Wang-Gillam A, Pastuszak I, Elbein A D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Biol Chem. 1998 Oct 16;273(42):27055-7. doi: 10.1074/jbc.273.42.27055.
We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147-13154). Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes in Escherichia coli. The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.
我们之前报道过从猪肝中纯化出一种UDP-N-乙酰己糖胺（UDP-HexNAc）焦磷酸化酶，它能催化从UTP和相应的己糖胺-1-磷酸合成UDP-GlcNAc和UDP-GalNAc（苏米洛，T.，曾，Y.，帕斯图扎克，I.，德雷克，R.，苏米洛，H.，和埃尔宾，A. D.（1996年）《生物化学杂志》271卷，13147 - 13154页）。两种糖核苷酸的合成速率几乎相同，尽管GalNAc-1-P的Km值比GlcNAc-1-P高约3倍。基于天然凝胶和SDS-聚丙烯酰胺凝胶电泳，该酶似乎是一个120 kDa的二聚体，由两个分别约为57 kDa和64 kDa的亚基组成。从64 kDa蛋白质中测序得到的三个肽段以及从57 kDa蛋白质中测序得到的两个肽段与AGX1具有100%的同一性，AGX1是一种来自人类精子的功能未知的57 kDa蛋白质。一种名为AGX2的同工型与AGX1序列相同，只是在羧基末端附近有一个17个氨基酸的插入片段。我们在大肠杆菌中表达了AGX1和AGX2基因。从AGX1克隆中分离出的蛋白质在SDS凝胶上与肝脏中的57 kDa焦磷酸化酶亚基迁移情况相同，并且对GalNAc-1-P的活性比对GlcNAc-1-P高2 - 3倍。另一方面，来自AGX2克隆的蛋白质与肝脏中的64 kDa焦磷酸化酶亚基迁移情况相同，并且对GlcNAc-1-P的活性比对GalNAc-1-P高8倍。这些结果表明，17个氨基酸肽段的插入将焦磷酸化酶的特异性从合成UDP-GalNAc改变为合成UDP-GlcNAc。
