The Hexokinases: Kinetic, Physical, and Regulatory Properties

Many cell types release ATP in response to mechanical or biochemical stimulation. The mechanisms responsible for this release, however, are not well understood and may differ among different cell types. In addition, there are numerous difficulties associated with studying the dynamics of ATP release immediately outside the cell membrane. Here, we report a new method that allows the visualization and quantification of ATP release by fluorescence microscopy. Our method utilizes a two-enzyme system that generates NADPH when ATP is present. NADPH is a fluorescent molecule that can be visualized by fluorescence microscopy using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The method is capable of detecting ATP concentrations <1 microM and has a dynamic range of up to 100 microM. Using this method, we visualized and quantified ATP release from human polymorphonuclear leukocytes and Jurkat T cells. We show that upon cell stimulation, the concentrations of ATP can reach levels of up to 80 microM immediately outside of the cell membrane. This new method should prove useful for the study of the mechanisms of release and functional role of ATP in various cell systems, including individual cells.

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“…2D). This may be a result of substrate inhibition (29). Based on the data from experiments shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

Corriden¹,

Insel²,

Junger³

2007

American Journal of Physiology-Cell Physiology

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“…Other tissues do not express aldolase B or ketohexokinase and are thought to metabolize fructose via fructose 6-phosphate (Fru-6-P) by the action of a hexokinase, as proposed for adipose [14]. The issue whether hexokinase has any appreciable activity toward fructose in vivo, in the presence of glucose, remains controversial [40].…”

Section: Introductionmentioning

confidence: 99%

Genes required for fructose metabolism are expressed in Purkinje cells in the cerebellum

Funari

Herrera

Freeman

et al. 2005

Molecular Brain Research

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“…It w^s reported in the early 1970s that the regulation of these hexokinases was different. While hexokinase I, II, and III are inhibited by glucose 5-phosphate, this inhibition is relieved by orthophosphate for hexokinase I alone (37). The glucose 5-phosphate inhibition of hexokinase II, and hexokinase III is not relieved by orthophosphate (38,39).…”

Section: Hexokinasementioning

confidence: 99%

“…The enzyme was judged pure by native(155,157) and SDS (158) gel electrophoresis. The correct identity of the hexokinase II was confirmed by kinetic analysis(37).Protein was assayed according to the method of Bradford (162). Purified hexokinase II, puri fied hexokinase I, and muscle extracts were subjected to electrophoresis (7% native polyacrylamide gels) and transferred to nitrocellulose paper.The assay was completed by two separate methods.…”

mentioning

confidence: 99%

The effect of diabetes and insulin on the turnover of hexokinase II in the skeletal muscle

Frank

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The Hexokinases: Kinetic, Physical, and Regulatory Properties

Cited by 69 publications

References 225 publications

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells

Genes required for fructose metabolism are expressed in Purkinje cells in the cerebellum

The effect of diabetes and insulin on the turnover of hexokinase II in the skeletal muscle

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