Veiga-Da-Cunha M, Detheux M, Watelet N, Van Schaftingen E
Laboratory of Physiological Chemistry, University of Louvain, Brussels, Belgium.
Eur J Biochem. 1994 Oct 1;225(1):43-51. doi: 10.1111/j.1432-1033.1994.00043.x.
Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.
非洲爪蟾肝脏中含有一种葡萄糖激酶的蛋白抑制剂，与哺乳动物的葡萄糖激酶调节蛋白不同，它对6-磷酸果糖和1-磷酸果糖不敏感[范德卡门A. & 范沙夫廷根，E.（1993年）《生物化学杂志》294卷，551 - 556页]。这项工作的目的是比较这种非洲爪蟾蛋白与其大鼠肝脏对应物的一级结构和其他特性。以编码大鼠肝脏调节蛋白的cDNA为探针，筛选非洲爪蟾肝脏cDNA文库。克隆得到的cDNA长2534 bp，编码一个619个氨基酸的蛋白，分子量为68695 Da，与大鼠肝脏调节蛋白的同源性为57%。在内部区域（氨基酸349 - 381）和70个羧基末端残基中，这种同源性仅约为30%。非洲爪蟾cDNA在大肠杆菌中表达，重组调节蛋白被纯化至近乎同质，发现其大小、与抗体的反应性以及对葡萄糖激酶动力学的影响与从非洲爪蟾肝脏纯化的蛋白相同。与大鼠肝脏调节蛋白不同，重组和天然的非洲爪蟾调节蛋白对6-磷酸果糖、1-磷酸果糖以及生理浓度的无机磷酸均不敏感，并且它们抑制非洲爪蟾葡萄糖激酶的亲和力比大鼠葡萄糖激酶更高。这些结果使人们能够得出结论，低等脊椎动物中对磷酸果糖不敏感的蛋白与哺乳动物中对6-磷酸果糖敏感和对1-磷酸果糖敏感的蛋白是同源的。
