Measurement of the ATP/ADP Ratio in Mitochondria and in the Extramitochondrial Compartment by Fractionation of Freeze-Stopped Liver Tissue in Non-aqueous Media

Elbers, Rembert; Heldt, Hans W.; Schmucker, P.; Soboll, Sibylle; Wiese, Helga

doi:10.1515/bchm2.1974.355.1.378

Cited by 113 publications

(32 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on the marker enzyme distribution in the fractions, metabolite contents and radioactivities in pure mitochondrial and cytosolic fractions were extrapolated according to [12]. Glucose, lactate and pyruvate concentrations in the perfusate, marker enzyme activities and adenine nucleotide contents in the fractions were determined as in [13].…”

Section: Methodsmentioning

confidence: 99%

Control of energy metabolism by glucagon and adrenaline in perfused rat liver

Soboll¹,

Scholz²

1986

FEBS Letters

Self Cite

View full text Add to dashboard Cite

Changes in subcellular distribution of adenine nucleotides, mitochondrial/cytosolic proton gradients, rates of respiration, gluconeogenesis (fasted state) and glycogenolysis (fed state) were studied in isolated perfused rat livers following addition of glucagon (IO-@ M) or adrenaline (lo-' M). Glucagon increased the gradient in all states. The cytosolic ATP/ADP ratio was increased in the fasted but decreased in the fed state which is consistent with a diminished futile cycling in gluconeogenesis (fasted state) or a decreased glycolytic rate (fed state). Adrenaline caused an increase in the proton gradient and the mitochondrial ATP/ADP ratio. The two effects are attributed to increased calcium entry into the matrix space. Subcellular distribution Adenine nucleotide Proton gradient Caz+ effect(Perfused rat liver) Glucagon Adrenaline

show abstract

Section: Methodsmentioning

confidence: 99%

Control of energy metabolism by glucagon and adrenaline in perfused rat liver

Soboll¹,

Scholz²

1986

FEBS Letters

Self Cite

View full text Add to dashboard Cite

show abstract

“…Metabolite distribution in the tissue was determined principally according to [6]. The dry tissue was homogenized in heptane/carbon tetrachloride mixture in a Biihbr homogenizer thermostated at -36°C.…”

Section: Tissue Sampling and Fraetionutionmentioning

confidence: 99%

“…Here, advantage has been taken of the method of tissue fractionation in non-aqueous media [6] to measure the cytosolic and m~tochondrial phospl~orylation potentials. The results show that the error in estimating the cytosolic phosphorylation potential from total tissue concentration of the reactants is quite small when calculated from the creatine kinase equilibrium.…”

Section: Introductionmentioning

confidence: 99%

Subcellular distribution of phosphagens in isolated perfused rat heart

1980

View full text Add to dashboard Cite

“…In order to avoid these difficulties and to keep the fractionation procedure as short as possible, we have worked out an alternative approach. Based on a procedure for the fractionation of liver developed in our laboratory (5), the fractionation of the dried tissue is carried out by a single density gradient centrifugation, resulting in an enrichment of the subcellular materials in the various fractions. In each of these fractions metabolites and marker enzymes are assayed, and from these data the subcellular distribution of the metabolites is evaluated.…”

mentioning

confidence: 99%

Measurement of Subcellular Metabolite Levels in Leaves by Fractionation of Freeze-Stopped Material in Nonaqueous Media

Gerhardt

Heldt²

1984

Plant Physiol.

Self Cite

246

188

View full text Add to dashboard Cite

ABSTRAC1This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia okracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifuption.Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated.As a frst application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole fails from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately I millimolar at the end of the night.An understanding of the metabolism of a plant cell requires information about the metabolite levels in the various metabolic compartments, such as the chloroplast, the mitochondria, the vacuole, and the cytosol. This demands a procedure for tissue fractionation during which the total content and the subcellular distribution of metabolites in these compartments is not altered. Recently, methods have been developed for the assay of subcellular metabolite levels in plant protoplasts in which the protoplasts were ruptured by passage through a nylon net or a capillary tube followed by immediate filtration of the particles either through a layer of silicone oil (8, 15, 18) or a combination of membrane filters (12). The measurement of subcellular metabolite levels in whole leaves is more difficult; one possible approach is to use a nonaqueous fractionation procedure similar to that developed in 1932 by Behrens (2) for the fractionation of nuclei. In this method the tissue is frozen and lyophilized, the dried material homogenized in nonaqueous solvents, and the resulting homogenate is fractionated by centrifugation. The absence of water prevents the enzymic interconversion of metabolites, and the polar enzymes and metabolites are not extracted during the treatment of the cell material with the nonpolar ' Supported by the Deutsche Forschungsgemeinschaft.solvents. This method has also been successfully applied to the separation of chloroplasts (9, 17). The general principle was to purify a chloroplast fraction by several centrifugation steps. Such procedure involves the loss of a large portion ofchloroplasts...

show abstract

Measurement of the ATP/ADP Ratio in Mitochondria and in the Extramitochondrial Compartment by Fractionation of Freeze-Stopped Liver Tissue in Non-aqueous Media

Cited by 113 publications

References 0 publications

Control of energy metabolism by glucagon and adrenaline in perfused rat liver

Control of energy metabolism by glucagon and adrenaline in perfused rat liver

Subcellular distribution of phosphagens in isolated perfused rat heart

Measurement of Subcellular Metabolite Levels in Leaves by Fractionation of Freeze-Stopped Material in Nonaqueous Media

Contact Info

Product

Resources

About