Frederiks W M, Marx F, van Noorden C J
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
Hepatology. 1991 Oct;14(4 Pt 1):634-9. doi: 10.1016/0270-9139(91)90050-6.
A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
已开发出一种定量组织化学方法，用于在大鼠肝脏中显示磷酸果糖激酶的活性，该酶被认为是糖酵解的限速酶之一。该方法基于以四唑盐作为最终电子受体的还原反应，以及使用外源性或内源性辅助酶醛缩酶、磷酸丙糖异构酶和甘油醛-3-磷酸脱氢酶的多步反应。当孵育介质含有17%（重量/体积）聚乙烯醇、100 mmol/L 马来酸三羟甲基氨基甲烷缓冲液（pH 8.4）、20 mmol/L 果糖-6-磷酸、2 mmol/L ATP、2 mmol/L 氯化镁、5.9 mmol/L NAD+、0.47 mmol/L 1-甲氧基吩嗪硫酸甲酯、5 mmol/L 叠氮化钠和5 mmol/L 硝基蓝四氮唑时，在大鼠肝脏的未固定低温恒温器切片中发现了最高活性。在大鼠肝脏中显示最大活性时，添加辅助酶并非必需。当在25 mmol/L 磷酸烯醇丙酮酸（磷酸果糖激酶的竞争性抑制剂）存在下进行孵育时，未出现任何特异性（试验减去对照）反应，从而证明了反应的特异性。细胞光度分析表明，形成的特异性反应产物的量与孵育时间和切片厚度之间存在线性关系。正常喂养或禁食24小时的大鼠肝脏的门静脉周围和中央静脉周围区域中，果糖-6-磷酸的Km值和Vmax值没有显著差异。肝脏腺泡中磷酸果糖激酶活性的均匀分布与使用从这两个不同区域分离的肝细胞的生化研究结果一致，表明这些细胞含有相似量的酶活性。（摘要截短于250字）
