scholarly journals Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro

1984 ◽  
Vol 221 (3) ◽  
pp. 685-695 ◽  
Author(s):  
K A Mitropoulos ◽  
S Venkatesan
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Covalent Modification ◽  
Time Dependent ◽  
Cholesterol Concentration ◽  
Acat Activity ◽  
Phospholipid Liposomes ◽  
Substrate Supply ◽  
Rate Of Increase ◽  
Liver Microsomal Fraction

The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.

scholarly journals On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone

1983 ◽  
Vol 215 (1) ◽  
pp. 191-199 ◽  
Author(s):  
S Synouri-Vrettakou ◽  
K A Mitropoulos
Keyword(s):  
Microsomal Fraction ◽  
Rate Of Change ◽  
Time Dependent ◽  
Cholesterol Concentration ◽  
Phosphatidylcholine Liposomes ◽  
Acat Activity ◽  
Rate Of Increase ◽  
A Site

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.

Influence of 1,4-benzdiazepin-2-ones on rat liver microsomal fraction and hepatocytes

1987 ◽  
Vol 21 (1) ◽  
pp. 5-8
Author(s):  
T. I. Davidenko ◽  
O. V. Sevast'yanov ◽  
L. N. Yakubovskaya
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Liver Microsomal Fraction

scholarly journals Loss of haem in rat liver caused by the porphyrogenic agent 2-allyl-2-isopropylacetamide

1971 ◽  
Vol 124 (4) ◽  
pp. 767-777 ◽  
Author(s):  
F. De Matteis
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Direct Evidence ◽  
Gradual Increase ◽  
Rapid Loss ◽  
Metabolizing Enzymes ◽  
Liver Microsomal Fraction ◽  
Green Pigments ◽  
Cytochrome P 450 ◽  
Increased Activity

1. The effect of a single dose of 2-allyl-2-isopropylacetamide on the cytochrome P-450 concentration in rat liver microsomal fraction was studied. The drug caused a rapid loss of cytochrome P-450 followed by a gradual increase to above the normal concentration. 2. The loss of cytochrome P-450 was accompanied by a loss of microsomal haem and by a brown–green discoloration of the microsomal fraction suggesting that a change in the chemical constitution of the lost haem had taken place. Direct evidence for this was obtained by prelabelling the liver haems with radioactive 5-aminolaevulate: the drug caused a loss of radioactivity from the haem with an increase of radioactivity in a fraction containing certain un-identified green pigments. 3. Evidence was obtained by a dual-isotopic procedure that rapidly turning-over haem(s) may be preferentially affected. 4. The loss of cytochrome P-450 as well as the loss of microsomal haem and the discoloration of the microsomal fraction were more intense in animals pretreated with phenobarbitone and were much less evident when compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) was given before 2-allyl-2-isopropylacetamide, suggesting that the activity of the drug-metabolizing enzymes may be involved in these effects. 5. The relevance of the destruction of liver haem to the increased activity of 5-aminolaevulate synthetase caused by 2-allyl-2-isopropylacetamide is discussed.

scholarly journals Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA

1984 ◽  
Vol 218 (2) ◽  
pp. 379-386 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Liver Mitochondria ◽  
Time Dependent ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Malonyl Coa ◽  
Rate Of Increase ◽  
Cpt I ◽  
Zero Time

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Binding of radioiodinated propylthiouracil to rat liver microsomal fractions. Stimulation by substrates for iodothyronine 5′-deiodinase

1979 ◽  
Vol 183 (1) ◽  
pp. 167-169 ◽  
Author(s):  
T J Visser ◽  
E Van Overmeeren
Keyword(s):  
Rat Liver ◽  
Enzyme Preparation ◽  
Microsomal Fraction ◽  
Sodium Sulphite ◽  
Enzyme Complex ◽  
Deiodinase Activity ◽  
Liver Microsomal Fraction ◽  
Thiouracil Derivatives

Previous studies have shown that 2-thiouracil derivatives are uncompetitive inhibitors of iodothyronine 5′-deiodinase activity of rat liver microsomal fraction. Therefore the interaction of radioiodinated 6-propyl-2-thiouracil with rat liver microsomal fraction and the effect of substrate, cofactor and other inhibitors of 5′-deiodinase activity activity were investigated. It was found that micromolar concentrations of, in order of increasing potency, 3,5-diiodotyrosine, thyroxine, 3,3′,5′-tri-iodothyronine and 3′,5′-di-iodothyronine significantly enhanced binding of 5-[125I]iodo-6-propyl-2-thiouracil to the enzyme preparation. This stimulation was not seen in the presence of 1 mM dithiothreitol, 0.1 mM-6-propyl-2-thiouracil, 0.1 mM-6-propyl-2-thiouracil, 0.1 M-2-mercapto-1-methylimidazole or 1 mM-sodium sulphite. These results support the hypothesis that thiouracil derivatives inhibit 5′-deiodinase activity by forming a mixed disulphide with an intermediate enzyme complex, probably a sulphenyl iodide.

scholarly journals Hydroperoxide-induced chemiluminescence of the perfused lung

1980 ◽  
Vol 192 (1) ◽  
pp. 303-309 ◽  
Author(s):  
E Cadenas ◽  
I D Arad ◽  
A B Fisher ◽  
A Boveris ◽  
B Chance
Keyword(s):  
Microsomal Fraction ◽  
Light Emission ◽  
Time Dependent ◽  
Butyl Hydroperoxide ◽  
Time Dependent Process ◽  
Perfused Lung ◽  
Liver Microsomal Fraction ◽  
Alveolar Oedema ◽  
Hydroperoxide Concentration

Light-emission of the perfused lung is induced by t-butyl hydroperoxide, giving chemiluminescence yields that oscillate between 800 and 1500 counts/s depending on the site and position of the lung. The response of the perfused lung to infusion with different hydroperoxides gives a pattern similar to that observed with the liver microsomal fraction; ethyl hydroperoxide shows a much higher chemiluminescence yield than the tertiary (t-butyl and cumene)hydroperoxides. Alveolar oedema affected the light-emission of the perfused lung depending on the time at which oedema developed, decreasing light emission on infusion of hydroperoxide in the oedematous lung and increasing it when oedema appeared after the maximal chemiluminescence yield was already achieved. Paraquat, administered in vivo, augmented light-emission by approximately 2-fold. The effect of paraquat was a time-dependent process. Lung chemiluminescence, compared with liver chemiluminescence, needed higher hydroperoxide concentration to induce light-emission.

Sign in / Sign up

Export Citation Format

Share Document