Smith C E
J Histochem Cytochem. 1980 Jul;28(7):689-99. doi: 10.1177/28.7.6771324.
Enamel organs were dissected from the labial surface of unfixed and glutaraldehyde-fixed rat incisors and assayed biochemically at pH 5.0 for acid phosphomonoester hydrolase activity using cytidine 5'-monophosphate (5'-CMP), beta-glycerophosphate (beta GP), phosphorylcholine (PC), and phosphoserine (PS) as substrates. Whole homogenates from unfixed enamel organs showed substantial enzyme activity toward 5'-CMP and beta GP, but 1/4 and 1/8 less activity toward PC and PS, respectively. Perfusion fixation with 2% glutaraldehyde resulted in a net loss of 80% of the enzyme activity toward each substrate. Lineweaver-Burk plots revealed that the fixative depressed the rate of hydrolysis of substrate (decrease in Vmax) and it also lowered the affinity of enzymes for substrate (increase in KM). Hence, fixed tissue generally required two or three times as much substrate to saturate the enzymes, but less substrate was hydrolyzed, as compared to unfixed tissue. Decalcification of fixed incisors with either formic citric acid, ethylenediaminetetraacetic acid (EDTA), or ethyleneglycoltetacetic acid (EGTA) did not further alter enzyme activity in the enamel organ as determined by Lineweaver-Burk plots, However, EDTA and EGTA were found to increase the susceptibility of fixed enzymes to inhibition by lead ions. This chelator-enhanced lead inhibition was greatest following decalcification with EGTA and using PC as substrate. Similar results were obtained for liver.
从未固定和经戊二醛固定的大鼠切牙唇面解剖牙釉质器官，并在pH 5.0条件下以胞苷5'-单磷酸（5'-CMP）、β-甘油磷酸酯（βGP）、磷酸胆碱（PC）和磷酸丝氨酸（PS）为底物，对酸性磷酸单酯水解酶活性进行生化分析。未固定牙釉质器官的全匀浆对5'-CMP和βGP显示出较高的酶活性，但对PC和PS的活性分别低1/4和1/8。用2%戊二醛进行灌注固定导致对每种底物的酶活性净损失80%。Lineweaver-Burk图显示，固定剂降低了底物的水解速率（Vmax降低），同时也降低了酶对底物的亲和力（KM增加）。因此，与未固定组织相比，固定组织通常需要两到三倍的底物才能使酶饱和，但水解的底物较少。用甲酸柠檬酸、乙二胺四乙酸（EDTA）或乙二醇四乙酸（EGTA）对固定的切牙进行脱钙，根据Lineweaver-Burk图测定，牙釉质器官中的酶活性没有进一步改变。然而，发现EDTA和EGTA会增加固定酶对铅离子抑制的敏感性。在用EGTA脱钙并以PC为底物后，这种螯合剂增强的铅抑制作用最为明显。肝脏也得到了类似的结果。
