植物性食品对尿激酶活性抑制的测定实验

目的 了解水果、蔬菜和茶叶等植物性食物对于促进细胞恶性转化的尿激酶活性抑制效果。方法 利用谷氨酸 甘氨酸 精氨酸肽为底物的尿激酶活性测定原理 ,分别用 2 5种水果、37种蔬菜的榨汁和 5 1种茶叶的水浸提液与尿激酶反应 ,再加入光活性尿激酶反应底物反应后用酶标仪测定剩余酶活性。结果 2 5种水果、37种蔬菜的测试样中有近一半的尿激酶活性抑制率为 2 0 %以上 ,其中 ,柠檬、猕猴桃、豌豆、菠菜、南瓜等的抑制率超过 80 % ,大蒜、萝卜、蜂斗菜、旱芹、李子、菠萝、葡萄等的抑制率为 5 0 %～ 79% ;5 1个茶叶样的平均抑制率为 83 2 % ,其中 2 8个茶叶样的抑制率 >90 % ,10个茶叶样为 80 %～ 89% ,10个茶样本为 70 %～ 79% ,绿茶类的平均抑制率比红茶类的抑制率高出10 %～ 15 % ;蔬菜的加工方式对抑制效果有影响 ,有刺激味的蔬菜以有机溶剂浸提效果好于水浸提效果 ,无刺激味的则以水浸提的效果为好 ;多数新鲜蔬菜经冷藏和热处理后的效果为减弱。结论 日常生活中的植物性食物有抑制尿激酶活性的作用。

抗人尿激酶单克隆抗体制备和鉴定

用人高分子量尿激酶(HMW-UK)为抗原,通过杂交瘤技术获得了两株阳性杂交瘤细胞(2B8、3A2)。采用葡萄球菌 A 蛋白(SPA)亲和层析或 Water's650快速蛋白分离系统纯化抗 UK 单克隆抗体(McAb).3A2McAb 为 IgG 1亚类,特异地识别 HMW-UK 和低分子量 LMW-UK,抑制 UK 活性,表明3A2McAb 所作用的位点为 UK 的 B 链,即活性中心。EIA 测定3A2McAb 与 UK 的亲和常数为8.43×10~8M^(-1)。2B_8McAb 亦为IgG_(?)亚类,特异地识别 HMW-UK 及其氨基端1～135氨基酸片段(ATF),不抑制活性,EIA 测定2B_8McAb 与UK 的亲和常数为2.8×10~9M^(-1)。2B_8McAb 可用于导向溶栓的研究以及 UK 与其受体间反应的研究。

共表达折叠调节因子对尿激酶功能性表达的影响

目的研究共表达折叠调节因子对真核蛋白尿激酶(urokinase,UK)功能性表达的影响,改善大肠杆菌中UK表达产物的活性。方法通过分离分子伴侣DnaK、DnaJ、GroEL-S及二硫键异构酶DsbA、DsbC的基因,构建折叠调节因子表达载体,与UK表达载体双质粒共转化同一个菌株,观察共表达与单独表达时UK的表达产物活性变化。结果共表达分子伴侣DnaK、GroEL-S和二硫键异构酶DsbC、DsbA对UK表达产物的活性有明显的提高,最高的是GroEL-S。结论在大肠杆菌中共表达分子伴侣能协助尿激酶蛋白的正确折叠,提高尿激酶表达产物的活性。

Kinetic method for enzymatic analysis by predicting background with uricase reaction as model

Objective：To investigate the reliability for kinetic assay of substance with background predicted by the integrated method using uricase reaction as model. Methods： Absorbance before uricase action （Δ0） was estimated by extrapolation with given lag time of steady-state reaction. With Km fixed at 12.5μmol/L, background absorbance （Δb） was predicted by nonlinearly fitting integrated Michaelis-Menten equation to Candida utilis uricase reaction curve. Uric acid in reaction solution was determined by the difference （ΔA） between Δ0 and Δb. Results .Ab usually showed deviation 〈3% from direct assay with residual substrate done fifth of initial substrate for analysis. ΔA showed CV 〈5% with resistance to common interferences except xanthine, and it linearly responded to uric acid with slope consistent to the absorptivity of uric acid. The lower limit was 2.0 μmol/L and upper limit reached 30 μmol/L in reaction solution with data monitored within 8 min reaction at 0. 015 U/ml uricase. Preliminary application to serum and urine gave better precision than the direct equilibrium method without the removal of proteins before analysis. Conclusion .This kinetic method with background predicted by the integrated method was reliable for enzymatic analysis, and it showed resistance to common interferences and enhanced efficiency at much lower cost.

Effects of vanadate on the activities of mice glucokinase and hexokinase

This study aimed at acquiring knowledge on the hypoglycemic mechanisms of sodium metavanadate (SMV) showed that the liver glucokinase and muscle hexokinase activities increased rapidly after oral SMV was given, and that the blood glucose level was correlated closely with the activities of the two enzymes but not with the insulin level; which indicated that SMV could improve the altered glucose phosphorylation in diabetic mice independently of stimulating insulin secretion. This was probably one of the mechanisms of hypoglycemic effects of SMV.