scholarly journals Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase

1979 ◽  
Vol 177 (1) ◽  
pp. 283-288 ◽  
Author(s):  
H G Nimmo
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Heat Inactivation ◽  
Mitochondrial Enzyme ◽  
Microsomal Enzyme ◽  
Glycerol Phosphate ◽  
Low Concentrations ◽  
High Concentrations

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.

scholarly journals Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1971 ◽  
Vol 125 (2) ◽  
pp. 557-562 ◽  
Author(s):  
A. Adinolfi ◽  
V. Guarriera-Bobyleva ◽  
S. Olezza ◽  
A. Ruffo
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Competitive Inhibition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Enzyme ◽  
Aconitate Hydratase ◽  
Inhibited Oxidation ◽  
Low Concentrations ◽  
High Concentrations

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1–0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01–0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the Ki values calculated were 1×10−6m for the mitochondrial and 2.5×10−6m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate→cis-aconitate more than that of the reaction isocitrate→cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.

scholarly journals Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups

1976 ◽  
Vol 157 (2) ◽  
pp. 479-482 ◽  
Author(s):  
T J Visser ◽  
I Does-Tobé ◽  
R Docter ◽  
G Hennemann
Keyword(s):  
Rat Liver ◽  
Essential Role ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Enzymic Activity ◽  
Marker Enzyme ◽  
Converting Enzyme ◽  
Thiol Groups ◽  
Liver Homogenates

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

scholarly journals The temporary postnatal decline in glucuronidation of certain phenols by rat liver

1983 ◽  
Vol 214 (2) ◽  
pp. 533-537 ◽  
Author(s):  
I Scragg ◽  
M Pollard ◽  
B Burchell ◽  
G J Dutton
Keyword(s):  
Rat Liver ◽  
Perinatal Period ◽  
Native Enzyme ◽  
Microsomal Enzyme ◽  
Early Weaning ◽  
Infant Rat ◽  
Udp Glucuronosyltransferase ◽  
High Concentrations ◽  
Tissue Fractionation

A temporary but marked postnatal decline in UDP-glucuronosyltransferase activity occurs in homogenates and microsomes from rat liver. The profile of this trough and its time of occurrence (maximal over 13-16 days) are almost identical with the two substrates 2-aminophenol and 1-naphthol, whose rates of glucuronidation differ 10-fold. The trough is greatest with digitonin-activated preparations, least with fresh latent (‘native’) enzyme and intermediate when the native enzyme is treated with its specific activator UDP-N-acetylglucosamine (UDP-GlcNAc). Less detailed evidence supports similar conclusions with 4-nitrophenol as substrate. The trough is not due to the presence of an inhibitor of the transferase in rat liver at 15 days of age. Over the whole perinatal period, including the time of the trough, the enzyme in homogenates can be activated by UDP-GlcNAc; the microsomal enzyme is activated to a rather lesser degree perinatally, and evidence suggests this may be due to artefacts introduced during tissue fractionation. When the overall process of glucuronidation is studied in snips of intact liver offered high concentrations of the two different phenols, the trough is again evident over the same period as observed with broken cells, and of equal depth for both substrates. The infant rat is therefore probably less able to glucuronidate hepatically these phenols over the suckling or early weaning period than are the adult, late foetus or newborn, and may be especially incompetent at 13-16 days of age.

scholarly journals Activation of a peroxisome-proliferating catabolite of cholic acid to its CoA ester

1993 ◽  
Vol 296 (1) ◽  
pp. 265-270 ◽  
Author(s):  
T Nishimaki-Mogami ◽  
A Takahashi ◽  
Y Hayashi
Keyword(s):  
Cholic Acid ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Liver Microsomes ◽  
Subcellular Fractionation ◽  
Fatty Acyl ◽  
Peroxisome Proliferator ◽  
Rat Liver Microsomes ◽  
Triton X ◽  
Long Chain

We have shown that a microbial cholic acid catabolite (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo- 1H-cyclopenta[f]quinolin-7 beta-yl)valeric acid (DCQVA), is a potent peroxisome proliferator. In this paper a possible key stage in DCQVA metabolism, the activation of DCQVA to its CoA ester, has been investigated in rat liver microsomes and particulate fractions. The microsomal reaction was dependent on CoA, ATP, DCQVA (0.2-1 mM) and protein content. The reaction was decreased by storage at 4 degrees C, preincubation of microsomes at 37 degrees C for 5 min, or inclusion of Triton X-100 in the reaction mixture. Such treatments also enhanced generation of long-chain fatty acyl-CoAs, as determined by h.p.l.c. analysis. The same effect was caused by exposing the microsomes to phospholipase A2, suggesting that endogenous fatty acids may compete with DCQVA for esterification with CoA. Subcellular fractionation of rat liver demonstrated that the activity of DCQVA-CoA synthesis was localized predominantly in the microsomal fraction, in contrast to long-chain fatty acyl-CoA synthetase, which was distributed among all particulate fractions. Administration of clofibrate of rats did not affect the distribution of DCQVA-CoA synthesis activity. In contrast to a 2-fold induction of long-chain fatty acyl-CoA synthetase by clofibrate treatment, the activity of DCQVA-CoA synthesis in the microsomal fraction decreased by 80%. These results suggest that DCQVA is activated by an enzyme distinct from long-chain fatty acyl-CoA synthetase. The resulting perturbation of fatty acid metabolism may be involved in the mechanism whereby DCQVA causes peroxisome proliferation.

scholarly journals Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria

1972 ◽  
Vol 127 (1) ◽  
pp. 271-283 ◽  
Author(s):  
R. G. Hansford
Keyword(s):  
Oxygen Uptake ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Slight Reduction ◽  
Glycerol Phosphate ◽  
Pyruvate Oxidation ◽  
Low Concentrations ◽  
High Concentrations ◽  
Kinetics Of ◽  
Very High

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl2 during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H+ ions, and inhibited by ATP, NADH and Ca2+. 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a `classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.

scholarly journals Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1974 ◽  
Vol 140 (3) ◽  
pp. 413-422 ◽  
Author(s):  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Protein Interactions ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Low Concentrations ◽  
High Concentrations ◽  
Lipid Protein Interactions

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid–protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30μm for 200μm-ATP and at 10μm for 200μm-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50μm) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250μm) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca2+ was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca2+. 4. The data provide further support for our hypothesis that lipid–protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.

A Rapidly Sedimenting Fraction of Rat Liver Endoplasmic Reticulum

1973 ◽  
Vol 13 (2) ◽  
pp. 447-459 ◽  
Author(s):  
J. A. LEWIS ◽  
J. R. TATA
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Close Association ◽  
Balance Sheet ◽  
Subcellular Fractionation ◽  
Electron Microscopic ◽  
Microsomal Preparation ◽  
Low Speed ◽  
Microscopic Studies

Balance-sheet experiments carried out to account for the distribution of endoplasmic reticulum fragments during subcellular fractionation of rat liver showed that a large proportion of these fragments are present in the pellets of low-speed centrifugation. Using glucose-6-phosphatase and RNA as markers we found that approximately 50% of the fragments of endoplasmic reticulum sedimented in the pellet of a 640-g centrifugation, 10% in that of a 6000-g centrifugation and 35% in the pellet of a 105000-g centrifugation. Starvation of the animals before use did not alter this distribution, nor did the use of more vigorous homogenization conditions. We have developed a procedure for removing nuclei and erythrocytes from the material sedimenting at 640g to give a fraction (rapidly sedimenting ER fraction or RS-ER) similar to the standard microsomal preparation. Centrifugation of this RS-ER fraction over 1.3 M sucrose yields subfractions of high and low RNA content analogous to the rough and smooth microsomal fractions. Electron-microscopic studies showed that, whereas the rough microsomal fraction consisted of ribosome-studded vesicles of varying size and content density, the rough RS-ER fraction contained a mixture of mitochondria and double lamellar membranes with ribosomes attached. These double lamellar membranes closely resemble the endoplasmic reticulum of intact rat liver. The double lamellar membranes are frequently observed grouped in stacks and in close association with the mitochondria. The significance of the association between endoplasmic reticulum and mitochondria of the RS-ER fraction and the relation between it and the standard microsomal preparation are discussed.

scholarly journals Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity

1976 ◽  
Vol 158 (2) ◽  
pp. 249-254 ◽  
Author(s):  
K S Bjerve ◽  
L N W Daae ◽  
J Bremer
Keyword(s):  
Fatty Acid ◽  
Phosphatidic Acid ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Acid Biosynthesis ◽  
Positional Specificity ◽  
Fatty Acid Specificity ◽  
High Concentrations

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.

scholarly journals Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

1972 ◽  
Vol 128 (2) ◽  
pp. 337-345 ◽  
Author(s):  
J. O. Dolly ◽  
K. S. Dodgson ◽  
F. A. Rose
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Competitive Inhibition ◽  
Similar Rate ◽  
Physiological Significance ◽  
Heat Inactivation ◽  
Mixed Substrate ◽  
Oestrone Sulphate ◽  
Foetal Liver ◽  
Hydrolysis Of

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17β 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17β. The physiological significance of this inhibition is discussed.

Hepatic Vitamin B12 Release and Transcobalamin II Synthesis in the Rat

1974 ◽  
Vol 47 (6) ◽  
pp. 531-545 ◽  
Author(s):  
W. G. E. Cooksley ◽  
J. M. England ◽  
L. Louis ◽  
M. C. Down ◽  
A. S. Tavill
Keyword(s):  
Vitamin B12 ◽  
Rat Liver ◽  
Plasma Concentrations ◽  
High Plasma ◽  
Free Form ◽  
Isolated Perfused Rat Liver ◽  
Low Concentrations ◽  
High Concentrations ◽  
Transcobalamin Ii

1. The release of 57Co-labelled vitamin B12 ([57Co]B12) and synthesis of transcobalamin II (TCII) by the isolated perfused rat liver were studied 10–42 days after the parenteral administration of a trace dose of 15 pmol (approximately 20 ng) of radioactive cyanocobalamin. 2. The rate of release of [57Co]B12 into plasma and bile was linear and constituted approximately 0.9% and 0.3% respectively of the initial hepatic radioactivity per hour of perfusion. 3. [57Co]B12 released into plasma was bound to TCII. Saturation of the total TCII by the addition of cyanocobalamin before perfusion resulted in the appearance of the hepatic [57Co]B12 in the free form. 4. These data were found to be compatible with the following observations in vivo: (i) rates of [57Co]B12 release as measured by urinary [57Co]B12 excretion after saturation of plasma binders with non-labelled cyanocobalamin; (ii) rates of biliary excretion of [57Co]B12. 5. Liver damage produced by hypoxaemia was associated with a fall in the rate of release of [57Co]B12. 6. TCII release occurred at a linear rate of almost twenty times that required for the binding of newly released hepatic vitamin B12. 7. Cycloheximide at a dose sufficient to inhibit release of TCII did not prevent the release of [57Co]B12 from the liver into plasma or bile. 8. Alteration of perfusate composition to contain either high plasma concentrations of vitamin B12 and low concentrations of unsaturated TCII or high plasma concentrations of vitamin B12 and high concentrations of unsaturated TCII had no effect on the rate of [57Co]B12 release into plasma or bile. 9. It is concluded that the fluxes of hepatic vitamin B12 and TCII are very rapid and that the release of vitamin B12 by the rat liver is controlled in the short term by factors other than the synthesis of TCII and the concentration of vitamin B12 or unsaturated transcobalamin in the plasma.

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