INTERACTION OF ATP ANALOGS WITH MYOSIN

肌球蛋白的两个巯基，当通过各种交联剂交联时， 试剂在活性位点捕获MgADP，将用放射性标记物标记， 通过标准品分离和鉴定含有这些硫醇的肽， 色谱和电泳技术。 如果合适的话， 将被测序并与已知的肽序列进行比较， 肌球蛋白的两个活性关键硫醇。 一种新的ATP光亲和标记物， 4-叠氮基，2-硝基苯胺乙基三磷酸，将被捕获的交联两个 随后共价结合到肌球蛋白的活性 通过光解作用。 含有（32 P）标记ATP的胰蛋白酶肽 类似物将通过凝胶色谱和电泳分离并比较 与其他人在用ATP类似物标记后分离的先前胰蛋白酶肽相比 含有与核糖环相连的对光不稳定的基团。 子单元 通过凝胶电泳确定核苷酸结合的位置。

The two thiols of myosin, which when cross-linked by a variety of cross-linking agents trap MgADP at the active site, will be tagged with radioactive labels and the peptides containing these thiols isolated and identified by standard chromatographic and electrophoretic techniques. If appropriate, the peptides will be sequenced and compared with known sequences of peptides which contain the two activity-critical thiols of myosin. A new ATP photoaffinity label, 4-azido,2-nitroanilinoethyl tri-phosphate, will be trapped by cross-linking two thiols of myosin and subsequently covalently incorporated into myosin's active site by photolysis. The tryptic peptides containing the (32P)-labeled ATP analog will be isolated by gel chromatography and electrophoresis and compared to previous tryptic peptides isolated by others after labeling with a ATP analog containing a photolabile group attached to the ribose ring. The subunit location of the nucleotide binding will be determined by gel electrophoresis.