Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, Taniguchi N
Department of Pediatric Dentistry, Hokkaido University School of Dentistry, Sapporo, Japan.
J Biol Chem. 1987 Dec 15;262(35):16969-72.
The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.
铜锌超氧化物歧化酶的非酶糖基化（糖基化）导致该酶逐渐失活（荒井，K.；饭冢，S.；多田，Y.；及川，K.；谷口，N.（1987年）《生物化学与生物物理学学报》924卷，292 - 296页）。从人红细胞中纯化得到的超氧化物歧化酶包含糖基化和非糖基化两种形式。通过硼酸亲和色谱法分离出非糖基化的铜锌超氧化物歧化酶。在体外将非糖基化的超氧化物歧化酶与D - [6 - ³H]葡萄糖一起孵育，导致酶蛋白中放射性逐渐积累，席夫碱加合物被硼氢化钠捕获。通过对胰蛋白酶处理后的肽段进行反相高效液相色谱分析，并经氨基酸分析确定了超氧化物歧化酶的糖基化位点。发现赖氨酸残基，即Lys3、Lys9、Lys30、Lys36、Lys122和Lys128发生了糖基化。其中三个糖基化位点位于Lys - Gly中，两个位于Lys - Ala中，一个位于Lys - Val中。超氧化物歧化酶糖基化时的失活主要归因于Lys122和Lys128的糖基化，这两个位点被认为位于活性位点配体环中。其余五个位点，如Lys - Glu、Lys - Asp、Lys - His和Lys - Thr，在形成席夫碱或阿马多里加合物方面相对不活跃。
