摘要
胆汁经Sephadex G-25分子筛层析和DEAE-纤维素吸附处理,除去其中直接胆红素及胆汁酸盐,然后对其中的β-葡萄糖醛酸苷酶(GUSB)的性质进行了详细研究:该酶的最适温度为56℃;最适pH为4.5;以4mu Gr为底物测得Km为0.68mmol/L;直接胆红素是其竞争性抑制剂,其k_i=2.04×10^(-3)mmol/L;PI_1=6.0—6.2,PI_2=7.2—7.4,MW=280kD。根据竞争性抑制的米氏方程式设计建立了一个在直接胆红素并存的条件下,定量测定胆汁GUSB的计算荧光法,准确性为97.97±1.79％,重复性CV=1.2％,测定时间大大缩短。利用本法在pH4.5和7.0条件下测定了20例胆红素胆结石和17例胆固醇胆结石患者的胆汁GUSB的真实活性。结果指出:不论在pH4.5或7.0条件下,前组活性均显著高于后组(P<0.01)。证明胆汁中高GUSB活性与胆红素胆结石的形成有密切关系。
In order to study the kinetics of biliary β-glucuronidase, direct bilirubin and bile salt were removed from bile by sephadex G-25 filtration and DEAE-cellulose absorption. The PI1,PI2 and MW of biliary β-glucuronidase are 6.0-6.2,7.2-7.4 and 280kD, respectively. Studies of the enzyme kinetics in a system using 4-nauGr as substrate revealed that the direct bilirubin acted as a competitive inhibitor for the enzyme with Ki= 2.04×10-3 mmol/L. Based on kinetic properties and equation for competitive inhibition, a fluoremetry for measuring the true activity of biliary β-glucuronidase (Vmax),in the presence of direct bilirubin, was established.Biles from 20 patients with bilirubin pigment gallstones,and biles from 17 cholesterol gallstones patients were assayed by this method. The results showed that the GUSB activity in biles of the former group of patients was much higher than that of the later. It is likely that the increased activity of biliary β-glucuronidase is the cause of bilirubin pigment gallstones.
关键词
胆汁
GUSB
荧光法
Bile, β-Glucuronidase, Fluoremetry, Direct Bilirubin, Gallstone
