Kobayashi S, Oguri K, Kobayashi K, Okayama M
J Biol Chem. 1983 Oct 10;258(19):12051-7.
Glomeruli isolated from rat kidney were incubated with [14C]glucosamine and [35S]sulfate. Linear incorporation of [14C]glucosamine into total glycosaminoglycans was observed during incubation up to 24 h. More than 95% of the 35S-labeled sulfated glycoconjugates were extracted from the tissue with 4 M guanidine HCl, 50 mM sodium acetate, pH 6.0, and 0.5% Triton X-100, and separated clearly on DEAE-Sephacel into three major fractions, i.e. sulfated glycoprotein (11% of the total radioactivity), proteoheparan sulfate (33%), and proteochondroitin sulfate (38%) fractions. The molecular weight of the 35S-labeled proteoheparan sulfate thus isolated was estimated to be about 185,000, whereas that released into the medium was estimated to be about 87,000. When the 35S-labeled heparan sulfate isolated on Sephadex G-75 after mild alkaline borohydride treatment was digested with a combination of heparitinase and heparinase, approximately 70% of the radioactivity was converted to 2-acetamido-2-deoxy-4-O-(alpha-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D- glucose.
从大鼠肾脏分离出的肾小球与[14C]葡糖胺和[35S]硫酸盐一起孵育。在长达24小时的孵育过程中，观察到[14C]葡糖胺线性掺入总糖胺聚糖。用4M盐酸胍、50mM乙酸钠（pH6.0）和0.5% Triton X-100从组织中提取超过95%的35S标记的硫酸化糖缀合物，并在DEAE-葡聚糖凝胶上清晰地分离成三个主要部分，即硫酸化糖蛋白（占总放射性的11%）、硫酸乙酰肝素蛋白聚糖（33%）和硫酸软骨素蛋白聚糖（38%）部分。如此分离出的35S标记的硫酸乙酰肝素蛋白聚糖的分子量估计约为185,000，而释放到培养基中的分子量估计约为87,000。当用肝素酶和肝素酶组合消化经温和碱性硼氢化物处理后在Sephadex G-75上分离出的35S标记的硫酸乙酰肝素时，大约70%的放射性转化为2-乙酰氨基-2-脱氧-4-O-（α-D-葡糖-4-烯吡喃糖醛酸）-6-O-磺基-D-葡萄糖。
