van den Heuvel L P, van den Born J, van de Velden T J, Veerkamp J H, Monnens L A, Schroder C H, Berden J H
Department of Biochemistry, University of Nijmegen, The Netherlands.
Biochem J. 1989 Dec 1;264(2):457-65. doi: 10.1042/bj2640457.
Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.
硫酸乙酰肝素蛋白聚糖通过胍提取法从人肾小球基底膜中溶解出来，并通过离子交换色谱法和凝胶过滤法进行纯化。蛋白聚糖的产量约为2毫克/克基底膜。该糖缀合物的表观分子量为200 - 400 kDa，约由75%的蛋白质和25%的硫酸乙酰肝素组成。其氨基酸组成的特点是甘氨酸、脯氨酸、丙氨酸和谷氨酸含量较高。用三氟甲磺酸水解产生了160 kDa和110 kDa的核心蛋白（以及90 kDa和60 kDa的少量条带）。用碱性硼氢化钠处理该蛋白聚糖释放出平均分子量为18 kDa的硫酸乙酰肝素链。这些链经亚硝酸氧化产生约5 kDa的寡糖，而肝素酶消化导致更完全的降解。数据表明N - 硫酸基团在糖胺聚糖链的周边区域聚集。针对完整蛋白聚糖产生的多克隆抗血清显示出对核心蛋白的反应性。在对人和各种哺乳动物物种的冰冻肾脏切片进行的免疫组织化学研究中，它以强烈的线性方式染色所有基底膜。
