Watanabe Yumiko, Makino Yasushi, Omichi Kaoru
Department of Environmental Sciences, Faculty of Science, Osaka Women's University, Sakai, Osaka 590-0035, Japan.
Anal Biochem. 2005 May 15;340(2):279-86. doi: 10.1016/j.ab.2005.02.016.
Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (B3), Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B4), Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5), Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B6), Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B7), and Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.
糖原脱支酶（GDE）与糖原磷酸化酶协同作用降解糖原。GDE具有两个不同的活性位点，分别用于麦芽寡糖转移酶和淀粉-1,6-葡萄糖苷酶活性。糖原的磷酸化酶极限糊精通过这两种活性的协同作用进行脱支。利用肺炎克雷伯菌支链淀粉酶的合成作用，以吡啶胺化麦芽八糖（PA-麦芽八糖）和麦芽四糖为原料制备了荧光分支糊精作为GDE的底物。它们的结构如下：Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA（B3）、Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA（B4）、Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA（B5）、Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA（B6）、Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA（B7）和Glcα1-4Glcα1-4Glcα1-4Glcα1-6Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA（B8）。将这些糊精与猪骨骼肌GDE一起孵育。在B8的消化产物中未发现荧光产物。B3、B4和B5的荧光产物仅为PA-麦芽八糖。PA-麦芽八糖、PA-麦芽十一糖和6(7)-O-α-葡萄糖基-PA-麦芽八糖来自B7。PA-麦芽八糖和6(6)-O-α-葡萄糖基-PA-麦芽八糖来自B6。这些结果表明，麦芽寡糖转移酶通过水解或分子内转糖基作用从麦芽四糖分支中去除麦芽三糖残基，以暴露6-O-α-葡萄糖基残基，然后淀粉-1,6-葡萄糖苷酶迅速水解产物的α-1,6-糖苷键。可能，来自B7和B6的6-O-α-葡萄糖基-PA-麦芽八糖比来自B3、B4和B5的6-O-α-葡萄糖基-PA-麦芽八糖对淀粉-1,6-葡萄糖苷酶的敏感性更低。考虑到这一点，B3、B4和B5是用于GDE测定的合适底物。
