声波刺激对铁皮石斛过氧化物酶同工酶基因表达的影响

从基因表达水平探索了植物适应物理应力刺激时可能作出的反应机制。以铁皮石斛组培苗为实验材料,以特定的声波(声强100dB、频率1000Hz)刺激为应力源,检测比较了声波刺激对铁皮石斛过氧化物酶(POD)同工酶酶谱及各酶带百分含量的影响;通过设计引物扩增出铁皮石斛POD基因片段,以此为探针,利用Northern点杂交技术检测分析了声波刺激对铁皮石斛POD基因表达的影响。结果显示,声波刺激没有引起铁皮石斛产生新的酶带,但促进了其POD同工酶酶量的提高;声波刺激对铁皮石斛POD基因表达的影响与其同工酶酶谱及其总RNA量的变化基本一致。表明声波刺激对植物POD同工酶基因表达有激活作用,可能引起植物某些基因转录和表达水平的变化。     

Characterization of an Isozyme of β-Glucosidase from Sweet Almond

A sweet almond β-glucosidase (EC 3.2.1.21) isozyme was purified from commercial crude product. The process of purification consisted of a Protein-Pak Q anion exchange chromatography following by a Superdex 75 HR gel filtration separation. The purified enzyme is a monomeric glycoprotein with molecular weight of 58 kDa and pI=4.55 which is distinguished from reported isozymes. The enzyme has apH optimum in the range of 5.2-5.6 when p-nitrophenyl-β-D-glycopyranosides are used as substrate and is stable up to 50 °C at that pH range. The purified protein also exhibits profound β-galactosidase and σ-L-arabinosidase activity. The study of substrate specificity revealed that lacking of hydroxymethyl group at C-5 of glycosides resulted in higher affinity for substrate binding to enzyme, whereas the chemical step of hydrolysis (k_cst) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-β-D-glycopyranosides with apparent pK₁ and pK₂ values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by δ-gluconolactone (K_i = 160 μM) and 4-phenylimidazole (K_i = 17.8 μM) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-β-D-glucosamine to the enzyme (K_l = 52 mM) was 6 times stronger than that of glucose and its epimers.