Klemperer N S, Pickart C M
Department of Biochemistry, State University of New York, Buffalo 14214.
J Biol Chem. 1989 Nov 15;264(32):19245-52.
Eukaryotic cells possess a multienzyme ATP-dependent proteolytic pathway in which the small, highly conserved protein ubiquitin (Ub) acts as a cofactor. In this pathway, formation of a covalent Ub-substrate conjugate precedes ATP-dependent degradation of the substrate. Inorganic arsenite (AsO2-) inhibited Ub-dependent protein degradation in a concentration-dependent fashion, both in intact rabbit reticulocytes and in a reticulocyte lysate (fraction II). Concentrations of arsenite causing half-maximal inhibition in fraction II varied with the substrate, ranging from 0.025 mM (bovine alpha-lactalbumin) to 3.3 mM (reduced/carboxymethylated bovine serum albumin). Inhibition was rapidly reversed upon addition of dithiothreitol. Arsenite inhibited the Ub-dependent proteolytic pathway at one or both of two steps, depending on the substrate. 1) Proteins with acidic amino termini must be amino terminally arginylated, in a tRNA-dependent reaction, prior to becoming conjugated to Ub (Ferber, S., and Ciechanover, A. (1987) Nature 326, 808-811). Arsenite inhibited substrate arginylation, and therefore also inhibited Ub conjugation. The inhibited species appeared to be arginyl aminoacyl-tRNA transferase, since arsenite was without strong effect on the rate or extent of arginyl-tRNA formation in fraction II, yet almost completely inhibited arginine transfer from arginyl-tRNA to reduced/carboxymethylated bovine serum albumin. 2) Arsenite also inhibited Ub-substrate conjugate turnover, as shown in pulse-chase experiments. For a given substrate, degradative (protease-dependent) and Ub regenerative (isopeptidase-dependent) components of conjugate turnover were similarly inhibited by arsenite. The potency of this inhibition varied for different substrates. Monosubstituted trivalent arsenicals such as arsenite typically interact with sites containing vicinal sulfhydryl groups. Inhibition by arsenite of two steps in the Ub-dependent proteolytic pathway suggests that the relevant pathway components could possess this kind of structural/catalytic feature.
真核细胞拥有一种多酶ATP依赖的蛋白水解途径，其中小的、高度保守的蛋白质泛素（Ub）作为辅因子发挥作用。在该途径中，共价Ub-底物缀合物的形成先于底物的ATP依赖降解。无机亚砷酸盐（AsO2-）在完整的兔网织红细胞和网织红细胞裂解物（组分II）中均以浓度依赖的方式抑制Ub依赖的蛋白质降解。在组分II中引起半数最大抑制的亚砷酸盐浓度因底物而异，范围从0.025 mM（牛α-乳白蛋白）到3.3 mM（还原/羧甲基化牛血清白蛋白）。加入二硫苏糖醇后抑制作用迅速逆转。根据底物的不同，亚砷酸盐在两个步骤中的一个或两个步骤抑制Ub依赖的蛋白水解途径。1）具有酸性氨基末端的蛋白质在与Ub缀合之前，必须在依赖tRNA的反应中在氨基末端进行精氨酸化（Ferber，S.和Ciechanover，A.（1987年）《自然》326，808 - 811）。亚砷酸盐抑制底物精氨酸化，因此也抑制Ub缀合。被抑制的物质似乎是精氨酰氨酰-tRNA转移酶，因为亚砷酸盐对组分II中精氨酰-tRNA形成的速率或程度没有强烈影响，但几乎完全抑制了精氨酸从精氨酰-tRNA转移到还原/羧甲基化牛血清白蛋白。2）如脉冲追踪实验所示，亚砷酸盐还抑制Ub-底物缀合物的周转。对于给定的底物，缀合物周转的降解（蛋白酶依赖）和Ub再生（异肽酶依赖）成分同样受到亚砷酸盐的抑制。这种抑制的效力因不同底物而异。单取代三价砷化合物如亚砷酸盐通常与含有邻位巯基的位点相互作用。亚砷酸盐对Ub依赖的蛋白水解途径中两个步骤的抑制表明，相关的途径成分可能具有这种结构/催化特征。
