Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

Magnani, Mauro; Stocchi, Vilberto; Serafini, Giordano; Chiarantini, Laura; Fornaini, G

doi:10.1016/0003-9861(88)90462-6

Cited by 35 publications

(24 citation statements)

References 34 publications

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“…D and Table ). Their affinities for glucose were 0.08 ± 0.01 ( K m1(glc) ) and 0.44 ± 0.2 m m ( K m2(glc) ), similar to the K m(glc) values reported for HKI and HKII of 0.03–0.06 and 0.13–0.3 m m , respectively . In contrast, in hyperglycemic cells, one predominant kinetic component was evident (Fig.…”

Section: Resultssupporting

confidence: 78%

Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms

et al. 2014

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The effect of hypoglycemia on the contents of glycolytic proteins, activities of enzymes/transporters and flux of HeLa and MCF-7 tumor cells was experimentally analyzed and modeled in silico. After 24 h hypoglycemia (2.5 mM initial glucose), significant increases in the protein levels of glucose transporters 1 and 3 (GLUT 1 and 3) (3.4 and 2.1-fold, respectively) and hexokinase I (HKI) (2.3-fold) were observed compared to the hyperglycemic standard cell culture condition (25 mM initial glucose). However, these changes did not bring about a significant increase in the total activities (V max ) of GLUT and HK; instead, the affinity of these proteins for glucose increased, which may explain the twofold increased glycolytic flux under hypoglycemia. Thus, an increase in more catalytically efficient isoforms for two of the main controlling steps was sufficient to induce increased flux. Further, a previous kinetic model of tumor glycolysis was updated by including the ratios of GLUT and HK isoforms, modified pyruvate kinase kinetics and an oxidative phosphorylation reaction. The updated model was robust in terms of simulating most of the metabolite levels and fluxes of the cells exposed to various glycemic conditions. Model simulations indicated that the main controlling steps were glycogen degradation > HK > hexosephosphate isomerase under hyper-and normoglycemia, and GLUT > HK > glycogen degradation under hypoglycemia. These predictions were experimentally evaluated: the glycolytic flux of hypoglycemic cells was more sensitive to cytochalasin B (a GLUT inhibitor) than that of hyperglycemic cells. The results indicated that cancer glycolysis should be inhibited at multiple controlling sites, regardless of external glucose levels, to effectively block the pathway. DatabaseThe mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at

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Section: Resultssupporting

confidence: 78%

Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms

et al. 2014

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“…5B dashed line). These features made CpHK differ from that of T. gondii (TgHK) that could use fructose at a much higher efficiency (i.e., 79% in comparison with glucose) (Saito et al 2002), or from humans that could use all three hexoses at similar efficiencies, e.g., 78% - 106% for mannose and 90% - 130% for fructose (Magnani et al 1988; Stocchi et al 1982). …”

Section: Resultsmentioning

confidence: 99%

“…5C). The ability for CpHK to use ATP and other NTPs (albeit with less efficiencies) was also unique, as T. gondii and human HKs were virtually incapable or with much less capabilities on other NTPs (i.e., <2% or <5% activity, respectively) (Magnani et al 1988; Saito et al 2002). Additionally, ATP displayed substrate inhibition on CpHK at physiological concentrations (i.e., peak activity at 2.5 mM and completely inhibited at 10 mM) (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Unique Hexokinase in Cryptosporidium parvum, an Apicomplexan Pathogen Lacking the Krebs Cycle and Oxidative Phosphorylation

Zhang

Guo

et al. 2014

Protist

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Cryptosporidium parvum may cause virtually untreatable infections in AIDS patients, and is recently identified as one of the top four diarrheal pathogens in children in developing countries. Cryptosporidium differs from other apicomplexans (e.g., Plasmodium and Toxoplasma) by lacking many metabolic pathways including the Krebs cycle and cytochrome-based respiratory chain, thus relying mainly on glycolysis for ATP production. Here we report the molecular and biochemical characterizations of a hexokinase in C. parvum (CpHK). Our phylogenetic reconstructions indicated that apicomplexan hexokinases including CpHK were highly divergent from those of humans and animals (i.e., at the base of the eukaryotic clade). CpHK displays unique kinetic features that differ from those in mammals and Toxoplasma gondii (TgHK) in the preference towards various hexoses and its capacity to use ATP and other NTPs. CpHK also displays substrate inhibition by ATP. Moreover, 2-deoxy-D-glucose (2DG) could not only inhibit the CpHK activity, but also the parasite growth in vitro at concentrations nontoxic to host cells (IC50 = 0.54 mM). While the exact action of 2-deoxy-D-glucose on the parasite is subject to further verification, our data suggest that CpHK and the glycolytic pathway may be explored for developing anti-cryptosporidial therapeutics.

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“…All isoenzymes, however, seem to exist in multiple molecular forms. Numerous reports provide evidence for a HK I microheterogeneity [Easterby, 1975;Ueda et al, 1975;Vowles and Easterby, 1979;Needels and Wilson, 1983;Magnani et al, 1988; for further references see Wilson (1985)]. Reports indicating multiple forms of the isoenzyme II are rare.…”

Section: Introductionmentioning

confidence: 99%

Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A

Rempel

Bannasch

Mayer

1994

Biochemical Journal

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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 was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.

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Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

Cited by 35 publications

References 34 publications

Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms

Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms

A Unique Hexokinase in Cryptosporidium parvum, an Apicomplexan Pathogen Lacking the Krebs Cycle and Oxidative Phosphorylation

Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A

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