Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates.
Ultraviolet photoinactivation of galactosyltransferase. Protection by substrates.
复制标题
半乳糖基转移酶的紫外线光灭活。
作者：
D. Clymer;C. Geren;K. Ebner
半乳糖转移酶在紫外线照射下不可逆失活，失活速率遵循一级动力学。在各种底物组合的存在下，观察到对失活的显著保护。udp半乳糖和Mn2+一起提供了最大的保护。氨基酸分析表明，在紫外线光失活过程中，每mol半乳糖转移酶损失1 mol色氨酸。通过光谱差异和2-羟基-5-硝基苄基溴的失活以及Mn2+和udp半乳糖对该试剂的保护，进一步证明了该试剂是必需的trypotphan。udp半乳糖和Mn2+的保护作用大于单独使用udp半乳糖。由于Mn2+本身不提供保护，这表明半乳糖转移酶-Mn2+- udp半乳糖复合物的形成引起了构象变化，这是观察到的必需色氨酸残基保护的原因。
Galactosyltransferase was irreversibly inactivated upon exposure to ultraviolet light and the rate of inactivation followed apparent first-order kinetics. Significant protection against inactivation was observed in the presence of various combinations of substrates. UDPgalactose and Mn2+ together gave the most protection. Amino acid analyses revealed the loss of 1 mol of tryptophan per mol of galactosyltransferase upon ultraviolet photoinactivation. Further evidence for an essential trypotphan was provided by difference spectra and by inactivation with 2-hydroxy-5-nitrobenzyl bromide and protection against this reagent by Mn2+ and UDPgalactose. The protection by UDPgalactose and Mn2+ was greater than that provided by UDPgalactose alone. Since Mn2+ provided no protection by itself, this suggested that the formation of the galactosyltransferase-Mn2+-UDPgalactose complex caused a conformational change which was responsible for the observed protection of the essential tryptophanyl residue.