Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer.
Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer.
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肌球蛋白亚片段的乙烯腺苷二磷酸结合位点的表征 1. 通过能量转移检测到的核苷酸两种状态之间平衡的能量。S1 和钒酸盐诱导的整体构象变化。
DOI：
10.1021/bi00428a058
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Cheung,HC
中科院分区：
文献类型：
--
作者：
Aguirre,R;Lin,SH;Gonsoulin,F;Wang,CK;Cheung,HC
材料和方法试剂和化学品。DDPM、eADP、ADP、ATP和NEM获自Sigma Chemical Co. (St.密苏里州路易斯）。这些试剂不经进一步纯化即使用。胰凝乳蛋白酶获自Worthington Diagnostic Systems（Freehold，NJ）。从Spex Industries（梅图陈，NJ）获得CoCl 2作为标准溶液。V205（Aldrich Chemical Co.，密尔沃基，WI）用于制备钒酸钠的储备溶液，如Goodno（1982）.Protein Preparations.如前所述从兔骨骼肌制备肌球蛋白（Aguirre等人，1986）通过Flamig和Cusanovich（1981）的方法。如Weeds和Taylor（1975）所述，使用新鲜制备的肌球蛋白通过胰凝乳蛋白酶消化制备SI。将DE-52柱分离的两种同工酶S1（A1）和S1（A2）合并，用乙酸铵和0.1mM DTT透析，并在0.1M蔗糖存在下冻干。根据Spudich和Watts（1971）的方法，从丙酮粉末中制备肌动蛋白。分子量为115 000，根据280 nm处的吸光度0.75 g^-cirf 1估算S1的浓度。假定肌动蛋白的单体分子量为42 000，在290 nm处的吸光度为0.63 g-Lcirf 1。
Materials and MethodsReagents and Chemicals. DDPM, eADP, ADP, ATP, and NEM were obtained from Sigma Chemical Co.(St. Louis, MO). These reagents were used without further purification. Chymotrypsin was obtained from Worthington Diagnostic Systems (Freehold, NJ). CoCl2 was obtained from Spex Industries (Metuchen, NJ) as a standard solution. V205 (Aldrich Chemical Co., Milwaukee, WI) was used to prepare a stock solution of sodium vanadate as described by Goodno (1982).Protein Preparations. Myosin was prepared from rabbit skeletal muscle as previously described (Aguirre et al., 1986) by the method of Flamig and Cusanovich (1981). Freshly prepared myosin was used to prepare SI by chymotryptic digestion as described by Weeds and Taylor (1975). Thetwo isozymes S1 (A1) and S1 (A2) isolated on a DE-52 column were pooled, dialyzed against ammonium acetateand 0.1 mM DTT, and lyophilized in the presence of 0.1 M sucrose. Actin was prepared from an acetone powder according to the method of Spudich and Watts (1971). A molecular weightof 115 000 was used to estimate the concentration of S1 from an absorbance of 0.75 g^-cirf1 at 280 nm. Actin was assumed to have a monomeric molecular weight of 42 000 and an absorbance of 0.63 g-Lcirf1 at 290 nm.