用S-（对叠氮苯甲酰）硫代肉碱对肉碱乙酰转移酶进行光亲和标记。
Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine.
作者信息
Mauro J M, Lewis R V, Barden R E
出版信息
Biochem J. 1986 Jul 15;237(2):533-40. doi: 10.1042/bj2370533.
A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.
合成了一种光不稳定试剂对叠氮苯甲酰-DL-硫代肉碱，并将其作为鸽胸肉中肉碱乙酰转移酶（EC 2.3.1.7）的光亲和标记物进行测试。对叠氮苯甲酰-DL-硫代肉碱是这种乙酰转移酶的活性位点导向试剂，因为它是肉碱的竞争性抑制剂（Ki为10 microM）。对叠氮苯甲酰-DL-硫代肉碱与酶的混合物经紫外线照射会产生不可逆抑制作用。乙酰-DL-肉碱可保护该酶免受光活化的对叠氮苯甲酰-DL-硫代肉碱的抑制。在存在30 mM 2-巯基乙醇作为清除剂的情况下，活性丧失与试剂的光掺入之间的关系表明，每分子受抑制的酶掺入一分子试剂。然而，用对叠氮苯甲酰[14C]硫代肉碱标记的酶的肽图表明，（可能的60 - 65个胰蛋白酶肽中）有几个（约六个）被修饰。5 mM乙酰-DL-肉碱的存在显著降低了试剂在每个标记的胰蛋白酶肽中的掺入。
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