scholarly journals Studies in vitro on the involvement of O-sulphate esters in the formation of O-methylated 3,4-dihydroxybenzoic acid by rat liver

1981 ◽  
Vol 193 (3) ◽  
pp. 869-874 ◽  
Author(s):  
E J M Pennings ◽  
G M J Van Kempen
Keyword(s):  
Rat Liver ◽  
High Speed ◽  
Simultaneous Action ◽  
Dihydroxybenzoic Acid ◽  
Rate Of Hydrolysis ◽  
Directing Effect ◽  
High Speed Supernatant ◽  
Assay Conditions ◽  
Methoxybenzoic Acid

The involvement of O-sulphate esters in the directed O-methylation was investigated in vitro with a dialysed ‘high-speed’ supernatant from rat liver as the enzyme preparation and the catechol compound 3,4-dihydroxybenzoic acid as the substrate. The enzyme reactions involved were studied separately with the O-methylated and O-sulphated derivatives. The rate of hydrolysis by arylsulphatase was 14.5 nmol/min per mg of protein for 3-methoxy-4-sulphonyloxybenzoic acid and 10.1 nmol/min per mg of protein for 4-methoxy-3-sulphonyloxybenzoic acid. The sulphotransferase activity towards the guaiacols 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid was 570pmol of 4-O-sulphated and 350pmol of 3-O-sulphated product formed/min per mg of protein. The 3-O- and 4-O-sulphate esters of 3,4-dihydroxybenzoic acid could not serve as substrates for the catechol O-methyltransferase reaction. When either ester was incubated in the presence of S-adenosyl-L-methionine, but without the arylsulphatase inhibitor KH2PO4, 3,4-dihydroxybenzoic acid was formed, which was subsequently O-methylated in a meta/para ratio of 4.6. It is concluded that O-methylation can precede O-sulphation but that O-sulphation prevents further metabolism by O-methylation. Also O-sulphate esters do not have a directing effect on O-methylation. From the study of the simultaneous action of sulphotransferase and catechol O-methyltransferase on 3,4-dihydroxybenzoic acid we conclude that O-sulphation and O-methylation proceed independently of each other under the assay conditions used, both directed preferentially to the 3-hydroxy group.

scholarly journals Studies on the meta and para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid by rat liver sulphotransferase in vitro

1980 ◽  
Vol 191 (1) ◽  
pp. 133-138 ◽  
Author(s):  
E J M Pennings ◽  
G M J Van Kempen
Keyword(s):  
Chemical Synthesis ◽  
Rat Liver ◽  
High Speed ◽  
Helix Pomatia ◽  
Dihydroxybenzoic Acid ◽  
High Speed Supernatant ◽  
Optimal Ph

The enzymic meta and para O-sulphation of 3,4-dihydroxybenzoic acid was investigated in vitro with a dialysed high-speed supernatant from rat liver. The O-sulphated products were identified by comparison with the reference compounds. The chemical synthesis and identification of the reference O-sulphate esters is described in detail. The sulphotransferase activity of the dialysed supernatant from rat liver towards 3,4-dihydroxybenzoic acid was 580 pmol of 3-O-sulphate and 120 pmol of 4-O-sulphate formed/min per mg of protein at the optimal pH of 7.4. The meta/para ratio of O-sulphation was independent of pH, time of incubation, concentration of enzyme and presence of dithiothreitol. The O-sulphate esters of 3,4-dihydroxybenzoic acid were found to be good substrates for the arylsulphatase reaction at pH 5.6. The arylsulphatase activity of a dialysed preparation from rat liver was 4.0 nmol of 3-O- and 5.7 nmol of 4-O-sulphate ester hydrolysed/min per mg of protein, respectively. Arylsulphatase from Helix pomatia had an activity of 620 pmol of 3-O-sulphate and of 16.6 nmol of 4-O-sulphate ester hydrolysed/min per unit (mumol/h) of sulphatase.

THE EFFECT OF DIPHENYLHYDANTOIN IN VITRO ON THE METABOLISM OF TESTOSTERONE BY RAT LIVER SUBCELLULAR FRACTIONS

1968 ◽  
Vol 57 (4) ◽  
pp. 649-656
Author(s):  
Leon J. Sholiton ◽  
Emile E. Werk ◽  
Joseph MacGee
Keyword(s):  
Rat Liver ◽  
High Speed ◽  
Metabolite Profile ◽  
Subcellular Fractions ◽  
Polar Metabolite ◽  
Metabolite Production ◽  
Liver Slices ◽  
High Speed Supernatant

ABSTRACT The effect of diphenylhydantoin (DPH) in vitro on the »metabolite profile« of testosterone-4-14C has been studied utilizing incubates of high speed supernatant and microsomal fractions of rat liver. In contrast to earlier experiments with rat liver slices, in which a standard amount of DPH resulted in increased non-polar metabolite production, in the present experiments, when separate subcellular fractions are incubated, no such DPH effect can be detected under the conditions utilized. When certain mixtures of supernatant and microsomal fractions are incubated, however, there is evidence that the DPH augmentation of non-polar metabolite production can again be manifest. An explanation for these results can only be speculated upon at present.

Effect of 2,4-D on the Hydrolysis of Diclofop-Methyl in Wild Oat (Avena fatua)

Weed Science ◽  
1980 ◽  
Vol 28 (6) ◽  
pp. 725-729 ◽  
Author(s):  
B. D. Hill ◽  
B. G. Todd ◽  
E. H. Stobbe
Keyword(s):  
Enzyme Preparation ◽  
Avena Fatua ◽  
Wild Oat ◽  
Enzymic Hydrolysis ◽  
Standard Assay ◽  
Rate Of Hydrolysis ◽  
Dichlorophenoxy Acetic Acid ◽  
Hydrolysis Of ◽  
Assay Conditions

The basis for 2,4-D [(2,4-dichlorophenoxy)acetic acid] antagonism of diclofop-methyl {methyl 2-[4-(2,4-dichlorophenoxy) phenoxy] propanoate} toxicity to wild oat (Avena fatuaL.) was investigated by studying changes in the metabolism of diclofop-methyl in vitro. An esterase from wild oat, which hydrolyzes diclofop-methyl to the acid diclofop, was extracted, partially purified, and the reaction characterized. The rate of hydrolysis of14C-diclofop-methyl was 0.14 ηmoles/2 h at standard assay conditions of 0.25 mg lyophilized enzyme preparation (19.6% protein) in 0.1 ml phosphate buffer (0.1 M, pH 7.0), substrate 5 μM. The addition of 2,4-D to this reaction did not inhibit14C-diclofop formation. Higher levels of 2,4-D stimulated enzymic hydrolysis.14C-diclofop-methyl was rapidly metabolized to14C-diclofop and polar14C-conjugates when vacuum-infiltrated into wild oat leaf segments. The addition of 2,4-D caused small increases in the rates of both14C-diclofop-methyl de-esterification and14C-diclofop conjugation. It is concluded that 2,4-D does not inhibit the in vitro de-esterification of diclofop-methyl.

Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions.

Endocrinology ◽  
1993 ◽  
Vol 133 (5) ◽  
pp. 2177-2186 ◽  
Author(s):  
T J Visser ◽  
E Kaptein ◽  
H van Toor ◽  
J A van Raaij ◽  
K J van den Berg ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Rat Liver ◽  
In Vitro Assay ◽  
Microsomal Enzyme ◽  
Vitro Assay ◽  
In Vivo Treatment ◽  
Microsomal Enzyme Inducers ◽  
Assay Conditions

‘Binding’ of [14C]phenol to rat liver high-speed supernatant

1980 ◽  
Vol 8 (1) ◽  
pp. 117-118
Author(s):  
H. PAUL A. ILLING ◽  
ESTHER S. A. HOUSE
Keyword(s):  
Rat Liver ◽  
High Speed ◽  
High Speed Supernatant

scholarly journals Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1977 ◽  
Vol 164 (3) ◽  
pp. 541-547 ◽  
Author(s):  
Ian H. Fraser ◽  
Sailen Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Column Chromatography ◽  
High Speed ◽  
Affinity Column ◽  
High Molecular Weight Fraction ◽  
Serum Enzyme ◽  
Membrane Enzyme ◽  
Membrane Bound ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn2+ that could not be replaced by Ca2+, Mg2+, Zn2+ or Co2+, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent Km for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.

scholarly journals Measurement and kinetic study of the formation of adrenaline sulphate in vitro

1978 ◽  
Vol 174 (3) ◽  
pp. 777-782 ◽  
Author(s):  
K P Wong
Keyword(s):  
Small Intestine ◽  
Guinea Pig ◽  
High Speed ◽  
Maximal Activity ◽  
Ph Optimum ◽  
Inorganic Sulphate ◽  
Measurable Activity ◽  
Small Intestines ◽  
High Speed Supernatant

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3′-phosphate 5′-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4–6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.

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