The Role of Mitochondrial Hexokinase in Neoplastic Phenotype and Its Sensitivity to Lonidamine a (original) (raw)
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The International journal of biochemistry, 1990
1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.
Glucose Catabolism in Cancer Cells: Amplification of the Gene Encoding Type II Hexokinase1
Hexokinase type II is highly overexpressed in many cancer cells, where it plays a pivotal role in the high glycolytic phenotype. Here we demon strate by Southern blot analysis and fluorescence in situ hybridization (FISH) that in the rapidly growing rat AS-30D hepatoma cell line, en hanced hexokinase activity is associated with at least a 5-fold amplification of the type II gene relativeto normalhepatocytes. This amplificationis located chromosomally, extends to the whole gene, and most likely occurs at the site of the residentgene. No rearrangement of the gene could be detected. Therefore, overexpression of hexokinase type II in AS-30D hepatoma cells may be based, at least in part, on a stable gene amplifica tion. This is the first report describing the amplification of a hexokinase gene in a tumorcell line expressingthe high glycolyticphenotype.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
Receivec tract "he activity, intracellular distribution and mRNA expression telial liver cells at different stages of neoplastic transformatio , all transformed cells exhibited only hexokinase I and II, whi~ abundant form. In parallel, the mRNA expression of the t~ ession. Hexokinase I and II were found in the cytosol and b¢ me activity increased with the grade of transformation from 3~ • The ratio of hexokinase I/II was higher in the membrane fr lved in two subtypes IIa and IIb by hydrophobic interaction c ificantly between normal liver (1:1) and transformed cells, onstrated the main activity in the more differentiated, IIb in t the intracellular glucose 6-phosphate concentration of the ceL rogressive alterations in the proportion and subcellular distrib which both showed significantly inc two isoenzymes was elevated, in(bound to mitochondrial membram 32% of total activity in normal li~ tembrane fraction than in the cytosol. In all chromatography. The relative pr(and among cells of different tr the less differentiated cell lines. cells. The data indicate that neopla distribution of hexokinase isoenzymes Microheterogeneity; Intracellular distribution; Hepatoma; Neoplastic cell account at least in part cancer cells [6,7]. In t~ y of key enzymes of glucose nantly localized on the ninon biochemical aberration The reversible associati transformation of liver cells represents an importan dyzes the first step of glycoly-enzyme activity, since ant control point in this path-sensitivity to glucose 6-1 rated activity in many tumor pared to the soluble fo tctivity shows a good correla-directly mitochondriall 3 malignancy of the tumor [4,5], the bound enzyme repre elevated levels of hexokinase ations in activity and su in neoplastic cells are isoenzyme profile, leadi ,'rum albumin; CFE, colony forming Mammalian hexokinase opropyl)dimethylammonio)-l-propan-(HK I-IV) with distinct idine: Glc6-P. ~lucose 6-nhosDhate: ti~,~ [1 1l lq'~nntnma~ u~ more expression. enzyme cells• resolved significantly demonstrated with by progl
Targeting of hexokinase 1 to liver and hepatoma mitochondria
Proceedings of the National Academy of Sciences, 1992
The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound.
Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A
The Biochemical journal, 1994
Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity ...
British journal of cancer, 1996
Loss of chromosome 10 was observed in 10 out of 12 xenografted glioblastomas studied. Chromosome 10 carries the gene coding the hexokinase type 1 isoenzyme (HK-I), which catalyses the first step of glycolysis, which is essential in brain tissue and glioblastomas. We investigated the relationships between the relative chromosome 10 number, the amount of HK-I mRNA, HK-I activity and its intracellular distribution, and glycolysis-related parameters such as the lactate-pyruvate ratio, lactate dehydrogenase (LDH) and ATP contents. Individual tumour HK-I mRNA amounts were 23-65% lower than that of normal human brain and reflected the relative decrease of chromosome 10 number (alpha < 0.01). Total HK activities of individual glioblastomas varied considerably but were constantly (a mean of seven times) lower than that of normal brain tissue. The mitochondria-bound HK-I fraction of individual tumours was generally over 50%, compared with that of normal brain tissue. As shown by lactate - ...
The Association of Brain Hexokinase with Mitochondrial Membranes and its Functional Implications
European Journal of Biochemistry, 1970
The intracellular location of brain hexokinase and the nature of its association with mitochondria were studied in calf and rat. Three different media were used in order to minimize possible artifacts in the homogenization process.Subcellular fractionation was followed testing, in addition to hexokinase, certain enzymes known as markers for the different cellular fractions. In the conditions used, hexokinase was almost entirely associated with mitochondria in calf, but only partially associated in rat. This is at variance with the rest of the enzymes involved in the glucose 6-phosphate crossroads. The use of a “physiological saline” homogenization medium resulted in a considerable increase in the proportion of soluble hexokinase in rat brain.Repeated washings of mitochondria from calf brain released little hexokinase, but in rat brain a rapid release was observed.Upon submitochondrial fractionation by Sottocasa's method, a substantial amount of hexokinase appeared bound to the inner membrane in the case of calf brain. In rat brain, the evidence found also points to the binding of the greater part of hexokinase to the inner membrane.Brain mitochondria were found to be sensitive to atractyloside, which made it possible to locate mitochondrial bound hexokinase as external to the ATP barrier, both in calf and rat.These results indicate that mitochondrially bound hexokinase is only active on substrates located in the cytosol.
The functional compartmentation of mitochondrial hexokinase
Archives of Biochemistry and Biophysics, 1974
These studies examined the functiona. relationship between rat hepatic mitochondria and associated hexokinase (ATP: o-hexose&phosphotransferase, 2.7.1.1) to determine whether the binding of hexokinase to mitochondria might provide a privileged interaction with sites of ATP production. Initial kinetic analysis followed the sequential flow of phosphate through ATP generated by the mitochondria into glucose-6-phosphate catalyzed by the bound hexokinase. Kinetics were compared with an identical bound hexokinase-mitochon drial system using externally supplied ATP. The hexokinase had lower apparent K, values for ATP generated in the mitochondria from supplied ADP than for ATP provided. Respiratory inhibitors blocked both the ADP-and ATP-mediated reactions. Tracer studies further documented that the mitochondrial hexokinase initially and preferentially utilized the internally generat.ed nucleotide. These studies demonstrate that the act,ive site of bound hexokinase is relatively inaccessible to extramitochondrial ATP. They provide evidence that bound hexokinase can sequentially accept mitochondrially generated ATP in a kinetically advantageous way. Finally, they support the assumption that mitochondrial binding of this acceptor enzyme may play a propitious role in cellular energy economy.