Rat brain hexokinase: Further studies on the specificity of the hexose and hexose 6-phosphate binding sites

Wilson, John E.; Chung, Vincent

doi:10.1016/0003-9861(89)90135-5

Cited by 40 publications

(22 citation statements)

References 28 publications

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“…We examined whether MJ is able to detach hexokinase from the mitochondrial fraction (MF) isolated from CT26 tumors by measuring hexokinase Figure 1). The detachment of both hexokinase types by MJ as well by the hexokinase reaction product, glucose-6 phosphate (G6P) (Wilson and Chung, 1989), was of a similar order of magnitude. However, there was a notable difference in the extent of hexokinase detachment as revealed by the activity assay versus western blotting.…”

Section: Resultsmentioning

confidence: 93%

“…Two main classes of agents affect mitochondrial hexokinase, that is hexokinase inhibitors and disruptors of hexokinase-mitochondria association. The first class includes competitive and/or allosteric antagonists of hexokinase activity (Wilson and Chung, 1989;Oudard et al, 1995;Brawer, 2005;Robey and Hay, 2005;Lampidis et al, 2006). The second class includes compounds that compete for VDAC-or hexokinase-binding sites, such as hexokinase-binding domain peptides (Majewski et al, 2004), VDAC1-based peptides (Arzoine L, Shoshan-Barmatz V (2006).…”

Section: Discussionmentioning

confidence: 99%

“…p15. www.ecdo.eu), agents covalently modifying VDAC (Robey and Hay, 2005), G6P and its analogs (Wilson and Chung, 1989), enzymes cleaving the N terminus of hexokinase that consists of the VDAC-binding site (Wilson, 2003) and agents with unknown mechanisms of action, such as hypericin and TH-070 (lonidamine) (Majewski et al, 2004;Robey and Hay, 2005). In comparison with the above-mentioned agents, only MJ was demonstrated to bind to hexokinase directly and detach it from VDAC.…”

Section: Discussionmentioning

confidence: 99%

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Methyl jasmonate binds to and detaches mitochondria-bound hexokinase

et al. 2008

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Section: Resultsmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Methyl jasmonate binds to and detaches mitochondria-bound hexokinase

et al. 2008

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“…Therefore, we substituted glucose with the glycolytic inhibitors 5-thio-glucose and 2-deoxy-glucose. 5-thio-glucose is a competitive inhibitor of hexokinase, it cannot be phosphorylated and therefore cannot be effectively used as a hexokinase substrate (Wilson and Chung 1989). In contrast, 2-deoxy-glucose can be phosphorylated but not further metabolized (Chi et al 1987).…”

Section: The First Committed Step Of Glycolysis Is Sufficient For Aktmentioning

confidence: 99%

“…HK activities were normalized to the protein content of the lysate. One milliunit of hexokinase activity represents the amount of enzyme activity required to phosphorylate 1 nmol of glucose in 1 minute at 25°C (Wilson and Chung 1989;Robey et al 2000).…”

Section: Kinase Activity Assaysmentioning

confidence: 99%

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Gottlob¹,

Majewski²,

Kennedy³

et al. 2001

Genes Dev.

866

704

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The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. We provide evidence that such coupling serves as a downstream effector function for Akt. First, Akt increases mitochondria-associated hexokinase activity. Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase. Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane. Extrinsic signals emanating from cell-surface growth factor and cytokine receptors are major determinants of mammalian cell survival. The transduction of these signals oppose both basal intrinsic proapoptotic activity, as well as external proapoptotic stimuli (Raff 1992;Raff et al. 1993). Analysis of downstream signaling pathways has shown that activation of the PI-3 kinase/Akt(PKB) pathway plays a major role in cell survival induced by cell surface receptors. Following the initial demonstration that activation of the serine/threonine kinase Akt promotes cell survival (Dudek et al. 1997; KauffmannZeh et al. 1997;Kennedy et al. 1997), mounting reports established Akt as a major determinant of cell survival. Akt has been reported to mediate cell survival by various growth factors and cytokines in a variety of cell types and blocks apoptosis induced by multiple apoptotic stimuli (for review, see Datta et al. 1999;Kandel and Hay 1999). Various specific targets of Akt have been proposed to mediate the antiapoptotic activity of Akt (for review, see Datta et al. 1999;Kandel and Hay 1999). However, because growth factors promote cell survival via maintenance of the metabolic function of mitochondria , it is likely that Akt exerts its effect through similar mechanisms, which may be more fundamental and generally conserved.In mammalian cells, apoptosis has been described as a multistep process that can be initiated by a variety of stimuli. Mitochondria play a major role in this process through the release of cytochrome c and other proapoptotic proteins that normally reside in the intermembrane space between the inner and outer mitochondrial membranes (for review, see Gross et al. 1999;Desagher and Martinou 2000). Cytochrome c release is considered an ea...

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Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures

Naik

Kumar

Reddy

et al. 2023

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by‐product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase‐2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6‐phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N‐glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2‐deoxy‐ d‐glucose (2DG) and 5‐thio‐ d‐glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO‐Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO‐Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N‐glycan analysis of EPO‐Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG‐supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi‐, tri‐, and tetra‐antennary structures and up to 50% lower EPO‐Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2‐deoxy‐hexose (2DH) on EPO‐Fc N‐glycans and addition of 5TG resulted in the first‐ever observed N‐glycan incorporation of 5‐thio‐hexose (5TH). Six percent to 23% of N‐glycans included 5TH moieties, most likely 5‐thio‐mannose and/or 5‐thio‐galactose and/or possibly 5‐thio‐N‐acetylglucosamine, and 14%–33% of N‐glycans included 2DH moieties, most likely 2‐deoxy‐mannose and/or 2‐deoxy‐galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N‐glycosylation processing, and formation of alternative glycoforms.

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Rat brain hexokinase: Further studies on the specificity of the hexose and hexose 6-phosphate binding sites

Cited by 40 publications

References 28 publications

Methyl jasmonate binds to and detaches mitochondria-bound hexokinase

Methyl jasmonate binds to and detaches mitochondria-bound hexokinase

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures

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