‘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 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.

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 Metabolism of the herbicide 2,6-dichlorobenzonitrile in rabbits and rats

1966 ◽  
Vol 101 (3) ◽  
pp. 698-706 ◽  
Author(s):  
JG Wit ◽  
H Van Genderen
Keyword(s):  
Oral Administration ◽  
Urinary Excretion ◽  
Rat Liver ◽  
High Speed ◽  
Liver Homogenate ◽  
Acid Formation ◽  
Thiol Compounds ◽  
Enzyme Preparations ◽  
Phenolic Metabolites ◽  
High Speed Supernatant

1. The metabolism of 2,6-dichlorobenzonitrile was studied in rabbits and rats. Oral administration caused an increased urinary excretion of glucuronides and ethereal sulphates. There was also an indication of mercapturic acid formation. 2,6-Dichloro-3-hydroxybenzonitrile and its 4-hydroxy analogue were identified as metabolites in the urine. A small amount of the unchanged substance was recovered from the faeces. 2. By using 2,6-dichlorobenzo[(14)C]nitrile the phenolic metabolites were determined quantitatively and some other possible metabolic routes were excluded. 3. Incubation of 2,6-dichlorobenzonitrile with enzyme preparations (papain and high-speed supernatant of rat-liver homogenate plus glutathione) gave no indications for a reaction with thiol compounds.

scholarly journals Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum

1976 ◽  
Vol 156 (2) ◽  
pp. 347-355 ◽  
Author(s):  
I H Fraser ◽  
S Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
High Speed ◽  
Broad Band ◽  
Chromatographic Technique ◽  
Appreciable Effect ◽  
Serum Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a ‘high-speed’ supernatant. The ‘high-speed’ supernatant was designated the ‘saline wash’ and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 × 10(-6) M. α-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.

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.

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 Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver

1989 ◽  
Vol 263 (2) ◽  
pp. 355-363 ◽  
Author(s):  
L Shaw ◽  
R Schauer
Keyword(s):  
Submandibular Gland ◽  
Mouse Liver ◽  
High Speed ◽  
Metal Salts ◽  
Supernatant Fraction ◽  
Inhibitory Influence ◽  
Hydroxylase Activity ◽  
Acetylneuraminic Acid ◽  
Liver Homogenates ◽  
High Speed Supernatant

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.

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