IMMUNOCHEMICAL CONFIRMATION OF THE PRIMARY STRUCTURE OF STREPTOCOCCAL HYALURONAN SYNTHASE AND SYNTHESIS OF HIGH-MOLECULAR-WEIGHT PRODUCT BY THE RECOMBINANT ENZYME
IMMUNOCHEMICAL CONFIRMATION OF THE PRIMARY STRUCTURE OF STREPTOCOCCAL HYALURONAN SYNTHASE AND SYNTHESIS OF HIGH-MOLECULAR-WEIGHT PRODUCT BY THE RECOMBINANT ENZYME
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DOI：
10.1021/bi00197a001
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发表时间：
1994-08-09
期刊：
影响因子：
2.9
通讯作者：
WEIGEL, PH
中科院分区：
文献类型：
--
作者：
DEANGELIS, PL;WEIGEL, PH
我们最近从 A 组链球菌中鉴定并克隆了乙酰透明质酸 (HA) 合酶基因 hasA [DeAngelis, P. L.、Papaconstantinou, J. 和 Weigel, P. H. (1993) J. Biol. 1993]。化学。 268, 19181-19184]。我们现在已经生成了两种针对与推导蛋白质部分相对应的合成肽的多克隆单特异性抗体。两种抗体均可识别来自野生型化脓性链球菌或来自质粒上含有克隆基因的大肠杆菌的表观分子量为 42 000 的蛋白质。固定化亲和纯化抗体以特定方式耗尽链球菌膜功能性洗涤剂提取物中的 HA 合酶活性。固定化蛋白显示出HA合酶活性，HasA是主要结合的多肽。在糖核苷酸特异性和多糖生产方面，重组HA合酶的行为与链球菌的HA合酶相同。只有真正的糖核苷酸UDP-葡萄糖醛酸和UDP-N-乙酰葡萄糖胺支持HA聚合。重组酶以持续的方式延长HA，并以约10-30个单糖/秒的速率、底物表观K-m的三倍快速产生大于或等于5 x 10(6) Da的聚合物。
We have recently identified and cloned the gene for hyaluronan (HA) synthase, hasA, from group A Streptococci [DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184]. We have now generated two polyclonal monospecific antibodies against synthetic peptides corresponding to portions of the deduced protein. Both antibodies recognize a protein with an apparent molecular weight of 42 000 either from wild-type Streptococcus pyogenes or from Escherichia coli containing the cloned gene on a plasmid. Immobilized affinity-purified antibody depleted HA synthase activity from functional detergent extracts of streptococcal membranes in a specific fashion. The immobilized protein displayed HA synthase activity, and HasA was the major bound polypeptide. The recombinant HA synthase behaves identically to that from Streptococci, with respect to sugar nucleotide specificity and polysaccharide production. Only the authentic sugar nucleotides UDP-glucuronic acid and UDP-N-acetylglucosamine support HA polymerization. The recombinant enzyme elongates HA in a processive manner and rapidly produces polymers on the order of greater than or equal to 5 x 10(6) Da at rates of about 10-30 monosaccharides/s at three times the apparent K-m of substrates.