Ajzannay A, Minassian C, Riou J P, Mithieux G
Institut National de la Santé et de la Recherche Médicale, U. 197, Faculté de Médecine Alexis Carrel, Lyon, France.
Eur J Biochem. 1993 Mar 1;212(2):335-8. doi: 10.1111/j.1432-1033.1993.tb17666.x.
With the aim of questioning the apparent loss of specificity of the microsomal glucose-6-phosphate phosphohydrolase after detergent-treatment, we performed competitive inhibition experiments among the four best substrates of the enzyme, i.e. the 6-phosphates of glucose (Glc6P), mannose-6 (Man6P), glucosamine (GlcN6P) and 2-deoxyglucose (dGlc6P). The Km and Vmax of glucose-6-phosphatase (Glc6Pase) and mannose-6-phosphatase (Man6Pase), assayed either by complex formation determination of P(i) produced or by radiometric determination of [U-14C]Glc or [U-14C]Man, were very close to 1 mM and 0.64 mumol.min-1.mg-1 microsomal protein, respectively. The Km of the enzyme for GlcN6P and for dGlc6P, determined by colorimetric assay of P(i), were equal to 1.53 +/- 0.07 mM and 2.35 +/- 0.15 mM, respectively, whilst the Vmax was not different from that of Glc6Pase and Man6Pase. Unexpectedly, the Ki of Man6P (1.61 +/- 0.22 mM), GlcN6P (2.24 +/- 0.17 mM) and dGlc6P (3.40 +/- 0.07 mM) for Glc6Pase, assayed by liberation of [U-14C]Glc, were significantly (50%) higher than their Km previously determined. The Ki of Glc6P (0.66 +/- 0.05 mM) for Man6Pase, assayed by liberation of [U-14C]Man, was significantly lower than its Km previously determined. In contrast, the Ki of GlcN6P (1.55 +/- 0.05 mM) for Man6Pase, assayed by the radiometric assay, was not different from its Km previously determined. It can be inferred from these data that Glc6P phosphohydrolase exhibits specific behaviour towards Glc6P after the detergent-treatment of the microsomal membrane.
为了探究微粒体葡萄糖-6-磷酸磷酸水解酶在去污剂处理后明显丧失特异性的问题，我们在该酶的四种最佳底物，即葡萄糖-6-磷酸（Glc6P）、甘露糖-6-磷酸（Man6P）、葡糖胺-6-磷酸（GlcN6P）和2-脱氧葡萄糖-6-磷酸（dGlc6P）之间进行了竞争性抑制实验。通过测定产生的无机磷酸（P(i)）的复合物形成或通过放射性测定[U-14C]葡萄糖或[U-14C]甘露糖来测定葡萄糖-6-磷酸酶（Glc6Pase）和甘露糖-6-磷酸酶（Man6Pase）的Km和Vmax，它们分别非常接近1 mM和0.64 μmol·min-1·mg-1微粒体蛋白。通过比色法测定P(i)确定该酶对GlcN6P和dGlc6P的Km分别等于1.53±0.07 mM和2.35±0.15 mM，而Vmax与Glc6Pase和Man6Pase的Vmax没有差异。出乎意料的是，通过释放[U-14C]葡萄糖测定，Man6P（1.61±0.22 mM）、GlcN6P（2.24±0.17 mM）和dGlc6P（3.40±0.07 mM）对Glc6Pase的抑制常数（Ki）比它们先前测定的Km显著高（50%）。通过释放[U-14C]甘露糖测定，Glc6P（0.66±0.05 mM）对Man6Pase的Ki显著低于其先前测定的Km。相反，通过放射性测定法测定，GlcN6P（1.55±0.05 mM）对Man6Pase的Ki与其先前测定的Km没有差异。从这些数据可以推断，在微粒体膜用去污剂处理后，Glc6P磷酸水解酶对Glc6P表现出特异性行为。
