersed form along the myofibrils and as large granules in the sarcoplasm of myocardium and skeletal muscle. The formazan produced specifically by creatine kinase (test minus control), as measured cytophotometrically at 585 nm, was completely inhibited by 2 mM 2,4-dinitrofluorobenzene, a specific inhibitor of creatine kinase activity. The control reaction was unaffected by the inhibitor. The results obtained with the present method are similar to results obtained with the far more complicated semipermeable membrane technique. The introduction of auxiliary enzymes in the polyvinyl alcohol method enables the development of histochemical methods for many enzymes by linking the reactions to a dehydrogenase reaction.
通过一种基于将低温恒温器切片与含聚乙烯醇的培养基孵育并使用辅助酶的方法，已在大鼠的心肌和骨骼肌中证明了肌酸激酶活性（EC 2.7.3.2.）。在安装待孵育的切片之前，将己糖激酶和葡萄糖-6-磷酸脱氢酶铺在载玻片上。通过这种方式，辅助酶置于载玻片和切片之间，从而防止切片内产生的甲臜损失。发现肌酸激酶活性以细分散的形式沿着肌原纤维分布，并以大颗粒形式存在于心肌和骨骼肌的肌浆中。通过在585nm处进行细胞光度测定，肌酸激酶特异性产生的甲臜（试验减去对照）被2mM 2,4-二硝基氟苯完全抑制，2,4-二硝基氟苯是肌酸激酶活性的特异性抑制剂。对照反应不受该抑制剂影响。用本方法获得的结果与用复杂得多的半透膜技术获得的结果相似。在聚乙烯醇方法中引入辅助酶能够通过将反应与脱氢酶反应联系起来，开发许多酶的组织化学方法。
