Gmeiner B, Wolf G
Cancer Res. 1987 May 1;47(9):2311-6.
We investigated the asialo-agalactofetuin galactosyltransferase solubilized by Triton X-100 from a human bladder transitional cell carcinoma line (cell-associated form). The specific activity of this enzyme was dependent on cell population density, being about 50% higher in cells from confluent than from sparse cultures. We compared the properties of this enzyme with those of a galactosyltransferase isoenzyme present in the culture medium (soluble form). Electrophoresis on nondenaturing polyacrylamide gels showed the two forms to be isoenzymes, in that the mobility of the soluble enzyme was greater than that of the cell-associated enzyme. The isoenzymes differed in that the Km for UDP-galactose of the cell-associated enzyme (1 X 10(-5) M) was one-half that of the soluble isoenzyme. The isoenzymes differed by 1 order of magnitude in their affinity for a fetuin-derived acceptor with a Km of 16 X 10(-5) M for the cell-associated and 1.2 X 10(-5) M for the soluble form, although the Km for ovalbumin and asialomucin as acceptor was similar for both. Both enzymes were active over a broad pH range and their response to divalent cations was the same: the most efficient cation was Mn2+; but modest activity was detected in the presence of either Cd2+ or Co2+. As determined by gel filtration on Sepharose 6B, the cell-associated galactosyltransferase showed a molecular weight of 66,000, whereas that of the soluble form was 51,000. Limited proteolysis of the cell-associated enzyme with thermolysin and subsequent analysis by nondenaturing polyacrylamide gel electrophoresis demonstrated that the cell-associated enzyme could be converted to an isoenzyme showing the same electrophoretic mobility as the soluble enzyme present in the culture medium, presumably by removal of a portion of the peptide chain. The same result was obtained by treating the cell-associated enzyme with a cell extract. This suggests but does not prove that the soluble enzyme secreted or shed into the medium is produced from the cell-associated form by an endogenous protease.
我们研究了用Triton X - 100从人膀胱移行细胞癌系（细胞相关形式）中溶解出来的去唾液酸-去半乳糖基胎球蛋白半乳糖基转移酶。这种酶的比活性取决于细胞群体密度，汇合培养的细胞中该酶的比活性比稀疏培养的细胞高约50%。我们将这种酶的特性与培养基中存在的半乳糖基转移酶同工酶（可溶性形式）的特性进行了比较。在非变性聚丙烯酰胺凝胶上进行电泳显示这两种形式是同工酶，因为可溶性酶的迁移率大于细胞相关酶的迁移率。这两种同工酶的不同之处在于，细胞相关酶对UDP - 半乳糖的Km值（1×10⁻⁵M）是可溶性同工酶的一半。这两种同工酶对胎球蛋白衍生受体的亲和力相差1个数量级，细胞相关形式的Km值为16×10⁻⁵M，可溶性形式的Km值为1.2×10⁻⁵M，尽管二者对卵清蛋白和去唾液酸粘蛋白作为受体的Km值相似。两种酶在较宽的pH范围内都有活性，并且它们对二价阳离子的反应相同：最有效的阳离子是Mn²⁺；但在Cd²⁺或Co²⁺存在时也检测到适度的活性。通过在Sepharose 6B上进行凝胶过滤测定，细胞相关的半乳糖基转移酶的分子量为66,000，而可溶性形式的分子量为51,000。用嗜热菌蛋白酶对细胞相关酶进行有限的蛋白水解，随后通过非变性聚丙烯酰胺凝胶电泳分析表明，细胞相关酶可以转化为一种同工酶，其电泳迁移率与培养基中存在的可溶性酶相同，推测是通过去除部分肽链实现的。用细胞提取物处理细胞相关酶也得到了相同的结果。这表明但并未证明分泌或释放到培养基中的可溶性酶是由细胞相关形式通过内源性蛋白酶产生的。
