Swann D A, Sotman S, Dixon M, Brooks C
Biochem J. 1977 Mar 1;161(3):473-85. doi: 10.1042/bj1610473.
The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.
通过在三个连续的氯化铯密度梯度中反复分级分离，去除牛滑液中痕量的透明质酸，从而制备出关节润滑组分。然后通过反复的凝胶渗透色谱法分离出主要的糖蛋白成分（LGP-I）。LGP-I组分的产量约为每升滑液20毫克。沉降平衡测量表明，这种糖蛋白是均一的，其分子量经计算为227500。氨基酸占该分子的43%（重量/重量），碳水化合物成分占44%（重量/重量）。苏氨酸、谷氨酸、脯氨酸和赖氨酸（分别为每1000个残基中有224、127、242和128个残基）是主要的氨基酸。半乳糖胺、半乳糖和N-乙酰神经氨酸（分别为每分子LGP-I组分中有202、162和114个残基）占总碳水化合物残基的98%。还存在少量的甘露糖和葡糖胺（分别为每摩尔LGP-I组分中有1和9摩尔）。用碱和硼氢化钠处理LGP-I组分后，放射性被掺入α-氨基丁酸和丙氨酸中，其摩尔比为4:1，并且通过离子交换色谱法从裂解的寡糖级分中分离出放射性半乳糖胺醇。这些数据证明存在苏氨酸和丝氨酸-O-半乳糖胺键，但通过β-消除反应仅裂解了涉及苏氨酸的理论键的25%。用链霉蛋白酶消化LGP-I组分，然后在DEAE-纤维素上进行色谱分离，得到糖肽级分，其氨基酸和碳水化合物组成与完整分子相似。用半乳糖氧化酶处理去唾液酸和完整的LGP-I组分，然后用硼氢化钠还原，结果表明完整分子中存在52摩尔的末端半乳糖，用神经氨酸酶处理后每摩尔LGP-I组分中有153摩尔的半乳糖。数据表明，LGP-I组分由一条含有150多个由O-GaINAc-Gal组成的寡糖侧链的单一多肽链组成，这些侧链分布在肽链的长度上，并且三分之二的这些侧链中的末端唾液酸残基与半乳糖相连。
