The study of metabolic turnover rates by means of isotopic tracers

Reiner, John M.

doi:10.1016/0003-9861(53)90170-2

Cited by 150 publications

(24 citation statements)

References 8 publications

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“…Cells filled with nonradioactive cholesterol complexes took up label to 80';~ of the control value. When labeled medium was withdrawn, and the washout followed, net flux would occur if ks2 --* Ks., > ks2, or k2a --* K2a < k2a, (18)(19)(20), which would produce net movement of cholesterol from compartment 3 to compartment 2. The consequence of this would be an increased loss of label from the cells.…”

Section: Ingestion and Intraeellular Fate Of Cholesterol-3h-albumin Cmentioning

confidence: 99%

Cholesterol Metabolism in the Macrophage

Werb

Cohn

1972

The Journal of Experimental Medicine

123

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Previous studies have described the exchange of free cholesterol molecules of macrophage membranes with those of serum lipoproteins (1). Exchangeable cholesterol was found in two compartments. The rapidly exchanging compartment was identified as the plasma membrane, and the slowly exchanging compartment was associated with intracellular membranes (2). Cholesterol esters (CE's) 1 were not exchanged under these conditions, a finding which is in keeping with prior studies employing lipoprotein classes and erythrocytes (3).Under physiological and pathological conditions macrophages are exposed to other forms of both free cholesterol and CE's. Effete erythrocytes, tissue cells, and chylomicrons are taken up by phagocytosis (4-6), and subsequently are localized within cytoplasmic phagolysosomes. Many of their constituents are then degraded by acid hydrolases (7-9).Similar events take place under in vitro culture conditions and may lead to the formation of an intralysosomal compartment of free and esterified cholesterol, clearly distinct from the membrane-associated intracellular pool. The processing and fate of cholesterol and CE's within the digestive vacuole is poorly understood.In this paper we describe the formation of intralysosomal pools of particulate-free and esterified cholesterol, their intracellular processing and excretion from the macrophage, and their influence on the kinetics of exchange. In addition, a lysosomal cholesterol esterase has been characterized and its function evaluated in cell lysates and in the intact macrophage. Materials and MethodsMethods for harvesting and cultivating macrophages, analytic procedures, and microscopy have been described previously (1, 2). For electron microscopy the propylene oxide steps were

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Section: Ingestion and Intraeellular Fate Of Cholesterol-3h-albumin Cmentioning

confidence: 99%

Cholesterol Metabolism in the Macrophage

Werb

Cohn

1972

The Journal of Experimental Medicine

123

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“…In 2 (4) where ln 2 is the natural logarithm of 2 and Kd is the rate constant of degradation (Reiner, 1953;Tarver, 1954). The data shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Turnover of adenosine 3′:5′-cyclic monophosphate in chicken erythrocytes

Gorin¹,

Dickbuch²

1979

Biochemical Journal

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Turnover of cyclic AMP was studied in intact chicken erythrocytes. Production of cyclic AMP was stimulated by adrenaline and then blocked by propranolol. The decline in the cyclic AMP concentration under these conditions is solely due to its intracellular degradation, whereas efflux of the nucleotide, although existing in these cells, does not contribute significantly to the change in its concentration. Intracellular degradation of cyclic AMP follows a first-order kinetics with a half-life of about 6min. Similar half-lives were obtained at widely different adrenaline concentrations or when the ratio of propranolol to adrenaline was varied by 25-fold. Theoretical equations were applied to calculate the rates of cyclic AMP synthesis and degradation in the intact cells under different experimental conditions. Maximal adrenaline concentrations raise the rate of cyclic AMP synthesis and its steady-state concentration by about 10-fold. The addition of caffeine causes a further 33 % increase in intracellular concentration of the nucleotide, which is in good agreement with the theoretical increase computed from its slowed-down degradation.The intracellular concentration of cyclic AMP is determined by the rates of its synthesis, degradation and efflux from the cells to the surrounding medium. The steady-state concentration within the cells can be regulated by modulating each one of these parameters.Numerous reports have demonstrated changes in cyclic AMP steady-state concentration in different animal tissues in response to various hormonal stimuli as well as to drug administration (Robison et al., 1971). Despite the intensive investigations on factors influencing cyclic AMP synthesis and degradation, very little is known at present about its turnover in intact cells. Measurements of adenylate cyclase activity or phosphodiesterase activity in cell-free preparations probably do not reflect the exact situation within the cell.In the present study an attempt was made to determine the turnover of cyclic AMP in adrenalinestimulated intact chicken erythrocytes. Kinetics of breakdown and efflux of cyclic AMP in situ were followed and the half-life of the nucleotide within the cell was determined. Methods Preparation ofchicken erythrocytesHeparinized chicken blood was diluted with Krebs-Ringer phosphate buffer (pH7.4) (Umbreit et al., 1959), containing 10mM-glucose, and was centrifuged at 10OOg for 5min at 5°C. The supernatant fraction and the upper buffy coat of the Vol. 184 precipitate were removed by aspiration. The erythrocytes were then resuspended in the buffer and centrifuged as above. After two similar washes, the cells were resuspended to give a 40 % suspension. Incubation procedureCells were incubated in the presence of various adrenaline concentrations, as detailed in the Results section, in a New Brunswick Gyrotory Water Bath Shaker at 37°C. At various times during the incubation, duplicate portions of 1.0ml were withdrawn, added to 1.5ml of boiling water and boiled for 5min. The samples were then centrifuged at 1000...

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“…However, neither group of researchers adequately considered how the isotopic enrichment of a metabolite pool changes with time. For example, Jolivet-Tournier and Gerster ( 17) derived their rate assuming that the isotopic content changes linearly with time, whereas in reality it changes in exponential fashion (22,28,36 …”

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confidence: 99%

“…The 180 isotope is a specific tracer for the glycolate pathway (5). The isotopic labeling kinetics curves of the intermediates can be used to determine rates of synthesis if they are coupled with the appropriate rate equations and nonlinear regression techniques (22). The amount of isotopic recycling by photosynthetic tissue is negligible (23) and isotopic discrimination is insignificant, at least for short-term studies (6,15).…”

mentioning

confidence: 99%

“…Pool sizes of the metabolites and the amount of excreted glycolate were determined by measuring the area under the gas chromatograph tracing and comparing it to the area under the tracing of known amounts of the respective compounds. The rate equations for all the compounds were derived from the equation relating the instantaneous change in concentration of a isotopically labeled metabolite over time as a function ofmetabolic rates offormation and degradation and the isotopic enrichment ofthe precursor and the production (22,28,36): dB*/dt = riEa-r2Eb where B* = concentration of isotopically labeled product; r, = rate of product synthesis; r2 = rate of product conversion to other metabolites; under steady state conditions r1 = r2; Ea = A*/A, the isotopic enrichment at time t, where A* is the concentration of isotopically labeled precursor and A is the total precursor concentration; Eb = B*/B, the isotopic enrichment at time t, where B* is the concentration of isotopically labeled precursor and B is the total precursor concentration.…”

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confidence: 99%

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Glycolate Metabolism in Low and High CO₂-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by ¹⁸O-Labeling

Veau

Burris²

1989

Plant Physiol.

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Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring '10-labeled intermediates of the glycolate pathway.Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from 180-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high C02-grown cells than in air-grown cells when both were assayed under the same 02 and CO2 concentrations. Synthesis of glycolate, for both types of cells, was sfimulated by high 02 levels and inhibited by high CO2 levels. Glycolate synthesis in 1.5% C02-grown Chlorella, when exposed to a 0.035% CO2 atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the 02 concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the 02 concentration was lowered to 2% or the CO2 concentration was raised to 1.5%. Glycolate excretion was also sensitive to 02 and CO2 concentrations in 1.5% C02-grown cells and the glycolate that was excreted was '10-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella.Photorespiratory 18O-labeling kinetics were determined for Pavlova Iutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.The existence and the amount of photorespiration in freshwater algae is uncertain (8 to photorespire when transferred to low C02 levels and to do so in a manner similar to terrestrial C3 plants (33). They have high C02 compensation points (14), evolve large amounts of C02 into C02-free air during illumination (14), and are photosynthetically inhibited by 21%°02(18, 25). They also excrete large amounts of glycolate (4, 7, 35). However, photorespira-I tion appears to be suppressed and glycolate excretion is negligible if the cells are grown under air-levels of 02 and C02 (3,4,14,25).It is thought that algae grown under air-levels of C02 actively pump in dissolved inorganic carbon, raising the intracellular level of inorganic carbon (2). This would presumably suppress the oxygenase function ofribulose bisphosphate carboxylase/oxygenase, thus lowering the rate of glycolate synthesis. However, it is not entirely clear ifglycolate synthesis is completely suppressed. It has been reported that if cells are treated with a glycolate metabolism inhibitor they excrete almost as much glycolate as if they had been grown under elevated C02 (31).The main objective of our research was to quantify and then compare the photorespiratory rate ofair-grown and 1.5% C02-in-air-grown Chlorella pyr...

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The study of metabolic turnover rates by means of isotopic tracers

Cited by 150 publications

References 8 publications

Cholesterol Metabolism in the Macrophage

Cholesterol Metabolism in the Macrophage

Turnover of adenosine 3′:5′-cyclic monophosphate in chicken erythrocytes

Glycolate Metabolism in Low and High CO₂-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by ¹⁸O-Labeling

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The study of metabolic turnover rates by means of isotopic tracers

Cited by 150 publications

References 8 publications

Cholesterol Metabolism in the Macrophage

Cholesterol Metabolism in the Macrophage

Turnover of adenosine 3′:5′-cyclic monophosphate in chicken erythrocytes

Glycolate Metabolism in Low and High CO2-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by 18O-Labeling

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Glycolate Metabolism in Low and High CO₂-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by ¹⁸O-Labeling