Moscarello M A, Mitranic M M, Vella G
Biochim Biophys Acta. 1985 Oct 4;831(2):192-200. doi: 10.1016/0167-4838(85)90035-4.
Purified bovine milk galactosyltransferase was stimulated by purified bovine colostrum N-acetylglucosaminyltransferase I by more than 10-fold. Only slight stimulation of the N-acetylglucosaminyltransferase I by galactosyltransferase was observed. Heat inactivation destroyed the ability of the N-acetylglucosaminyltransferase I to stimulate the galactosyltransferase. The stimulation of galactosyltransferase was accompanied by a decrease in Km of this enzyme from 9.7 to 3.3. mM and an increase in Vmax from 1.87 to 3.71 nmol galactose transferred/min per mg galactosyltransferase when GlcNAc was the substrate. When the Km for UDPgalactose was determined, it increased from 0.19 to 0.42 mM in the presence of N-acetylglucosaminyltransferase I and the Vmax increased from 0.66 to 2.76 nmol galactose transferred/min per mg galactosyltransferase. In phosphatidylcholine vesicles, no effect on Km values with GlcNAc as substrate was noted, while an increase in the Km of UDPgalactose was observed. The Vmax values were generally higher in the lipid vesicles. Complex formation between galactosyltransferase and N-acetylglucosaminyltransferase I was demonstrated both by glycerol density gradient centrifugation and Bio-Gel P-100 column chromatography. An approximate molecular weight for the complex was obtained on a calibrated Sephadex G-200 column and found to be about 75 000, consistent with a 1:1 complex. The stimulation of galactosyltransferase involved the N-acetyllactosamine synthetase activity of this enzyme and not the lactose synthetase activity, since the latter activity was only slightly affected. Since N-acetylglucosaminyltransferase I is not involved in the lactose synthetase reaction, the stimulation is consistent with the known biosynthetic role of N-acetylglucosaminyltransferase I in the biosynthesis of asparagine-linked oligosaccharides.
纯化的牛初乳N-乙酰葡糖胺基转移酶I对纯化的牛乳半乳糖基转移酶的刺激作用超过10倍。而半乳糖基转移酶对N-乙酰葡糖胺基转移酶I仅有轻微的刺激作用。热失活破坏了N-乙酰葡糖胺基转移酶I刺激半乳糖基转移酶的能力。半乳糖基转移酶受到刺激时，该酶的米氏常数（Km）从9.7 mM降至3.3 mM，最大反应速度（Vmax）从1.87 nmol半乳糖转移/（分钟·毫克半乳糖基转移酶）增至3.71 nmol半乳糖转移/（分钟·毫克半乳糖基转移酶），此时底物为N-乙酰葡糖胺（GlcNAc）。当测定尿苷二磷酸半乳糖（UDPgalactose）的Km时，在N-乙酰葡糖胺基转移酶I存在的情况下，其从0.19 mM增至0.42 mM，Vmax从0.66 nmol半乳糖转移/（分钟·毫克半乳糖基转移酶）增至2.76 nmol半乳糖转移/（分钟·毫克半乳糖基转移酶）。在磷脂酰胆碱囊泡中，以GlcNAc为底物时未观察到对Km值有影响，而观察到UDPgalactose的Km增加。脂质囊泡中的Vmax值通常更高。通过甘油密度梯度离心和生物凝胶P - 100柱色谱法均证明了半乳糖基转移酶与N-乙酰葡糖胺基转移酶I之间形成了复合物。在经校准的葡聚糖凝胶G - 200柱上获得了该复合物的近似分子量，发现约为75000，与1:1复合物相符。半乳糖基转移酶的刺激作用涉及该酶的N-乙酰乳糖胺合成酶活性，而非乳糖合成酶活性，因为后者的活性仅受到轻微影响。由于N-乙酰葡糖胺基转移酶I不参与乳糖合成酶反应，这种刺激作用与N-乙酰葡糖胺基转移酶I在天冬酰胺连接的寡糖生物合成中已知的生物合成作用一致。
