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.

Improvement in D-E Hysteresis Curve Squareness of PZT Thin Films - Effect of Oxygen Partial Pressure in High-Pressure Post-Annealing

2003 ◽  
Vol 52 (1) ◽  
pp. 137-145
Author(s):  
Junichi Karasawa ◽  
Taku Aoyama ◽  
Takeshi Kijima ◽  
Eiji Natori ◽  
Tatsuya Shimoda
Keyword(s):  
Thin Films ◽  
High Pressure ◽  
Oxygen Partial Pressure ◽  
Partial Pressure ◽  
Hysteresis Curve ◽  
Pzt Thin Films ◽  
Post Annealing ◽  
Effect Of Oxygen

Purine metabolism in isolated rat hepatocytes

1977 ◽  
Vol 55 (11) ◽  
pp. 1134-1139 ◽  
Author(s):  
Camilla M. Smith ◽  
Liisa M. Rovamo ◽  
Martti P. Kekomäki ◽  
Kari O. Raivio
Keyword(s):  
Cell Function ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Purine Metabolism ◽  
Nucleotide Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Collagenase Perfusion ◽  
Adenine Nucleotide Pool

The metabolism of adenine, hypoxanthine, guanine, and adenosine was studied in rat liver cell suspensions, prepared by collagenase perfusion. Oxygen supply was a critical variable in the preparation and subsequent incubation of the cells, as judged on the basis of the ratio of radioactivity in ATP to that in ADP after incubation with [14C]adenine. This ratio is suggested as an additional criterion of cell function. Adenine nucleotides synthesized from [14C]adenine were slowly catabolized to allantoin, with little incorporation of radioactivity into other purine compounds. [14C]Adenine is thus suitable for prelabelling the adenine nucleotide pool. [14C]Guanine and [14C]hypoxanthine were rapidly catabolized to allantoin, whereas nucleotide synthesis was low. [14C]Adenosine was initially phosphorylated and deaminated at about equal rates, but with continued incubation catabolic products predominated. Isolated hepatocytes were found suitable for studies of purine metabolism, in which the liver has important functions for the whole organism.

scholarly journals Measurement of Nucleotide Pools in Platelets and Red Blood Cells Using High Pressure Liquid Chromatography

1977 ◽  
Author(s):  
L. D’Souza ◽  
H. I. Glueck
Keyword(s):  
High Pressure ◽  
Platelet Aggregation ◽  
Adenine Nucleotide ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Single Step ◽  
Small Sample ◽  
Normal Donor ◽  
Chromatographic Technique ◽  
Nucleotide Pools

Release of adenine nucleotides during platelet aggregation is well established. The released adenosine diphosphate (ADP) is thought to cause secondary platelet aggregation. A high pressure liquid chromatographic technique has been used to measure accurately the various nucleotide pools in intact platelets, as well as their levels during various stages of aggregation. Accurate and highly reproducible measurements of 10-12 moles of all nucleotides are possible using the bonded microparticulate, Partisil −10 SAX column, a strong anion exchanger (Whatman Inc., Clifton, N. J.) and an electronic integrator. The method is a modification of an earlier method of P. Brown (1973), and G. Rao et al (1975). Using this technique we are able to obtain within 75 minutes a single step gradient separation of cyclic nucleotides (cAMP, cCMP, cGMP) as well as mono-, di-, and tri-phosphonucleosides. Sample sizes of 0.5 ml of platelet rich plasma are adequate for at least 3 estimations. Normal donor platelets show the following adenine nucleotide levels;* AMP 0.13 (0.14); ADP 1.64 (0.81); and ATP 4.90 (0.42) nanomoles per 1011 platelets. The method has certain advantages over the enzymatic or luciferase assays, since all the nucleotides can be simultaneously measured in a single step from a very small sample and there is no cross reactivity with any of the nucleotides. Similar studies of plasma and red blood cell nucleotides are possible with this technique.*Mean (standard deviation)

Regulation of the mitochondrial ATP-Mg/Pi carrier in isolated hepatocytes

1993 ◽  
Vol 264 (3) ◽  
pp. C663-C670 ◽  
Author(s):  
D. T. Dransfield ◽  
J. R. Aprille
Keyword(s):  
Subcellular Distribution ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Isolated Hepatocytes ◽  
Cellular Regulation ◽  
Intact Cells ◽  
Nucleotide Content ◽  
Dose Dependent

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.

Adenine and Adenosine Metabolism in Intact Erythrocytes Deficient in Adenosine Monophosphate-Pyrophosphate Phosphoribosyltransferase: A Study of Two Families

1978 ◽  
Vol 55 (4) ◽  
pp. 407-412 ◽  
Author(s):  
B. M. Dean ◽  
D. Perrett ◽  
H. Anne Simmonds ◽  
A. Sahota ◽  
K. J. Van Acker
Keyword(s):  
High Performance ◽  
Adenine Nucleotide ◽  
Intact Cell ◽  
Adenine Nucleotides ◽  
Adenosine Monophosphate ◽  
Cell System ◽  
Adenine Phosphoribosyltransferase ◽  
Phosphoribosyl Pyrophosphate ◽  
Control Activity ◽  
Adenine Nucleotide Pool

1. We have studied the metabolism of adenine and adenosine in intact erythrocytes from three children and four adults of two separate families. The children were homozygotes for a deficiency of AMP-pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, EC 2.4.2.7), and the parents heterozygotes. 2. In this intact cell system at physiological concentrations of inorganic phosphate (Pi) and adenine heterozygote adenine phosphoribosyltransferase activity was normal, whereas at higher adenine concentrations (1.0–10 μmol/l) the four heterozygotes showed approximately 50% of control activity. At high concentrations of Pi (18 mmol/l) and adenine (10 μmol/l) and with extended incubation heterozygote activity was again indistinguishable from normal. Activity of the enzyme in homozygotes was negligible under all these conditions. 3. Formation of inosine 5′-monophosphate from [8-14C]hypoxanthine at high Pi concentrations was normal in both homozygotes and heterozygotes. Thus no abnormality of either of the enzymes IMP—pyrophosphate phosphoribosyltransferase (hypoxanthine phosphoribosyltransferase, EC 2.4.2.8) or phosphoribosyl pyrophosphate synthetase (ribose phosphate pyrophosphokinase, EC 2.7.6.1) was found. 4. To ascertain whether deficiency of adenine phosphoribosyltransferase was linked with enhanced conversion of adenosine into adenine nucleotides at the expense of its deamination pathways, the complete metabolism of [8-14C]adenosine was studied over the range 0.1–9.0 μmol/l. No such increase was observed for either heterozygotes or homozygotes. 5. Insignificant formation of either [8-14C]-adenine from [8-14C]adenosine or vice versa in the erythrocytes of homozygotes lacking adenine phosphoribosyltransferase but capable of forming hypoxathine from adenosine, supports the concept that adenine and adenosine are not directly interconvertible and do not utilize purine nucleoside phosphorylase (EC 2.4.2.1) in erythrocytes. 6. Measurement of erythrocyte nucleotide concentrations by high-performance liquid chromatography revealed no obvious abnormalities. ATP:ADP:AMP proportions were normal (approximately 10:1:0.1) and the total adenine nucleotide pool was at the lower limit of the normal range for both homozygotes and heterozygotes. These observations imply that deficiency of adenine phosphoribosyltransferase does not grossly affect adenine nucleotides synthesis in the erythrocyte.

The Purine Nucleotide Profile in Mouse, Chicken and Human Dystrophic Muscle: An Abnormal Ratio of Inosine plus Adenine Nucleotides to Guanine Nucleotides

1982 ◽  
Vol 62 (1) ◽  
pp. 113-115 ◽  
Author(s):  
R. J. Shuttlewood ◽  
J. R. Griffiths
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Purine Nucleotide ◽  
Dystrophic Muscle ◽  
Human Patient ◽  
Abnormal Ratio

1. AMP, ADP, ATP, IMP, GDP, GTP and adenylosuccinate have been measured by high pressure liquid chromatography in three types of animal muscular dystrophy and in a human patient with Duchenne muscular dystrophy. 2. Abnormalities in nucleotide content varied from one dystrophy to another. 3. In each case, however, the ratio [total adenine nucleotide + IMP]/[total guanine nuclotides] was lower in dystrophic muscle, even when severely exercised or ischaemic muscles were used. 4. The practical advantages of this assay for diagnosis of muscular dystrophy are discussed.

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