Takahashi S, Takahashi K, Kaneko T, Ogasawara H, Shindo S, Kobayashi M
Department of Bioengineering, Akita Research Institute of Food and Brewing, Sanuki, Arayamachi, Akita, 010-1623, Japan.
J Biochem. 1999 Feb;125(2):348-53. doi: 10.1093/oxfordjournals.jbchem.a022293.
The existence of human renin-binding protein (RnBP) in the kidney has been shown by the isolation and characterization of a complex of porcine renin-human RnBP [S. Takahashi et al. (1985) J. Biochem. 97, 671-677]. However, the properties of the free form of human RnBP had not been understood, because of the limitation of materials. In the present study, we have expressed human RnBP in Escherichia coli JM 109 cells under the transcriptional control of taq promoter and purified it by conventional column chromatographies. The purified recombinant human RnBP (rhRnBP) exists as a dimer and inhibits porcine renin activity through formation of a complex of porcine renin with rhRnBP, the so-called high-molecular-weight renin. Moreover, the rhRnBP catalyzes the interconversion between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-Mannosamine (ManNAc) with the apparent Km values of 21.3 mM for GlcNAc and 12.8 mM for ManNAc, and 0.13 mM for effector ATP. ATP is essential for the GlcNAc 2-epimerase activity of human RnBP. These results indicate that the human RnBP is a GlcNAc 2-epimerase.
通过对猪肾素-人肾素结合蛋白（RnBP）复合物的分离和特性鉴定，已证明人肾素结合蛋白（RnBP）存在于肾脏中[S. Takahashi等人（1985年）《生物化学杂志》97卷，671 - 677页]。然而，由于材料的限制，人RnBP游离形式的特性尚未明确。在本研究中，我们在Taq启动子的转录控制下，在大肠杆菌JM 109细胞中表达了人RnBP，并通过常规柱色谱法对其进行了纯化。纯化的重组人RnBP（rhRnBP）以二聚体形式存在，并通过形成猪肾素与rhRnBP的复合物（即所谓的高分子量肾素）来抑制猪肾素活性。此外，rhRnBP催化N - 乙酰 - D - 葡萄糖胺（GlcNAc）和N - 乙酰 - D - 甘露糖胺（ManNAc）之间的相互转化，对GlcNAc的表观Km值为21.3 mM，对ManNAc为12.8 mM，对效应物ATP为0.13 mM。ATP对人RnBP的GlcNAc 2 - 表异构酶活性至关重要。这些结果表明人RnBP是一种GlcNAc 2 - 表异构酶。
