Lohse D L, Linhardt R J
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
J Biol Chem. 1992 Dec 5;267(34):24347-55.
Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.
肝素裂解酶I已从肝素黄杆菌中纯化出来，并已进行了部分特性鉴定（杨，V.C.，林哈特，R.J.，伯恩斯坦，H.，库尼，C.L.，和兰格，R.（1985年）《生物化学杂志》260，1849 - 1857）。尚未有关于从该生物体中纯化其他多糖裂解酶的报道。尽管这三种肝素/硫酸乙酰肝素裂解酶都被广泛使用，但除了肝素裂解酶I外，关于它们的纯度以及物理和动力学特性均无相关信息。缺乏纯的肝素裂解酶以及对最佳催化条件和底物特异性缺乏了解，阻碍了将这些酶用作试剂，将肝素和硫酸乙酰肝素特异性解聚为寡糖以进行结构和活性研究。本文描述了一种单一、可重复的方案，用于同时从肝素黄杆菌中纯化所有三种肝素裂解酶至表观均一性。肝素裂解酶I（肝素酶，EC 4.2.2.7）、肝素裂解酶II（无EC编号）和肝素裂解酶III（类肝素酶，EC 4.2.2.8）的分子量（通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳）和等电点（通过等电聚焦）分别为M(r) 42,800，pI 9.1 - 9.2、M(r) 84,100，pI 8.9 - 9.1、M(r) 70,800，pI 9.9 - 10.1。它们的氨基酸分析和肽图表明，虽然这些蛋白质是不同的基因产物，但它们密切相关。已确定了肝素裂解酶的动力学性质以及优化其活性和稳定性的条件。这些数据应能改善这些重要酶在肝素和硫酸乙酰肝素研究中的应用。
