scholarly journals Validity of the digitonin method for metabolite compartmentation in isolated hepatocytes

1980 ◽  
Vol 188 (1) ◽  
pp. 207-212 ◽  
Author(s):  
D G Brocks ◽  
E A Siess ◽  
O H Wieland
Keyword(s):  
Low Temperature ◽  
Adenine Nucleotides ◽  
Isolated Hepatocytes ◽  
Cell Fractionation ◽  
Conventional Procedure ◽  
Cellular Content ◽  
Temperature Method

1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the ‘convaentional’ method) was developed that allows the fractionation of isolated hepatocytes at −5 degrees C (i.e. ‘low-temperature’ method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.

scholarly journals Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate

1977 ◽  
Vol 166 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Elmar A. Siess ◽  
Dietrich G. Brocks ◽  
Herbert K. Lattke ◽  
Otto H. Wieland
Keyword(s):  
Adenine Nucleotides ◽  
Isolated Hepatocytes ◽  
Cell Fractionation ◽  
Acetyl Coa ◽  
Isolated Rat Liver ◽  
Rat Liver Cells ◽  
The Matrix ◽  
Isolated Rat ◽  
Reduced State ◽  
Stimulation Of

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40–50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD+]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP+]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD+]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.

RETRACTED: Low temperature method for synthesis of ZnS quantum dots and its luminescence characterization studies

2013 ◽  
Vol 264 ◽  
pp. 17-20 ◽  
Author(s):  
K. Senthilkumar ◽  
T. Kalaivani ◽  
S. Kanagesan ◽  
V. Balasubramanian
Keyword(s):  
Quantum Dots ◽  
Low Temperature ◽  
Temperature Method ◽  
Characterization Studies

scholarly journals A low-temperature method for the isolation of small-intestinal epithelium along the crypt-villus axis

1991 ◽  
Vol 280 (2) ◽  
pp. 331-334 ◽  
Author(s):  
N Flint ◽  
F L Cove ◽  
G S Evans
Keyword(s):  
Small Intestine ◽  
Low Temperature ◽  
Intestinal Epithelium ◽  
Sequential Fractionation ◽  
Potential Value ◽  
Temperature Method ◽  
Small Intestinal ◽  
The Comparative Study ◽  
Cell Phenotypes ◽  
Proliferative Cell

A variety of enzymic and non-enzymic methods to isolate epithelium from the small intestine have been previously published. Sequential fractionation of cells from the villus to the crypt has been reported in some of these papers, which allows the comparative study of terminally differentiated and proliferative cell phenotypes. However, these methods often involve the incubation of tissues at 37 degrees C, which may affect the structural and biochemical integrity of the cells. We have developed a rapid low-temperature (4 degrees C) method for isolating purified populations of crypt and villus cells from mouse and rat intestines. The fractionated cells have been partially characterized, and the potential value of the procedure has been indicated by the ability to analyse the comparative protein and mRNA expression along the crypt-villus axis.

Structural and optical characterization of hemimorphite with flower-like morphology synthesized by a novel low-temperature method

2012 ◽  
Vol 85 ◽  
pp. 138-141 ◽  
Author(s):  
Ivana Lj. Validžić ◽  
Miodrag Mitrić ◽  
Bojan M. Jokić ◽  
Mirjana I. Čomor
Keyword(s):  
Low Temperature ◽  
Optical Characterization ◽  
Temperature Method

Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins

1993 ◽  
Vol 264 (5) ◽  
pp. G880-G885 ◽  
Author(s):  
Y. Yamaguchi ◽  
E. Dalle-Molle ◽  
W. G. Hardison
Keyword(s):  
Horseradish Peroxidase ◽  
Parenchymal Cell ◽  
Fluid Phase ◽  
Mannose Receptor ◽  
Isolated Hepatocytes ◽  
Cell Counting ◽  
Cell Fractionation ◽  
Morphological Studies ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation

1988 ◽  
Vol 250 (3) ◽  
pp. 641-645 ◽  
Author(s):  
H Hummerich ◽  
H de Groot ◽  
T Noll ◽  
S Soboll
Keyword(s):  
High Pressure ◽  
Oxygen Partial Pressure ◽  
Partial Pressure ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Adenine Nucleotides ◽  
Strong Dependence ◽  
Isolated Hepatocytes ◽  
Pressure Filtration ◽  
Filtration Technique

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.

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