Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities.
Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities.
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人结直肠癌细胞中路易斯岩藻脂抗原的生物合成。
DOI：
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
G. Larson
中科院分区：
文献类型：
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作者：
V. Liepkans;G. Larson
检测纯化的糖脂作为GDP-[14 C]岩藻糖的[14 C]岩藻糖受体的能力，所述[14 C]岩藻糖由在无血清培养基中培养的人结肠直肠癌细胞SW 1116的无细胞提取物和纯化的膜组分催化。纯化的乳糖四糖基神经酰胺（Gal β 1-3GlcNAc β 1-3Gal β 1-4Glc-Cer或LcOse 4Cer）和H-1糖脂（Fuc α 1-2Gal β 1-3GlcNAc β 1-3Gal β 1-4Glc-Cer或IV 2 Fuc α LcOse 4Cer）刺激放射性以大于莱亚糖脂10倍的速率掺入脂溶性糖脂[Gal β 1-3（Fuc α 1-4）GlcNAc β 1-3Gal β 1-4Glc-Cer或III4 Fuc α LcOse4Cer]。在粗和纯化的膜组分的酶活性进行了优化的底物浓度（糖脂和GDP-岩藻糖），洗涤剂的要求（牛磺胆酸盐），pH值，时间和蛋白质。H-1岩藻糖基化的放射性产物在高效薄层色谱图（HPTLC）上迁移为离散且不同的条带。提供了与先前描述的Leb岩藻糖脂[Fuc α 1-2Gal β 1-3（Fuc α 1-4）GlcNAc β 1-3Gal β 1 -4Glc-Cer或III 4 IV 2（Fuc α）LcOse 4Cer]相同的证据。LcOse 4 Cer岩藻糖基化的放射性产物主要是莱亚岩藻脂，这是通过在三种HPTLC系统中与真实的莱亚岩藻脂（天然衍生物和乙酰化衍生物）共迁移确定的。我们的研究结果还表明，从LcOse 4Cer的H-1和Leb糖脂合成的低水平。在此基础上，采用蔗糖梯度三个峰，对LcOse 4Cer和H-1岩藻糖基转移酶受体活性进行了12-19倍的纯化。具有最高比活性的峰（峰3）在密度和Na+、K+、ATP酶比活性方面最高，尽管NADH-细胞色素-c还原酶和UDP-GalNac转移酶也存在于峰3中。通过统计检验，峰3中LcOse 4Cer受体活性和H-1受体活性的表观Km值显著不同（p小于0.01），分别为2.4 μ M和0.5 μ M。这些表观Km值（10（3）X）和最适pH值（4.8-5.3）比从人乳中纯化的α 1- 3/α 1- 4岩藻糖基转移酶报道的相应性质低得多。我们的研究结果表明，这些岩藻糖基转移酶活性的受体和/或组织特异性或原始表达的非糖苷部分的作用。
Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.
DOI：
10.1016/s0021-9258(19)45389-1
发表时间：
1982-12
期刊：
The Journal of biological chemistry
影响因子：
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作者：
John L. MagnaniS;Bo Nilssong;Manfred BrockhausSV;David Zopfl;Zenon SteplewskiJI;Hilary Koprowskill;V. Ginsburg
通讯作者：
John L. MagnaniS;Bo Nilssong;Manfred BrockhausSV;David Zopfl;Zenon SteplewskiJI;Hilary Koprowskill;V. Ginsburg
中国仓鼠卵巢糖基化突变体 LEC11 和 LEC12 表达两种新型 GDP-岩藻糖:N-乙酰氨基葡萄糖 3-α-L-岩藻糖基转移酶。
DOI：
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发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Campbell,C;Stanley,P
通讯作者：
Stanley,P
针对人类 Leb 血型抗原的单克隆抗体。
DOI：
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发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Brockhaus,M;Magnani,JL;Blaszczyk,M;Steplewski,Z;Koprowski,H;Karlsson,KA;Larson,G;Ginsburg,V
通讯作者：
Ginsburg,V