Nakashima R A, Paggi M G, Scott L J, Pedersen P L
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Cancer Res. 1988 Feb 15;48(4):913-9.
Recent studies from this laboratory have demonstrated that a form of hexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a Mr 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904-10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015-1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase. In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction. Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a Km for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding. Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).
本实验室最近的研究表明，一种在快速生长、高度糖酵解的肿瘤细胞中特有的己糖激酶形式，与一种含有分子量为35000的孔蛋白的线粒体外膜受体复合物结合（D.M.帕里和P.L.佩德森，《生物化学杂志》，258:10904 - 10912,1983；R.A.中岛等人，《生物化学》，25:1015 - 1021,1986）。在此报道的新研究中，确定了这种受体复合物结合己糖激酶的特异性，并描述了一种纯化方案，该方案可得到肿瘤己糖激酶的均一且可结合的形式。在AS - 30D肝癌中，己糖激酶活性相对于肝脏组织升高了100多倍。己糖激酶活性的相对增加比任何其他糖酵解酶大8倍。己糖激酶是AS - 30D细胞中唯一一种线粒体/细胞质比活性大于1的糖酵解酶，在线粒体部分升高了3.5倍。己糖激酶的纯化是通过用6 - 磷酸葡萄糖优先溶解线粒体结合的酶，然后在凝胶渗透和阴离子交换柱上进行高效液相色谱来完成的。最终组分的比活性为每毫克蛋白质144单位，对葡萄糖的Km为0.13 mM，对ATP的Km为1.4 mM。纯化的肿瘤酶在十二烷基硫酸钠：聚丙烯酰胺凝胶电泳上迁移为单一物种，表观分子量为98000。值得注意的是，纯化的肿瘤酶保留了其与线粒体结合的活性。色谱、多克隆抗体和氨基酸分析研究的其他结果表明，主要的大鼠肝癌己糖激酶种类与通常称为II型的酶的同工酶形式关系最密切，与肝脏IV型同工酶（葡萄糖激酶）关系最不密切。
