An improved assay for hexokinase activity in human tissue homogenates

Scheer, W D; Lehmann, H.; Beeler, Myrton F.

doi:10.1016/0003-2697(78)90531-6

Cited by 37 publications

(16 citation statements)

References 11 publications

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“…Hexokinase was measured by the method of Scheer et al [30]. The supernatant was prepared by homogenizing the tissue in 9 volumes of ice-cold 150 mmol/L potassium chloride/5 mmol/L MgCl 2 /5 mmol/L EDTA/5 mmol/L 2-mercaptoethanol for 1 minute and centrifuging at 14,500 Â g for 20 minutes at 48C.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Effect of hind limb muscle unloading on liver metabolism of rats

Stein

Schluter

Galante³

et al. 2005

The Journal of Nutritional Biochemistry

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Section: Enzyme Assaysmentioning

confidence: 99%

Effect of hind limb muscle unloading on liver metabolism of rats

Stein

Schluter

Galante³

et al. 2005

The Journal of Nutritional Biochemistry

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“…This difference presumably arises because acinar hexokinase activities are so low that, during the first 8 rain, NADP + reduction in the 6PGDH reaction is limited by the availability of the 6PGA produced in the coupled reaction. It may also reflect cellular variations in endogenous 6PGDH activity since previous biochemical studies have shown a significant relationship between tissue 6PGDH activity and the overestimation of hexokinase activity in homogenates (Scheer et al, 1978;Lawrence & Trayer, 1985;Lawrence et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity

1989

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The effect of endogenous 6-phosphogluconate dehydrogenase (6PGDH) on the histochemical quantification of low-Km hexokinase activity in rat submandibular salivary glands has been investigated using a Seescan Solitaire Plus image analysis system and a modified black and white Newvicon TV camera. Absorbance readings of neutral density filters were close to their known absorbance values. A significant correlation was found between the absorbance of Nitro BT reduction products within sections, with and without the use of a 588 nm interference filter. Furthermore, absorbance readings obtained from 8 microns-thick sections were 1.92 and 2.22 times greater than values obtained from 4 microns-thick sections using 'white' and 588 nm light respectively. The level of background illumination was not critical for absorbance measurements provided it was below the level that saturated the Newvicon camera and was not changed between background and image capture. The greatest variations in absorbance readings on tissue sections were associated with changes in zoom and objective combinations. Our studies indicate that this relatively low cost image analysis system can give reproducible absorbance readings from various structures defined in digitized, captured images of tissue sections. Results from continuous monitoring studies indicated that the high levels of 6PGDH activity in excretory ducts caused a 1.67-fold overestimation of hexokinase activity as assessed by absorbance readings performed throughout a 22-minute reaction period. By contrast, overestimation of hexokinase activity in salivary gland acini only became apparent after 8 min incubation with a 1.4-fold overestimation being seen at 22 min. This difference appears to reflect the relatively low hexokinase and 6PGDH activities present in acini compared with excretory ducts.

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“…HK activity was measured enzymatically in mitochondrial fractions of control and PAF-activated human neutrophils by the method of Scheer in which the downstream product of reaction (NADPH) is quantified spectrophotometrically [30]. Cell fractions from isolated human neutrophils were diluted to 400 ug protein/ml.…”

Section: Methodsmentioning

confidence: 99%

18 F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury

Rodrigues

Bozza

Hanrahan

et al. 2017

Nuclear Medicine and Biology

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Introduction Molecular imaging of the earliest events related to the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) could facilitate therapeutic development and patient management. We previously reported that 18F-fluoro-2-deoxyglucose (18F-FDG) PET identifies ALI/ARDS prior to radiographic abnormalities. The purpose of this study was to establish the time courses of 18F-FDG uptake, edema and neutrophil recruitment in an endotoxin-induced acute lung injury model and to examine molecular events required for 14C-2DG uptake in activated neutrophils. Methods Lung uptake of 18F-FDG was measured by PET in control male Sprague Dawley rats and at 2, 6 and 24 hours following the intraperitoneal injection of 10 mg/kg LPS. Lung edema (attenuation) was measured by microCT. Neutrophil influx into the lungs was measured by myeloperoxidase assay. Control and activated human donor neutrophils were compared for uptake of 14C-2DG, transcription and content of hexokinase and GLUT isoforms and for hexokinase (HK) activity. Results Significant uptake of 18F-FDG occurred by 2h following LPS, and progressively increased to 24h. Lung uptake of 18F-FDG preceded increased CT attenuation (lung edema). Myeloperoxidase activity in the lungs, supporting neutrophil influx, paralleled 18F-FDG uptake. Activation of isolated human neutrophils resulted in increased uptake of 14C-2DG, expression of GLUT 3 and GLUT 4 and expression and increased HK1 activity. Conclusion Systemic endotoxin-induced ALI results in very early and progressive uptake of 18F-FDG, parallels neutrophil accumulation and occurs earlier than lung injury edema. Activated neutrophils show increased uptake of 14C-2DG, expression of specific GLUT3, GLUT4 and HK1 protein and HK activity. Advances in knowledge and implications for patient care 18F-FDG pulmonary uptake is an early biomarker of neutrophil recruitment in ALI and is associated with specific molecular events that mediate 14C-2DG uptake in activated neutrophils. 18F-FDG PET may provide a potential mechanism for early diagnosis and therapeutic assessment of ALI/ARDS.

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An improved assay for hexokinase activity in human tissue homogenates

Cited by 37 publications

References 11 publications

Effect of hind limb muscle unloading on liver metabolism of rats

Effect of hind limb muscle unloading on liver metabolism of rats

The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity

18 F-fluoro-2-deoxyglucose PET informs neutrophil accumulation and activation in lipopolysaccharide-induced acute lung injury

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