Terashima M, Mishima K, Yamada K, Tsuchiya M, Wakutani T, Shimoyama M
Department of Biochemistry, Shimane Medical University, Izumo, Japan.
Eur J Biochem. 1992 Feb 15;204(1):305-11. doi: 10.1111/j.1432-1033.1992.tb16638.x.
We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394]. In the present study, we obtained evidence that chicken non-muscle beta/gamma-actin, skeletal muscle alpha-actin and smooth-muscle gamma-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP-ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP-ribose/mol of beta/gamma-actin, alpha-actin and gamma-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 microM with beta/gamma-actin, 35 microM with alpha-actin and 20 microM with gamma-actin. The Km values for the actin isoforms were 15 microM for beta/gamma-actin, 2.5 microM for alpha-actin and 10 microM for gamma-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluorescence intensity with N-(1-pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of beta/gamma-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.
我们报道了鸡外周多形核白细胞（异嗜性粒细胞）颗粒中精氨酸特异性ADP核糖基转移酶和受体蛋白p33的纯化及特性[三岛，K.，寺岛，M.，小原，S.，山田，K.，今井，K. & 岛山，M.（1991年）《生物化学杂志》（东京）110，388 - 394]。在本研究中，我们获得证据表明鸡非肌肉β/γ - 肌动蛋白、骨骼肌α - 肌动蛋白和平滑肌γ - 肌动蛋白被异嗜性粒细胞ADP核糖基转移酶进行了ADP核糖基化。ADP核糖掺入这些肌动蛋白的化学计量分别为每摩尔β/γ - 肌动蛋白、α - 肌动蛋白和γ - 肌动蛋白1.2摩尔、1.0摩尔和2.0摩尔ADP核糖。ADP核糖基化的最适pH值对于各自的肌动蛋白为8.5。计算得出β/γ - 肌动蛋白的NAD的Km值为30微摩尔，α - 肌动蛋白为35微摩尔，γ - 肌动蛋白为20微摩尔。肌动蛋白同工型的Km值对于β/γ - 肌动蛋白为15微摩尔，α - 肌动蛋白为2.5微摩尔，γ - 肌动蛋白为10微摩尔。如用N - （1 - 芘基）碘乙酰胺标记的肌动蛋白荧光强度增加所测定，肌动蛋白的ADP核糖基化抑制了其聚合能力。用各自的肌动蛋白同工型聚合的丝状肌动蛋白（F - 肌动蛋白）也被ADP核糖基化，尽管F - 肌动蛋白的修饰程度低于球状肌动蛋白（G - 肌动蛋白）。用皂素通透处理的鸡外周异嗜性粒细胞证明了β/γ - 肌动蛋白的原位ADP核糖基化。因此，异嗜性粒细胞中肌动蛋白的内源性ADP核糖基化可能参与吞噬作用、分泌和迁移等细胞过程。
