scholarly journals Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver

1983 ◽  
Vol 216 (3) ◽  
pp. 701-707 ◽  
Author(s):  
J E Seely ◽  
A E Pegg
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Enzyme Protein ◽  
Rapid Loss ◽  
Immunoreactive Protein ◽  
Full Effect

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.

scholarly journals Induction of carnitine acetyltransferase by clofibrate in rat liver

1981 ◽  
Vol 194 (1) ◽  
pp. 249-255 ◽  
Author(s):  
B Mittal ◽  
C K R Kurup
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Time Lag ◽  
Mitochondrial Protein ◽  
Carnitine Acetyltransferase ◽  
Enzyme Protein ◽  
General Protein Synthesis ◽  
Liver And Kidney ◽  
General Protein

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine O-acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

scholarly journals Diamine-induced inhibition of liver ornithine decarboxylase

1979 ◽  
Vol 177 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Arja Kallio ◽  
Monica Löfman ◽  
Hannu Pösö ◽  
Juhani Jänne
Keyword(s):  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Decarboxylase Activity ◽  
Enzyme Protein ◽  
Intracellular Degradation ◽  
Ornithine Decarboxylase Activity ◽  
Normal Rat

Re!peated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ‘antienzymes’ [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858–1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme–inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

scholarly journals A macromolecular inhibitor of the antizyme to ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 647-652 ◽  
Author(s):  
K Fujita ◽  
Y Murakami ◽  
S Hayashi
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Molecular Size ◽  
Antizyme Inhibitor ◽  
Heat Labile

A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, ‘antizyme inhibitor’, was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.

scholarly journals Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

1977 ◽  
Vol 168 (1) ◽  
pp. 1-8 ◽  
Author(s):  
J C Ramsey ◽  
W J Steele
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Size Structure ◽  
Large Fraction ◽  
Liver Homogenate ◽  
Structure And Function ◽  
Structural And Functional Properties ◽  
Membrane Bound ◽  
And Function

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

scholarly journals Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2C11 Enzymes in Rat Liver Microsomes: In Vitro Inhibitory Effect of Salicylic Acid on CYP2C11 Enzyme

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4294 ◽  
Author(s):  
Salhab ◽  
Naughton ◽  
Barker
Keyword(s):  
High Performance Liquid Chromatography ◽  
Enzyme Activity ◽  
Salicylic Acid ◽  
Liquid Chromatography ◽  
Rat Liver ◽  
High Performance ◽  
Inhibitory Effect ◽  
Metabolic Reaction ◽  
Rat Liver Microsomes

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%–120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug–drug interactions.

scholarly journals Biotin-mediated protein biosynthesis

1974 ◽  
Vol 140 (3) ◽  
pp. 549-556 ◽  
Author(s):  
R. L. Boeckx ◽  
K. Dakshinamurti
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Liver ◽  
Intestinal Mucosa ◽  
Protein Biosynthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.

scholarly journals Studies on sex-organ development. Oestrogenic effect on ornithine decarboxylase activity in the differentiating Müllerian ducts and other organs of the chick embryo

1978 ◽  
Vol 176 (1) ◽  
pp. 143-149 ◽  
Author(s):  
C S Teng ◽  
C T Teng
Keyword(s):  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Mullerian Duct ◽  
Stages Of Development ◽  
Müllerian Duct ◽  
Mullerian Ducts ◽  
The Right ◽  
The Brain

The activity of ornithine decarboxylase in the differentiating left and right Müllerian ducts was assayed and compared with that in other embryonic organs, i.e. the liver and the brain throughout the stages of development. In general the enzyme activity was high in the early stages and decreased extensively in the late stages of development. Specifically, in the left and righ Müllerian ducts, the enzyme activity was high from day 8 to day 9 of incubation. In the right duct the enzyme activity started to decline on day 9 and then continuously decreased to an almost undetectable value on day 18 of incubation. In the left duct the enzyme activity also decreased slightly from day 9 to day 12; however, it increased from day 13 to day 15 and finally decreased to a constant value from day 18 until hatching. The alteration in enzyme activity in the Müllerian duct as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. When the enzyme activity was subjected to oestrogen stimulation, an increase of 5–10-fold for the left duct and of 5–3-fold for the right duct was observed during the course of development. No such stimulation was observed with the treatment of progesterone. Testosterone consistently caused a 25–30% inhibition of the enzyme activity in the Müllerian duct. Oestrogen slightly stimulated the enzyme activity in the developing liver but inhibited that of the brain. The concentration of the three polyamines measured in the Müllerian duct corresponds to the activity of the enzyme determined.

scholarly journals The dependence on vitamin E and selenium of drug demethylation in rat liver microsomal fractions

1975 ◽  
Vol 146 (2) ◽  
pp. 339-350 ◽  
Author(s):  
A S M Giasuddin ◽  
C P J Caygill ◽  
A T Diplock ◽  
E H Jeffery
Keyword(s):  
Enzyme Activity ◽  
Vitamin E ◽  
Rat Liver ◽  
Selenium Deficiency ◽  
Wide Range ◽  
Km Value ◽  
Demethylase Activity ◽  
In Vitro Induction ◽  
Substrate Concentrations

1. The effects of vitamin E deficiency, and of vitamin E and selenium deficiency, on rat liver microsomal aminopyrine demethylase activity were investigated. It was found that, over a wide range of substrate concentrations, the enzyme activity in preparations from deficient animals was significantly lower than that in controls. 2. Addition of antioxidants in vitro, either to the homogenization or to the assay media, was without significant effect on the depressed enzyme activity. Castration and alteration in dietary protein concentration were also without effect. The rate of oxidation of NADPH was however, lower in preparations from deficient animals. 3. Lineweaver-Burk plots of the reciprocal of enzyme activity and substrate concentration showed a higher Km value in preparations from vitamin E-deficient animals, irrespective of whether selenium was present; the Vmax. was unaffected. These parameters were unchanged when antioxidants were added in vitro. Induction with phenobarbitone and 3-methylcholanthrene showed large changes in Km value which, for preparations from vitamin E-deficient animals, was higher than that for corresponding controls. 4. Examination of the synergism between NADH and NADPH as donors of reducing equivalents for aminopyrine demethylation showed that vitamin E and selenium were only minimally involved in the phenomenon. However, both the initial rate and the extent of demethylation were significantly lower in vitamin E- and selenium-deficient preparations and both nutrients were required for the restoration of full activity. 5. The significance of these results is discussed in the light of our working hypothesis.

scholarly journals Intracellular enzymes of collagen biosynthesis in rat liver as a function of age and in hepatic injury induced by dimethylnitrosamine. Purification of rat prolyl hydroxylase and comparison of changes in prolyl hydroxylase activity with changes in immunoreactive prolyl hydroxylase

1976 ◽  
Vol 158 (2) ◽  
pp. 369-376 ◽  
Author(s):  
J Risteli ◽  
L Tuderman ◽  
K I Kivirikko
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Hepatic Injury ◽  
Specific Activity ◽  
Prolyl Hydroxylase ◽  
Newborn Rats ◽  
Hydroxylase Activity ◽  
Immunoreactive Protein ◽  
Molecular Weights ◽  
Polypeptide Chains

Prolyl hydroxylase was purified from newborn rats by affinity chromatography using poly(L-proline), and antiserum to the enzyme was prepared in rabbits. The rat prolyl hydroxylase was similar to the chick and human enzymes with respect to specific activity, molecular weight and molecular weights of the polypeptide chains. The activity of prolyl hydroxylase and the content of immunoreactive enzyme were measured in rat liver as a function of age in experimental hepatic injury. Active prolyl hydroxylase comprised about 13.2% of the total immunoreactive protein in the liver of newborn rats and the value decreased to about 3.6% at the age of 420 days. This decrease was due to a decrease in the enzyme activity, whereas only minor changes were found in the content of the immunoreactive protein. In hepatic injury, a significant increase was found in the ratio of active enzyme to total immunoreactive protein, owing to an increase in the enzyme activity. The data indicate that prolyl hydroxylase activity in rat liver is controlled in part by a mechanism which does not involve changes in the content of the total immunoreactive protein.

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