Lammers G, Jamieson J C
Department of Chemistry, University of Manitoba, Winnipeg, Canada.
Biochem J. 1988 Dec 1;256(2):623-31. doi: 10.1042/bj2560623.
Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
高尔基体膜结合的β-1,4-半乳糖基-N-乙酰葡糖胺α-2,6-唾液酸转移酶（CMP-N-乙酰神经氨酸：β-半乳糖苷α-2,6-唾液酸转移酶，EC 2.4.99.1）表现为一种急性期反应物，在患有炎症的大鼠血清中增加约5倍。其从高尔基体膜释放的机制尚不清楚。在本研究中发现，通过超声振动（超声处理）然后在降低的pH下孵育，可以使唾液酸转移酶从膜上释放出来。最大释放发生在pH 5.6时，炎症大鼠的膜比对照大鼠的膜释放出更多的酶。半乳糖基转移酶（UDP-半乳糖：N-乙酰葡糖胺半乳糖基转移酶；EC 2.4.1.38）是另一种位于高尔基体的酶，它不表现为急性期反应物，在相同条件下仍与膜结合。胃蛋白酶抑制剂A（一种组织蛋白酶D样蛋白酶的有效抑制剂）可显著抑制α-2,6-唾液酸转移酶从高尔基体膜的释放。在用针对大鼠肝脏溶酶体组织蛋白酶D产生的抗血清预孵育超声处理的高尔基体膜后，也发生了唾液酸转移酶释放的抑制。将牛脾脏组织蛋白酶D添加到超声处理的高尔基体膜的孵育混合物中会导致唾液酸转移酶的释放增强。完整的高尔基体膜在存在胃蛋白酶抑制剂A的情况下于降低的pH下孵育，以抑制胞质面的任何蛋白酶活性；随后的超声处理表明唾液酸转移酶已被释放，这表明该蛋白酶在高尔基体的腔内面是有活性的。高尔基体膜含有低水平的组织蛋白酶D活性（EC 3.4.23.5）；该酶主要与膜结合，因为它只能通过用Triton X-100提取或用5 mM甘露糖6-磷酸孵育超声处理的高尔基体膜来释放。免疫印迹分析表明，在降低的pH下从超声处理的高尔基体膜释放的转移酶的表观Mr约为42,000，而膜结合酶的表观Mr约为49,000。结合和释放的酶活性的Km值相当，并且与先前报道的肝脏和血清酶的值相似。这项工作表明，含有催化位点的唾液酸转移酶的主要部分是由位于高尔基体腔内面的组织蛋白酶D样蛋白酶从膜锚定物上释放出来的，这解释了该酶的急性期行为。
