Taylor C, Cox A J, Kernohan J C, Cohen P
Eur J Biochem. 1975 Feb 3;51(1):105-15. doi: 10.1111/j.1432-1033.1975.tb03911.x.
Debranching enzyme was purified 150-fold from rabbit skeletal muscle by a three-step procedure which utilised ammonium sulphate precipitation, ion-exchange chromatography on DEAE-cellulose and "hydrophobic" chromatography on Sepharose-NH(CH2)4NH2. The preparation was completed within three days, and 200 mg enzyme was isolated from 1000 g muscle, which represented an overall yield of 60%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation coefficient, s20,w, was 8.1 S. The amino acid composition was determined, and the absorption coefficient, A280 1%, measured refractometrically was 17.5. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 166000 and this value was supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride (1550oo). The molecular weight of the native enzyme measured by high-speed sedimentation equilibrium was 164000, showing that the debranching enzyme is a monomeric protein at the concentrations which exist in muscle (0.7 mg/ml). The results indicate that the two different enzyme activities which are associated with debranching enzyme, 1,4-glucan-4-glycosyltransferase and amylo-1,6-glucosidase, reside on the same polypeptide chain. Protein-glycogen particles isolated from skeletal muscle showed seven major protein-staining components by polyacrylamide gel electrophoresis, one of which was identified as debranching enzyme. ,our of the other components were the alpha and beta subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase. A new titrimetric assay for debranching enzyme was developed; it was used to demonstrate that the maximum potential activity of debranching enzyme is only 5--10% that of phosphorylase at the concentrations of the two enzymes in skeletal muscle. Since the activity of debranching enzyme is unaffected by every mechanism which leads to the activation of glycogen phosphorylase and phosphorylase kinase, the evidence suggests that the hormonal control of muscle glycogenolysis by adrenalin might be confined to a stimulation of rate of degradation of the outermost branches of glycogen.
采用三步法从兔骨骼肌中纯化脱支酶，纯化倍数达150倍。该方法包括硫酸铵沉淀、DEAE - 纤维素离子交换色谱以及Sepharose - NH(CH2)4NH2上的“疏水”色谱。整个制备过程在三天内完成，从1000克肌肉中分离得到200毫克酶，总产率为60%。根据聚丙烯酰胺凝胶电泳和超速离心分析的标准，该制剂是均一的。沉降系数s20,w为8.1 S。测定了氨基酸组成，通过折射法测得的吸收系数A280 1%为17.5。在十二烷基硫酸钠存在下通过凝胶电泳测定的亚基分子量为166000，在6 M氯化胍存在下的沉降平衡也支持该值（155000）。通过高速沉降平衡测得的天然酶分子量为164000，表明在肌肉中存在的浓度（0.7毫克/毫升）下，脱支酶是一种单体蛋白。结果表明，与脱支酶相关的两种不同酶活性，即1,4 - 葡聚糖 - 4 - 糖基转移酶和淀粉 - 1,6 - 葡萄糖苷酶，存在于同一条多肽链上。从骨骼肌中分离的蛋白质 - 糖原颗粒通过聚丙烯酰胺凝胶电泳显示出七个主要的蛋白质染色成分，其中之一被鉴定为脱支酶。其他成分中的四个是磷酸化酶激酶的α和β亚基、糖原磷酸化酶和糖原合成酶。开发了一种新的脱支酶滴定分析法；用于证明在骨骼肌中两种酶的浓度下，脱支酶的最大潜在活性仅为磷酸化酶的5 - 10%。由于脱支酶的活性不受导致糖原磷酸化酶和磷酸化酶激酶激活的任何机制的影响，证据表明肾上腺素对肌肉糖原分解的激素控制可能仅限于刺激糖原最外层分支的降解速率。
