Swedlow J R, Matteri R L, Papkoff H
Proc Soc Exp Biol Med. 1986 Mar;181(3):432-7. doi: 10.3181/00379727-181-42277.
A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.
一种从脑膜败血黄杆菌中纯化得到的市售内切糖苷酶（N - 聚糖酶，Genzyme公司，马萨诸塞州波士顿），其特异性为切割糖蛋白中天冬酰胺连接的碳水化合物部分，以几种垂体和绒毛膜促性腺激素作为底物进行了测试。所有测试的完整激素以及各自促性腺激素的所有β亚基均对该酶的作用具有抗性。然而，所有α亚基对该酶敏感，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）检测时，其分子大小减小可证明这一点。用绵羊促黄体生成素亚基（oLHα）进行的制备实验表明，N - 聚糖酶处理后仅35 - 40%的碳水化合物被去除，这表明在所用条件下，两个碳水化合物部分中可能只有一个是可切割的。经酶修饰的亚基（DG - oLHα）能够与未处理的oLHβ重新组合。体外类固醇生成生物测定法（大鼠睾丸间质细胞）表明，重组体（DG - oLHα - oLHβ）的效力约为天然oLH的22%，但在LH的睾丸膜结合测定中，它在与放射性配体竞争时与天然激素的效力相当。
