Studies on two high-affinity enolase inhibitors. Reaction with enolases.
Studies on two high-affinity enolase inhibitors. Reaction with enolases.
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两种高亲和力烯醇化酶抑制剂的研究。
DOI：
10.1021/bi00801a010
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发表时间：
1971
期刊：
影响因子：
2.9
通讯作者：
F. Wold
中科院分区：
文献类型：
--
作者：
T. Spring;F. Wold
材料和方法抑制剂。d-TSP和AEP按照试验前所述制备（Springand Wold，1971 b）。纯化后，将TSP冷冻储存在Dowex 1色谱柱上，并在使用前立即解冻。AEP由TSP在1 M NH 4Cl（pH 9.5）中制备，并且未经纯化而冷冻。因此，AEP储备溶液含有一些污染性TSP，但TSP不干扰这些实验。酶和酶测定。丙酮酸激酶（兔骨骼肌，I型）购自Sigma Chemical Co.，并通过Pon和Bondar（1967）的方法使用三环己基烯醇丙酮酸磷酸铵（Sigma Chemical Co.）和NazADP（加州生物化学公司）通过Westhead和麦克兰（1964）的方法制备的酵母烯醇化酶A是Paul Hargrave博士的礼物。用Win-stead和Wold（1966）的方法制备结晶兔肌肉烯醇化酶。Coho salmonenolase是1967年通过Ruth等人的方法制备的结晶样品。（1970年）。E.用Spring和Wold（1971 a）的方法制备大肠杆菌烯醇化酶。所有的烯醇化酶样品具有高比活性，并通过圆盘凝胶电泳是均匀的。通过瓦尔堡和Christian（1942）的分光光度法进行烯醇酶测定，使用三环己基甘油酸铵2-磷酸（d-G2-P）作为底物（由钡盐（Sigma ChemicalCo.）Winstead和Wold（1966）的方法。测定缓冲液含有0.2mKCl、10-3 mMgSO 4、10-6 mEDTA和0. 05 mTris-HCl（pH8.0）（E.大肠杆菌和酵母烯醇化酶）或0.05M咪唑-HCl（pH 7.0）（兔和coho肌肉烯醇化酶）。在TSP或AEP作为抑制剂的试验中，在通过加入底物引发酶促反应之前，将酶与抑制剂在试验系统中预孵育2 min。
Materials and MethodsInhibitors. d-TSP and AEP were prepared as described pre-viously (Springand Wold, 1971b). TSP was stored frozen after purification on a Dowex 1 column and was thawed immediately prior to use. AEP was prepared in 1 m NH4C1 (pH 9.5) from TSP and frozenwithout purification. The AEP stock solution thus contained some contaminating TSP, but the TSP but did not interfere with these experiments. Enzymes and Enzyme Assays. Pyruvate kinase (rabbit skele-tal muscle, type I) was purchased from Sigma Chemical Co. and assayed by the method of Pon and Bondar (1967) using tricyclohexylammonium enolpyruvate phosphate(Sigma Chemical Co.) and NazADP (California Biochemical Corp.) as substrates.Yeast enolase A, prepared by the method of Westhead and McLain (1964), was a gift of Dr. Paul Hargrave. Crystalline rabbit muscle enolase was prepared by the method of Win-stead and Wold (1966). Coho salmonenolase was a crystalline sample prepared in 1967 by the method of Ruth et al.(1970). E. coli enolase was prepared by the method of Spring and Wold (1971a). All enolase samples were of high specific activity and were homogeneous by disc gel electrophoresis. Enolase assays were performed by the spectrophotometric method of Warburg and Christian (1942), using tricyclohexylammonium glycerate 2-phosphate (d-G2-P) as substrate (prepared from the barium salt (Sigma ChemicalCo.) by the method of Winstead and Wold (1966)). The assay buffer con-tained 0.2 m KC1, 10-3 m MgS04, 10-6 m EDTA, and either 0.05 m Tris-HCl (pH 8.0)(E. coli and yeast enolase) or 0.05 m imidazole-HCl (pH 7.0)(rabbit and coho muscle enolase). In the assays for TSP or AEP as inhibitors, the enzyme was preincubated with the inhibitor in the assay system for 2 min prior to initiation of the enzymatic reaction by the addition of substrate.