Akerström B, Lögdberg L, Babiker-Mohamed H, Lohmander S, Rask L
Department of Physiological Chemistry, University of Lund, Sweden.
Eur J Biochem. 1987 Dec 30;170(1-2):143-8. doi: 10.1111/j.1432-1033.1987.tb13678.x.
Rabbit alpha 1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100, affinity chromatography on concanavalin-A--Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit alpha 1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. Alpha 1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of alpha 1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human alpha 1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit alpha 1-microglobulin, with a gap between each band of 2.6--2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig alpha 1-microglobulin. Our results indicate that human alpha 1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other alpha 1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of alpha 1-microglobulin.
通过在葡聚糖凝胶G - 100上进行凝胶色谱、在伴刀豆球蛋白A - 琼脂糖上进行亲和色谱以及在二乙氨基乙基葡聚糖凝胶上进行离子交换色谱，从经铬酸钠处理的动物尿液中纯化出兔α1 - 微球蛋白。在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳中，兔α1 - 微球蛋白的分子量为25.6 kDa。此前已通过类似方法从人、豚鼠和大鼠的尿液中纯化出α1 - 微球蛋白，并通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和在变性介质中的凝胶色谱对这四种同源物的分子量进行了比较。通过这两种方法，人源同源物分别比其他三种蛋白质大6 kDa和3 kDa。对α1 - 微球蛋白进行内切糖苷酶F消化，随后进行十二烷基硫酸钠/聚丙烯酰胺凝胶电泳，结果显示人α1 - 微球蛋白样品中有三条蛋白带，而豚鼠、大鼠和兔α1 - 微球蛋白中只有两条带，每条带之间的间隔为2.6 - 2.9 kDa。测定了这四种同源物的氨基末端氨基酸序列，同源性在72%至81%之间。豚鼠α1 - 微球蛋白中缺少其他物种中存在的五个氨基末端氨基酸。我们的结果表明，人α1 - 微球蛋白被两个N - 连接寡糖取代，而其他α1 - 微球蛋白每种仅连接一个，并且人蛋白质中额外的糖胺连接寡糖连接到第17位的天冬酰胺上。最后表明，所有四种同源物均抑制人淋巴细胞的抗原刺激，这一发现与我们之前提出的N - 连接寡糖具有α1 - 微球蛋白免疫抑制活性的观点一致。
