scholarly journals Effect of dexamethasone on mannolipid synthesis by hepatocytes prepared from control and inflamed rats

1984 ◽  
Vol 219 (2) ◽  
pp. 429-436 ◽  
Author(s):  
M Sarkar ◽  
S Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Microsomal Fraction ◽  
Fold Increase ◽  
Cell Homogenate

Hepatocytes were prepared from control and inflamed rats. Mannose incorporation into dolichol monophosphate mannose in homogenate and microsomal fraction of the hepatocytes was increased 2-fold over the controls 24 h after induction of inflammation by turpentine injection. Incubation of hepatocytes from both control and inflamed rats with 0.1-10 microM-dexamethasone produced a 1.5-fold increase of dolichol phosphate mannose formation, whereas, 100 microM-dexamethasone decreased its formation. The increase in the ratio of dolichol phosphate mannose formation in inflamed over controls was virtually eliminated when the cell homogenate assay mixtures included 30 nmol of exogenous dolichol phosphate. This supports the earlier suggestion that the increase in the enzyme activity in inflammation could be due to higher concentrations of endogenous dolichol phosphate [Coolbear & Mookerjea (1981) J. Biol. Chem. 256, 4529-4535]. In contrast, the increase in the ratio of dolichol phosphate mannose formation between dexamethasone-treated and untreated hepatocytes remained unchanged when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the increase in glycosylation of dolichol phosphate in dexamethasone-treated hepatocytes is probably due to the increased mannosyltransferase activity, rather than due to higher concentrations of endogenous dolichol phosphate in these cells.

scholarly journals Adrenal microsomal C19-steroid 5α-reductase activity in the Snell transplantable rat adrenocortical tumour 494 and the effect of oestradiol, testosterone propionate and adrenocorticotrophin in intact and gonadectomized rats

1973 ◽  
Vol 132 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Paul V. Maynard ◽  
Euan H. D. Cameron
Keyword(s):  
Enzyme Activity ◽  
Steroid Hormones ◽  
Microsomal Fraction ◽  
Testosterone Propionate ◽  
Reductase Activity ◽  
Intact Control ◽  
Adrenal Tissue ◽  
Males And Females ◽  
Adrenocortical Tumour ◽  
Hormonal Treatments

The C19-steroid 5α-reductase activity in the microsomal fraction of rat adrenal tissue under various hormonal treatments was examined. In intact control rats the activity is similar in both males and females, and after gonadectomy it is markedly increased. Treatment with oestradiol (150μg/day per animal for 7 days) or testosterone propionate (2mg/day per animal for 7 days) lowered the activity of 5α-reductase in castrated animals to approximately the values for intact animals in both sexes, and in intact animals the activity was also decreased by these treatments. The enzyme activity was also decreased by adrenocorticotrophin treatment but to a lesser extent than by the steroid hormones. The activity of the 5α-reductase enzyme in the Snell adrenocortical tumour 494 is very low when incubated as a whole homogenate, but the activity in microsomal material of the tumour was measured and unexpectedly found to be similar to that in intact controls.

scholarly journals Identification and Characterization ofMAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

1998 ◽  
Vol 180 (11) ◽  
pp. 2875-2882 ◽  
Author(s):  
Eckhard Boles ◽  
Patricia de Jong-Gubbels ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Malic Enzyme ◽  
Fold Increase ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Oxidative Decarboxylation ◽  
Mrna Levels ◽  
Malic Enzyme Activity

ABSTRACT Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression ofYKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures,MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

Hydroxylation of the B-Ring of Flavonoids in the 3′-and 5′-Position with Enzyme Extracts from Flowers of Verbena hybrida

1982 ◽  
Vol 37 (1-2) ◽  
pp. 19-23 ◽  
Author(s):  
G. Stotz ◽  
G. Forkmann
Keyword(s):  
Enzyme Activity ◽  
Microsomal Fraction ◽  
Enzyme Preparations ◽  
Flavonoid Compounds

Enzyme preparations from flowers of Verbena hybrida do not only catalyse hydroxylation of the B-ring of flavanones and dihydroflavonols in the 3'-position but also in the 5'-position. Enzyme activity for 3',5'-hydroxylation was found to be localized in the microsomal fraction and required NADPH as cofactor. Evidence is provided that the formation of the 3',4',5'- hydroxylated flavanone (5,7,3',4',5'-pentahydroxyflavanone) and dihydroflavonol (dihydromy- ricetin), respectively, proceeds via the corresponding 3',4'-hydroxylated compounds eriodictyol and dihydroquercetin, respectively, which are most probably formed by action of the same enzyme. Enzyme activity for 3',5'-hydroxylation was found to be strictly correlated with the prescence of 3',4',5'-hydroxylated flavonoid compounds in the flowers.

scholarly journals Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification

1983 ◽  
Vol 214 (1) ◽  
pp. 69-75 ◽  
Author(s):  
P B Moore ◽  
N Kraus-Friedmann
Keyword(s):  
Affinity Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fold Increase ◽  
Partial Purification ◽  
Dependent Atpase

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.

scholarly journals Modulation of polyamine-biosynthetic activity by S-adenosylmethionine depletion

1988 ◽  
Vol 249 (2) ◽  
pp. 581-586 ◽  
Author(s):  
D L Kramer ◽  
J R Sufrin ◽  
C W Porter
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Inhibitors ◽  
Fold Increase ◽  
Negative Control ◽  
Biosynthetic Activity ◽  
Drug Induced ◽  
Polyamine Biosynthesis ◽  
Sequence Characterization ◽  
Adomet Synthetase

The methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), was used to study the early effects of AdoMet depletion on polyamine biosynthesis. In the presence of decreased methionine (30 microM) in the medium, treatment of cultured L1210 cells with 1 mM-L-cisAMB resulted in a near-total (95%) depletion of cellular AdoMet pools by 4 h. This was accompanied by a 3-fold increase in ornithine decarboxylase (ODC) activity, a 2.5-fold increase in AdoMet decarboxylase (AdoMetDC) activity and a 20% decrease in spermidine and spermine pools. The increase in enzyme activities seemed to be partially due to prolongation of enzyme activity half-life, since that of ODC was extended from 30 to 50 min and that of AdoMetDC from 65 to 310 min. By temporal sequence characterization (0-4 h), the onset of elevations of enzyme activity (0.5-1 h) seemed to be causally related to an earlier (0-0.5 h) decline in AdoMet pools, as opposed to the 20% decrease in spermidine and spermine pools, which occurred much later (2-4 h); the latter are known to regulate decarboxylase activities negatively. Drug-induced elevations in ODC and, to a lesser extent, AdoMetDC activities were reversed by later treatment with exogenous AdoMet. However, because the latter also increased spermine pools (which could not be prevented with various enzyme inhibitors), the reversal of elevations in enzyme activities could not be directly linked to AdoMet. Although not definitive, the data raise the interesting possibility that, in addition to being negatively regulated by polyamines, ODC and AdoMetDC activities may also be subject to negative control by cellular AdoMet (or an AdoMet metabolite). The net effect of either or both of these influences would be to conserve polyamine-biosynthetic activity in the face of declining AdoMet supplies.

scholarly journals The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1970 ◽  
Vol 119 (3) ◽  
pp. 437-445 ◽  
Author(s):  
B. P. F. Adlard ◽  
G. H. Lathe
Keyword(s):  
Enzyme Activity ◽  
Glucuronic Acid ◽  
Liver Microsomes ◽  
Fold Increase ◽  
Rat Liver Microsomes ◽  
Uridine Diphosphate ◽  
Competitive Effects ◽  
Solubilized Enzyme ◽  
High Concentrations ◽  
Solubilized Form

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis–Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The Km for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca2+ were inhibitory in the absence of Mg2+ but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3β-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.

scholarly journals The effects of 24R,25-dihydroxycholecalciferol and of 1 α,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum

1983 ◽  
Vol 214 (2) ◽  
pp. 293-298 ◽  
Author(s):  
D Sömjen ◽  
I Binderman ◽  
Y Weisman
Keyword(s):  
Vitamin D ◽  
Enzyme Activity ◽  
Ornithine Decarboxylase ◽  
Thymidine Incorporation ◽  
Single Injection ◽  
Fold Increase ◽  
Decarboxylase Activity ◽  
Long Bones ◽  
Vitamin D Metabolites ◽  
Ornithine Decarboxylase Activity

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.

Oriented Immobilization of Penicillin G Acylase on the Epoxydized Magnetic Hydroxyl Particles by Phenyl Acetic Acid

2012 ◽  
Vol 550-553 ◽  
pp. 1345-1351 ◽  
Author(s):  
Wei Wang ◽  
Zhong Hai Li ◽  
Ji Lie Li ◽  
Yao Hui Wu
Keyword(s):  
Enzyme Activity ◽  
Acetic Acid ◽  
Magnetic Particles ◽  
Suspension Polymerization ◽  
Reaction System ◽  
Fold Increase ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Phenyl Acetic Acid ◽  
Immobilized Penicillin G Acylase

Magnetic hydroxyl microspheres were prepared by suspension polymerization and activated by epoxy chloropropane. The magnetic particles were characterized in terms of chemical composition, particle size and electrophoretic mobility. These epoxy-activated magnetic particles were assessed as a new carries for immobilized penicillin G acylase (PGA) by covalent coupling. The oriented PGA immobilization was achieved by employing the interaction electrostatic repulsion between PGA and magnetic supports through adding the phenyl acetic acid (PAA), which resulted in a 1.194-fold increase in the enzyme activity yield as compared to that of untreated PGA. No activity of immobilized PGA was lost after 20 cycles and about 94.28% enzyme activity was retained at the end of the 80th cycle in the batch reaction system.

scholarly journals Metabolism of palmitate in perfused rat liver. Isolation of subcellular fractions containing triacylglycerol

1979 ◽  
Vol 184 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J Kondrup
Keyword(s):  
Rat Liver ◽  
Lipid Droplets ◽  
Microsomal Fraction ◽  
Fold Increase ◽  
Water Soluble ◽  
Particulate Fraction ◽  
Isotopic Equilibrium ◽  
Precursor Pool ◽  
Pass Perfusion ◽  
Fat Fraction

1. The metabolism of [1-14C]palmitate in rat liver was studied in a single-pass perfusion system at concentrations of 0.2 or 1 mM. 2. After the perfusion the liver was homogenized and the floating fat was isolated. The incorporation of [1-14C]palmitate into triacylglycerol in this pool increased 9-fold when the palmitate concentration in the medium was increased from 0.2 to 1 mM. In time studies with 1 mM-[1-14C]palmitate 75% of the total accumulation of triacylglycerol occurred in this pool. Our results support the concept that the floating-fat fraction contains the storage pool of triacylglycerol, i.e. the cytoplasmic lipid droplets. 3. In a particulate preparation consisting mainly of mitochondria and microsomal fraction the incorporation of [1-14C]palmitate into triacylglycerol was proportional to the fatty acid concentration. Triacylglycerol in the perfusate medium and in the particulate fraction was in isotopic equilibrium, which indicates that the particulate fraction contained the precursor pool for secreted triacylglycerol, i.e. the pool in endoplasmic reticulum and Golgi apparatus. 4. The oxidation to labelled water-soluble products and to CO2 was increased 14-fold by the 5-fold increase in palmitate concentration.

scholarly journals The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1970 ◽  
Vol 117 (2) ◽  
pp. 319-324 ◽  
Author(s):  
G. J. Mulder
Keyword(s):  
Enzyme Activity ◽  
Density Gradient ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Male Rats ◽  
Uridine Diphosphate ◽  
Triton X

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The Km of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.

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