Okayama E, Oguri K, Kondo T, Okayama M
Clinical Research Institute, National Nagoya Hospital, Japan.
Blood. 1988 Aug;72(2):745-55.
The authors' previous studies showed that the glycosaminoglycans present in rabbit bone marrow were composed of chondroitin 6-sulfate (79%) and hyaluronic acid (16%). Immunohistochemically the chondroitin 6-sulfate was demonstrated to be in bone marrow matrix constructing hematopoietic microenvironment. In this study the authors isolated and characterized these glycosaminoglycans in their macromolecular form (i.e., proteoglycans). Bone marrow of 3-month-old rabbits was defatted with organic solvents containing proteinase inhibitors at -20 degrees C, and proteoglycans were extracted from the defatted tissue with 4 mol/L guanidine HCl containing the proteinase inhibitors. After extensive dialysis of the extract against 7 mol/L urea, more than 90% of hexuronate-containing materials was recovered in the urea-soluble fraction. The proteoglycans were purified from the urea-soluble fraction by diethyl aminoethyl (DEAE)-Sephacel chromatography, CsCl density-gradient centrifugation, and Bio-Gel A-5m gel filtration, then were rechromatographed on DEAE-Sephacel and on Bio-Gel A-5m. The proteoglycans were separated into three molecular species with different mol wts that were assessed to be 46,000, 16,000, and 8,300 by sedimentation-equilibrium centrifugation. Amino acid analyses of these proteoglycans revealed that serine and glycine accounted for approximately 60% of the total amino acids common to the three proteoglycans. The glycosaminoglycan side chains of these proteoglycans were converted stoichiometrically into unsaturated 6-sulfated disaccharide by digestion with chondroitinase AC-II, indicating that they were fully sulfated chondroitin 6-sulfate. Their apparent mol wts were estimated by gel filtration on Bio-Gel A-0.5m to be 10,900, 14,400, and 7,700. Computation of these results, taken together with their biochemical composition, revealed that the largest proteoglycan, PG-II, consisted of four chains of the chondroitin 6-sulfate and the core peptide with mol wt of approximately 4,000. The smaller two proteoglycan subpopulations, PG-I and PG-III, consisted of a single glycosaminoglycan chain linked to a small peptide with mol wts of approximately 500 and 1,000, respectively.
作者之前的研究表明，兔骨髓中存在的糖胺聚糖由6-硫酸软骨素（79%）和透明质酸（16%）组成。免疫组织化学显示，6-硫酸软骨素存在于构建造血微环境的骨髓基质中。在本研究中，作者以大分子形式（即蛋白聚糖）分离并鉴定了这些糖胺聚糖。用含蛋白酶抑制剂的有机溶剂在-20℃下对3月龄兔的骨髓进行脱脂，然后用含蛋白酶抑制剂的4mol/L盐酸胍从脱脂组织中提取蛋白聚糖。将提取物用7mol/L尿素进行广泛透析后，超过90%的含己糖醛酸物质在尿素可溶部分中回收。通过二乙氨基乙基（DEAE）-Sephacel层析、CsCl密度梯度离心和Bio-Gel A-5m凝胶过滤从尿素可溶部分中纯化蛋白聚糖，然后在DEAE-Sephacel和Bio-Gel A-5m上再次层析。这些蛋白聚糖被分离成三种不同分子量的分子种类，通过沉降平衡离心法评估其分子量分别为46,000、16,000和8,300。对这些蛋白聚糖的氨基酸分析表明，丝氨酸和甘氨酸约占这三种蛋白聚糖共有的总氨基酸的60%。这些蛋白聚糖的糖胺聚糖侧链经软骨素酶AC-II消化后化学计量转化为不饱和6-硫酸化二糖，表明它们是完全硫酸化的6-硫酸软骨素。通过在Bio-Gel A-0.5m上进行凝胶过滤，估计它们的表观分子量分别为10,900、14,400和7,700。综合这些结果及其生化组成进行计算，结果显示最大的蛋白聚糖PG-II由四条6-硫酸软骨素链和分子量约为4,000的核心肽组成。较小的两个蛋白聚糖亚群PG-I和PG-III分别由一条糖胺聚糖链与分子量约为500和1,000的小肽相连组成。
