Parry D M, Pedersen P L
J Biol Chem. 1984 Jul 25;259(14):8917-23.
The subcellular location of hexokinase was investigated in rat kidney. Both soluble and particulate locations are indicated by differential centrifugation. The particulate form is predominant, representing about 80% of the total activity. None of the activity is latent. Density gradient centrifugation followed by marker enzyme analysis reveals the presence of two populations of mitochondria with distinct densities. Hexokinase is associated primarily with the mitochondrial population having the lower density. Association of hexokinase with brush border, plasma membrane, lysosomes, and endoplasmic reticulum is considered unlikely on the basis of density gradient centrifugation and enzyme analysis. About 95% of the hexokinase activity associated with the mitochondrial fraction can be released in soluble form by repeated incubations with glucose 6-phosphate. An incubation time of about 4 min at 30 degrees C is required to achieve a maximal solubilizing effect. Release is accomplished without disrupting the mitochondrial compartments. Hexokinase is released also by treatment of the mitochondrial fraction with increasing concentrations of digitonin. This technique disrupts and differentially releases the mitochondrial compartments. As observed with liver, but in contrast to that observed with tumor (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912), the release of hexokinase from the mitochondrial fraction of kidney does not correlate with the release of enzymes known to mark the mitochondrial membranes or compartments. These studies provide the first critical evidence about the subcellular location of hexokinase in kidney. They show that in this tissue hexokinase is associated primarily with low density mitochondria, a finding that adds credibility to the existence of this discrete population of mitochondria in vivo. Significantly, this association of hexokinase with kidney mitochondria appears unique in that its release on submitochondrial fractionation does not correlate with the release of known mitochondrial marker enzymes. These results are directly relevant to those cells in the kidney which utilize glucose as an energy source. It is suggested that the enhanced glycolytic capacity of these cells may be due, at least in part, to an association of hexokinase with low density mitochondria.
对大鼠肾脏中己糖激酶的亚细胞定位进行了研究。通过差速离心法表明其存在于可溶性部分和颗粒部分。颗粒形式占主导，约占总活性的80%。没有活性是潜伏的。密度梯度离心后进行标记酶分析，发现存在两种密度不同的线粒体群体。己糖激酶主要与密度较低的线粒体群体相关。基于密度梯度离心和酶分析，认为己糖激酶与刷状缘、质膜、溶酶体和内质网的关联不太可能。与线粒体部分相关的约95%的己糖激酶活性可通过与6-磷酸葡萄糖反复孵育以可溶性形式释放。在30℃下孵育约4分钟可达到最大溶解效果。释放过程不会破坏线粒体区室。用浓度递增的洋地黄皂苷处理线粒体部分也可释放己糖激酶。该技术会破坏并差异性地释放线粒体区室。正如在肝脏中观察到的，但与在肿瘤中观察到的情况相反（帕里，D.M.，和佩德森，P.L.（1983年）《生物化学杂志》258，10904 - 10912），肾脏线粒体部分中己糖激酶的释放与已知标记线粒体膜或区室的酶的释放不相关。这些研究提供了关于肾脏中己糖激酶亚细胞定位的首个关键证据。它们表明在该组织中己糖激酶主要与低密度线粒体相关，这一发现为体内这种离散线粒体群体的存在增加了可信度。值得注意的是，己糖激酶与肾脏线粒体的这种关联似乎是独特的，因为其在亚线粒体分级分离时的释放与已知线粒体标记酶的释放不相关。这些结果与肾脏中利用葡萄糖作为能量来源的细胞直接相关。有人提出，这些细胞增强的糖酵解能力可能至少部分归因于己糖激酶与低密度线粒体的关联。
