Tomana M, Zikan J, Kulhavy R, Bennett J C, Mestecky J
Department of Medicine, University of Alabama, Birmingham 35294.
Mol Immunol. 1993 Feb;30(3):277-86. doi: 10.1016/0161-5890(93)90056-h.
Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)
β-1,4-半乳糖基转移酶（β-1,4-GT）活性检测表明，除血清、乳汁、初乳、羊水、脑脊液及恶性积液外，该酶也存在于眼泪和唾液中。对人初乳清和血清进行分子筛层析，随后检测β-1,4-GT活性，结果显示β-1,4-GT以游离酶（55 kDa）形式存在，并与较大摩尔质量的成分相关联。当在不含或富含β-1,4-GT活性辅因子Mn2+的缓冲液中进行层析时，洗脱模式没有变化。然而，当在螯合剂（EDTA）存在下进行层析时，与大摩尔质量成分相关的活性消失。在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳（SDS-PAGE）以及免疫扩散对洗脱物质进行分析，结果表明含β-1,4-GT活性组分中的主要初乳成分是分泌型IgA（S-IgA）；此外，在含有乳铁蛋白和α-乳白蛋白的组分中也检测到了β-1,4-GT活性。通过对初乳清与UDP-[3H]-半乳糖孵育后进行免疫电泳和放射自显影获得的沉淀线中的放射性检测，也证明了初乳中β-1,4-GT与S-IgA和乳铁蛋白之间的相互作用。此外，当分别在非还原和还原条件下通过SDS-PAGE分析与UDP-[3H]-半乳糖孵育的初乳清时，检测到了放射性标记的S-IgA和α链。在血清中，分子筛层析后各组分中鉴定出的β-1,4-GT结合成分是IgG、IgA、IgM和转铁蛋白。通过检测与固定在琼脂糖-4B上的各种同种型和分子形式的Ig相关的β-1,4-GT活性，也证明了β-1,4-GT与免疫球蛋白（Ig）的结合，这些Ig在Mn2+存在下与初乳β-1,4-GT孵育。通过酶活性测定的β-1,4-GT与这些Ig的结合顺序为：聚合IgA2 > 单体IgA1 = 聚合IgA1 = 分泌型IgA = 五聚体IgM > IgG。固定化的成分链，即α、μ和J链，比天然Ig更有效地结合β-1,4-GT。在UDP[3H]-半乳糖和Mn2+存在下，将IgA1骨髓瘤蛋白与粗制人初乳半乳糖基转移酶孵育，导致N-和O-连接的碳水化合物侧链均发生半乳糖基化。（摘要截断于400字）
