Pyrimidine reducing enzymes of rat liver

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4.Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH-dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH.Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.

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Analytical Subcellular Fractionation Studies on Rat Liver and on Isolated Jejunal Enterocytes with Special Reference to the Separation of Lysosomes, Peroxisomes and Mitochondria

Clinical Science ◽

10.1042/cs0500355 ◽

1976 ◽

Vol 50 (5) ◽

pp. 355-366 ◽

Cited By ~ 2

Author(s):

T. J. Peters ◽

H. Shio

Keyword(s):

Rat Liver ◽

Sucrose Density Gradient ◽

Subcellular Fractionation ◽

Low Molecular Weight ◽

Rat Jejunum ◽

Marker Enzymes ◽

Good Separation ◽

Subcellular Organelles ◽

Rat Liver Cells ◽

Isopycnic Centrifugation

1. Enterocytes were isolated from rat jejunum and characterized morphologically. 2. Attempts to separate the enterocyte subcellular organelles, characterized by their marker enzymes, with isopycnic centrifugation were unsuccessful but good separation of peroxisomes, lysosomes and mitochondria was achieved by sedimentation through a shallow sucrose density gradient with a superimposed inverse gradient of low-molecular-weight dextran. 3. The properties and enzyme activities of the principal subcellular organelles in rat liver cells and enterocytes were compared.

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Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

Biochemical Journal ◽

10.1042/bj1980457 ◽

1981 ◽

Vol 198 (3) ◽

pp. 457-466 ◽

Cited By ~ 20

Author(s):

Govind S. Rao ◽

Marie Luise Rao ◽

Astrid Thilmann ◽

Hans D. Quednau

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Liver Homogenate ◽

Membrane Vesicles ◽

Liver Cells ◽

Free Form ◽

Plasma Membrane Vesicles ◽

Cytosol Fraction ◽

Low Concentrations ◽

Rat Liver Cells

1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[125I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.

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Methionine Adenosyltransferase, Cystathionine β-Synthase and Cystathionine γ-Iyase Activity of Rat Liver Subcellular Particles, Human Blood Cells and Mixed White Cells from Rat Bone Marrow

Clinical Science ◽

10.1042/cs0480509 ◽

1975 ◽

Vol 48 (6) ◽

pp. 509-513

Author(s):

Jennifer Allsop ◽

R. W. E. Watts

Keyword(s):

Bone Marrow ◽

Rat Liver ◽

Liver Homogenate ◽

Methionine Adenosyltransferase ◽

Cell Nuclei ◽

Cytoplasmic Fraction ◽

Cytosol Fraction ◽

Human Blood Cells ◽

Rat Liver Cells ◽

Methionine Adenosyl Transferase

1. Methionine adenosyltransferase (ATP:l-methionine-S-adenosyl transferase, EC 2.5.1.6), cystathionine β-synthase [l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and cystathionine γ-lyase [l-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] activities were found only in the cytosol fraction of rat liver cells. None was found in the mitochondrial or endoplasmic reticulum fractions as judged by the distribution of marker enzymes on a density gradient after centrifugation of the cytoplasmic fraction of a liver homogenate, or in a preparation of liver cell nuclei. 2. Polymorphs, lymphocytes (with admixed monocytes) and mixed bone marrow white cells contained no methionine adenosyl transferase, cystathionine β-synthase or cystathionine γ-lyase activities. 3. The possible bearing of these results on the problem of abnormal cystine storage in cystinosis is briefly discussed.

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DISTRIBUTION OF RADIOACTIVE CARBON DIOXIDE INCORPORATED INTO RAT LIVER GLYCOGEN

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)65896-x ◽

1956 ◽

Vol 218 (1) ◽

pp. 327-333 ◽

Cited By ~ 12

Author(s):

Paul A. Marks ◽

B.L. Horecker

Keyword(s):

Carbon Dioxide ◽

Rat Liver ◽

Liver Glycogen ◽

Radioactive Carbon

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Histone synthesis during development of Triturus embryos

Development ◽

10.1242/dev.30.1.73 ◽

1973 ◽

Vol 30 (1) ◽

pp. 73-82

Author(s):

Hiroshi Imoh ◽

Izumi Kawakami

Keyword(s):

Carbon Dioxide ◽

Gel Electrophoresis ◽

Fast Moving ◽

Basic Protein ◽

Disc Electrophoresis ◽

Gastrula Stage ◽

Tail Bud ◽

Blastula Stage ◽

Radioactive Carbon ◽

Histone Synthesis

Synthesis of histone fractions and one basic protein fraction, which moved fast on gel electrophoresis and had been reported to increase in nuclei accompanying a decrease in cytoplasm during development, were studied with radioactive carbon dioxide as a tracer. Acid-extractable proteins of nuclei or cytoplasm, isolated from labelled embryos, were fractionated by polyacrylamide disc electrophoresis and the histone fractions and the fast-moving basic protein were identified. Radioactivities in these fractions and DNA were determined. Synthesis of the fast-moving basic protein was not detected throughout the period of development studied and this fraction was thought to move in from the cytoplasm to the nucleus during development. Syntheses of histone fractions were observed as early as the blastula stage. Rates of syntheses of four histone fractions (f3, f2b, f2a2 and f2al) per embryo increased thereafter, keeping pace with the increase in the rate of DNA synthesis with advancing development. The rate of the very lysine-rich fl histone synthesis per embryo did not increase after the gastrula stage and the rate remained almost constant until the late tail-bud stage. Compositions of newly synthesized histones, calculated from the radioactivities incorporated into histone fractions, were almost the same during development and among different regions of neurula or tail-bud-stage embryos, with the exception of the f1 fraction, which varied depending on the stage and region of the embryos. The results are discussed in relation to the possible roles of the histone fractions in developing embryos.

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Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver

Biochemical Journal ◽

10.1042/bj3040577 ◽

1994 ◽

Vol 304 (2) ◽

pp. 577-584 ◽

Cited By ~ 12

Author(s):

I Niot ◽

F Pacot ◽

P Bouchard ◽

J Gresti ◽

A Bernard ◽

...

Keyword(s):

Rat Liver ◽

Marker Enzyme ◽

Outer Membranes ◽

Physiological Importance ◽

Malonyl Coa ◽

Mitochondrial Fractions ◽

Mitochondrial Population ◽

Carnitine Palmitoyltransferase I ◽

Cpt I ◽

Carnitine Palmitoyl Transferase I

Liver mitochondrial fractions as normally isolated contain only 10-20% of total mitochondria and may not be representative of the whole mitochondrial population. This study was designed to evaluate the dependence of the sensitivity of carnitine palmitoyl-transferase I (CPT I) to malonyl-CoA inhibition in mitochondrial fractions that are not normally studied. Four fractions prepared from rat liver were found to be contaminated to different extents by microsome vesicles, on the basis of marker-enzyme activities and micrographic data. Purification of mitochondrial fractions on a Percoll gradient decreased to some extent the microsomal contamination, which was due in part to the existence of close bonds between microsomes and the outer membranes of mitochondria. A greater degree of contamination of mitochondrial fractions by microsomes was correlated with a greater sensitivity of CPT I to malonyl-CoA inhibition. Attempts were made to enhance the sensitivity of CPT I to malonyl-CoA with the use of microsomes. Measurements performed by adding mitochondria and microsomes in the same CPT I assay failed to demonstrate any significant enhancement of malonyl-CoA inhibition. However, addition of ATP to a mixture of mitochondria and microsomes was shown to trigger the binding of both particles, as assessed by enzymic and micrographic data, and to increase the sensitivity of CPT I to malonyl-CoA inhibition. These results demonstrated that the binding of microsomes to mitochondria, unlike the simple mixing of both particles, was capable of altering the sensitivity of CPT I to malonyl-CoA. The data also suggest that this process could be of physiological importance, owing to the frequency of contiguous zones between mitochondria and endoplasmic reticulum observed in sections of intact liver cells.

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Effects of Hypophysectomy on the Fine Structure of Rat Liver Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060799 ◽

1967 ◽

Vol 25 ◽

pp. 156-157

Author(s):

Robert R. Cardell

Keyword(s):

Electron Microscopy ◽

Fine Structure ◽

Rat Liver ◽

Liver Cell ◽

Anterior Pituitary ◽

Liver Cells ◽

Biochemical Studies ◽

Liver Functions ◽

Rat Liver Cells ◽

And Control

Hypophysectomy of the rat renders this animal deficient in the hormones of the anterior pituitary gland, thus causing many primary and secondary hormonal effects on basic liver functions. Biochemical studies of these alterations in the rat liver cell are quite extensive; however, relatively few morphological observations on such cells have been recorded. Because the available biochemical information was derived mostly from disrupted and fractionated liver cells, it seemed desirable to examine the problem with the techniques of electron microscopy in order to see what changes are apparent in the intact liver cell after hypophysectomy. Accordingly, liver cells from rats which had been hypophysectomized 5-120 days before sacrifice were studied. Sham-operated rats served as controls and both hypophysectomized and control rats were fasted 15 hours before sacrifice.

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