Magnus J H, Stenstad T, Husby G, Kolset S O
Department of Rheumatology, University of Tromsø, Norway.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):225-31. doi: 10.1042/bj2880225.
Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody.
通过用胍提取，然后进行三氯乙酸沉淀、DEAE - Sephacel离子交换色谱和Sepharose CL - 6B凝胶色谱，从人淀粉样变性肝脏中分离出蛋白聚糖。发现该物质的很大一部分是游离的硫酸软骨素/硫酸皮肤素链（30%），而主要部分是硫酸乙酰肝素蛋白聚糖（HSPG）（70%）。通过凝胶电泳和凝胶色谱测定，HSPG的近似分子量为200 kDa。在硫酸乙酰肝素链进行脱氨基裂解后，通过SDS/PAGE显示核心蛋白的分子量为60 kDa。通过碱处理从核心蛋白中释放出硫酸乙酰肝素链，通过Sepharose CL - 6B凝胶色谱发现其分子量约为35 kDa。通过免疫印迹法显示，核心蛋白与抗牛基底膜HSPG的单克隆抗体发生反应。使用相同抗体对淀粉样变性肝脏组织切片进行免疫组织化学，进一步证实了淀粉样沉积物中存在HSPG。
