Wilson J R, Deinhart J A, Weiser M M
Biochim Biophys Acta. 1987 May 19;924(2):332-40. doi: 10.1016/0304-4165(87)90031-6.
UDPgalactose: N-acetylgalactosamine mucin galactosyltransferase activity of the rat intestine was studied and purified using asialo-ovine submaxillary mucin as the acceptor substrate and inhibitors to suppress UDPgalactose breakdown by pyrophosphatase activities particularly prevalent in the duodenal-jejunal regions. Despite adequate suppression of UDPgalactose breakdown, significant intestinal region differences of mucin galactosyltransferase activity were observed. Elevations of activity were observed in the duodenum and distal ileum of the small intestine and the cecum and proximal colon; these elevations in activity correspond to areas of increased mucin production. Similarly, mucin galactosyltransferase activity of duodenal cells isolated along a crypt-to-villus axis showed a moderate increase (67.7%) in activity associated with cells in the crypt region. Small intestine mucin galactosyltransferase activity was purified 800-fold using a series of ion exchange (DEAE-Sepharose), gel filtration (S-200 Sephacryl) and affinity chromatographic steps to isolate the mucin galactosyltransferase activity from a Triton X-100/Nonidet P-40 extract of homogenized cells obtained by scraping everted intestines. The partially purified enzyme showed two distinct protein bands of 81.5 and 50 kDa and a faint band at 53.3 kDa. Kinetic analysis gave an apparent Km of 152 microM for UDPgalactose. The enzyme showed optimal activity with Mn2+ (20 mM) and partial activities using a number of other divalent cations. Higher concentrations of Mn2+ were slightly inhibitory. Mucin galactosyltransferase activity was inhibited by more then 90% in the presence of Zn2+ (4 mM) and this inhibition could not be reversed by additional Mn2+. Addition of Zn2+ (4 mM) to assays containing Mn2+ (20 mM) did not cause appreciable UDPgalactose breakdown, as measured by high-voltage paper electrophoresis, suggesting that Zn2+ inhibition is not a result of pyrophosphatase activation. In addition, Zn2+ does not appear to activate a protease or glycosidase activity in the partially purified enzyme preparation which could hydrolyze the galactosylated product prior to isolation.
以去唾液酸的羊下颌粘蛋白作为受体底物，并使用抑制剂来抑制十二指肠-空肠区域特别普遍的焦磷酸酶活性对UDP-半乳糖的分解，在此条件下，对大鼠肠道的UDP-半乳糖：N-乙酰半乳糖胺粘蛋白半乳糖基转移酶活性进行了研究和纯化。尽管对UDP-半乳糖的分解有足够的抑制作用，但仍观察到粘蛋白半乳糖基转移酶活性存在明显的肠道区域差异。在小肠的十二指肠和回肠末端以及盲肠和近端结肠中观察到活性升高；这些活性升高与粘蛋白产生增加的区域相对应。同样，沿隐窝-绒毛轴分离的十二指肠细胞的粘蛋白半乳糖基转移酶活性显示，与隐窝区域的细胞相关的活性适度增加（67.7%）。通过一系列离子交换（DEAE-琼脂糖）、凝胶过滤（S-200琼脂糖凝胶）和亲和色谱步骤，从小肠外翻肠刮取的匀浆细胞的Triton X-100/Nonidet P-40提取物中分离粘蛋白半乳糖基转移酶活性，将小肠粘蛋白半乳糖基转移酶活性纯化了800倍。部分纯化的酶显示出两条明显的蛋白带，分子量分别为81.5 kDa和50 kDa，以及一条53.3 kDa的淡带。动力学分析得出UDP-半乳糖的表观Km为152 microM。该酶在Mn2+（20 mM）存在下显示出最佳活性，使用其他一些二价阳离子时也有部分活性。较高浓度的Mn2+有轻微抑制作用。在Zn2+（4 mM）存在下，粘蛋白半乳糖基转移酶活性被抑制90%以上，并且这种抑制不能通过额外添加Mn2+来逆转。通过高压纸电泳测量，在含有Mn2+（20 mM）的测定中添加Zn2+（4 mM）不会导致明显的UDP-半乳糖分解，这表明Zn2+抑制不是焦磷酸酶激活的结果。此外，Zn2+似乎不会激活部分纯化的酶制剂中的蛋白酶或糖苷酶活性，这些酶活性可能在分离之前水解半乳糖基化产物。
