Sugumaran G, Katsman M, Silbert J E
Connective Tissue Research Laboratory, Building 70, Edith Nourse Rogers Memorial Veterans Hospital, 200 Springs Road, Bedford, MA 01730, USA.
Biochem J. 1998 Jan 1;329 ( Pt 1)(Pt 1):203-8. doi: 10.1042/bj3290203.
Microsomal membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose-density-gradient centrifugation and assayed for GlcA (glucuronic acid) transferase I (the enzyme that transfers GlcA from UDP-GlcA to Gal-Gal-Xyl of proteochondroitin linkage region), for comparison with GlcA transferase II (the GlcA transferase of chondroitin polymerization). Gal(beta1-3)Galbeta1-methyl (disaccharide) and GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4) GlcA(beta1-3)GalNAc(pentasaccharide) were used respectively as acceptors of [14C]GlcA from UDP-[14C]GlcA. Distributions of the two GlcA transferase activities in the sucrose-density-gradient fractions were compared with each other and with the previously reported distribution of the activities of Gal transferases (UDP-Gal to ovalbumin, and to xylose of the proteochondroitin linkage region) and GalNAc (N-acetylgalactosamine) transferase II of chondroitin polymerization. The linkage-region GlcA transferase I had a dual Golgi distribution similar to that of chondroitin-polymerizing GlcA transferase II and distinctly different from the distribution of linkage-region Gal transferases I and II, which were found exclusively in the heavier fractions. Solubilized GlcA transferase I was partly purified by sequential use of Q-Sepharose, heparin-Sepharose and wheatgerm agglutinin-agarose and was accompanied at each step by some of the GlcA transferase II activity. Both GlcA transferase I and II bound to the Q-Sepharose as though they were highly anionic. However, treatment with chondroitin ABC lyase eliminated the binding while markedly decreasing enzyme stability. The enzyme activities could not be reconstituted by adding chondroitin or chondroitin pentasaccharide to the chondroitin ABC lyase-treated enzymes. Incubation of the partly purified enzymes with both UDP-GlcA and UDP-GalNAc resulted in a 40-fold greater incorporation than with just one sugar nucleotide, indicating the presence of bound, nascent proteochondroitin serving as the acceptor for chondroitin polymerization. These results, together with the membrane co-localization, indicate that GlcA transferase I and GlcA transferase II occur closely together with nascent proteochondroitin at the site of synthesis and that this complex with the nascent proteochondroitin stabilizes both enzymes during purification.
通过平衡蔗糖密度梯度离心法对鸡胚骨骺软骨的微粒体膜进行分级分离，并检测葡萄糖醛酸（GlcA）转移酶I（该酶将UDP-GlcA中的GlcA转移至蛋白聚糖连接区的Gal-Gal-Xyl）的活性，以便与软骨素聚合反应中的GlcA转移酶II进行比较。分别使用Gal(β1-3)Galβ1-甲基（二糖）和GalNAc(β1-4)GlcA(β1-3)GalNAc(β1-4)GlcA(β1-3)GalNAc（五糖）作为UDP-[14C]GlcA中[14C]GlcA的受体。将两种GlcA转移酶活性在蔗糖密度梯度级分中的分布相互比较，并与先前报道的Gal转移酶（UDP-Gal转移至卵清蛋白以及蛋白聚糖连接区的木糖）和软骨素聚合反应中的GalNAc（N-乙酰半乳糖胺）转移酶II的活性分布进行比较。连接区GlcA转移酶I具有与软骨素聚合GlcA转移酶II相似的双重高尔基体分布，与连接区Gal转移酶I和II的分布明显不同，后两者仅存在于较重的级分中。通过依次使用Q-琼脂糖凝胶、肝素-琼脂糖凝胶和麦胚凝集素-琼脂糖对溶解的GlcA转移酶I进行部分纯化，且在每一步中都伴随着一些GlcA转移酶II的活性。GlcA转移酶I和II都像具有高度阴离子性一样与Q-琼脂糖凝胶结合。然而，用软骨素ABC裂解酶处理会消除这种结合，同时显著降低酶的稳定性。向经软骨素ABC裂解酶处理的酶中添加软骨素或软骨素五糖无法恢复酶活性。将部分纯化的酶与UDP-GlcA和UDP-GalNAc一起孵育，其掺入量比仅使用一种糖核苷酸时高40倍，表明存在结合的新生蛋白聚糖作为软骨素聚合反应的受体。这些结果与膜共定位一起表明，GlcA转移酶I和GlcA转移酶II在合成位点与新生蛋白聚糖紧密共存，并且这种与新生蛋白聚糖的复合物在纯化过程中稳定了两种酶。
