Biosynthesis of 1,2-3H and 4-14C steroid sulfates of high specific activity

1970 ◽  
Vol 48 (1) ◽  
pp. 148-150 ◽  
Author(s):  
J. Torday ◽  
G. Hall ◽  
M. Schweitzer ◽  
C. J. P. Giroud
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Homogenate ◽  
High Specific Activity ◽  
Supernatant Fraction ◽  
Metabolic Studies

A supernatant fraction of rat liver homogenate enriched with ATP was used for the biosynthesis of the ester sulfates of several 3H and 14C steroids of the pregn-4-ene series. The method provides a simple means to prepare steroid sulfates of high specific activity for use in either metabolic studies or as reference compounds in the quantification of such conjugates by isotope assays.

scholarly journals Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination.

1976 ◽  
Vol 70 (3) ◽  
pp. 660-670 ◽  
Author(s):  
H Cheng ◽  
M G Farquhar
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Liver Homogenate ◽  
Supernatant Fraction ◽  
Golgi Membranes ◽  
Microsomal Membranes

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.

METHYLATION OF THE 3-OH POSITION OF CATECHOL ACIDS BY RAT LIVER AND KIDNEY PREPARATIONS

1958 ◽  
Vol 36 (5) ◽  
pp. 491-497 ◽  
Author(s):  
J. Pellerin ◽  
A. D'Iorio
Keyword(s):  
Enzymatic Activity ◽  
Rat Liver ◽  
Paper Chromatography ◽  
Reduced Glutathione ◽  
Liver Homogenate ◽  
Supernatant Fraction ◽  
Hydroxybenzoic Acid ◽  
Dihydroxybenzoic Acid ◽  
Kidney Homogenate ◽  
Liver And Kidney

3,4-Dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxycinnamic acid were separately incubated with L-methionine-methyl-C14 in the presence of rat liver or kidney homogenate. In each case, the radioactive metabolite separated by paper chromatography was found to have migrating properties similar to those of the 3-methoxy-4-hydroxyphenolic acid. This reaction was enhanced by the addition of ATP, Mg++, and reduced glutathione. When 3-hydroxybenzoic acid was incubated in this medium no methylated derivative was obtained. Preliminary experiments indicated that the enzymatic activity was contained mostly in the supernatant fraction. It was also noted that liver homogenate was much more active than kidney homogenate in methylating catechol acids.

scholarly journals The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate

1970 ◽  
Vol 117 (5) ◽  
pp. 951-956 ◽  
Author(s):  
P. C. Jocelyn
Keyword(s):  
Uric Acid ◽  
Xanthine Oxidase ◽  
Rat Liver ◽  
Liver Homogenate ◽  
No Oxidation ◽  
Urate Oxidase ◽  
Supernatant Fraction ◽  
Endogenous Substrate ◽  
Additional Protein ◽  
Microsome Fraction

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.

METHYLATION OF THE 3-OH POSITION OF CATECHOL ACIDS BY RAT LIVER AND KIDNEY PREPARATIONS

1958 ◽  
Vol 36 (1) ◽  
pp. 491-497 ◽  
Author(s):  
J. Pellerin ◽  
A. D'Iorio
Keyword(s):  
Enzymatic Activity ◽  
Rat Liver ◽  
Paper Chromatography ◽  
Reduced Glutathione ◽  
Liver Homogenate ◽  
Supernatant Fraction ◽  
Hydroxybenzoic Acid ◽  
Dihydroxybenzoic Acid ◽  
Kidney Homogenate ◽  
Liver And Kidney

3,4-Dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxycinnamic acid were separately incubated with L-methionine-methyl-C14 in the presence of rat liver or kidney homogenate. In each case, the radioactive metabolite separated by paper chromatography was found to have migrating properties similar to those of the 3-methoxy-4-hydroxyphenolic acid. This reaction was enhanced by the addition of ATP, Mg++, and reduced glutathione. When 3-hydroxybenzoic acid was incubated in this medium no methylated derivative was obtained. Preliminary experiments indicated that the enzymatic activity was contained mostly in the supernatant fraction. It was also noted that liver homogenate was much more active than kidney homogenate in methylating catechol acids.

scholarly journals The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

1986 ◽  
Vol 239 (1) ◽  
pp. 185-190 ◽  
Author(s):  
P Fritzson ◽  
T B Haugen ◽  
H Tjernshaugen
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Specific Activity ◽  
Ph Dependence ◽  
Gel Permeation Chromatography ◽  
Total Activity ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Distinct Fraction ◽  
Membrane Bound

An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

scholarly journals Stereospecificity of hepatic l-tryptophan 2,3-dioxygenase

1980 ◽  
Vol 189 (3) ◽  
pp. 393-405 ◽  
Author(s):  
Y Watanabe ◽  
M Fujiwara ◽  
R Yoshida ◽  
O Hayaishi
Keyword(s):  
Rat Liver ◽  
Mouse Liver ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Assay Method ◽  
Supernatant Fraction ◽  
Subunit Structure ◽  
Apparent Homogeneity ◽  
Specific Activities ◽  
Assay Conditions

Tryptophan 2,3-dioxygenase [L-tryptophan–oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.

scholarly journals Pathways of fructose conversion into glucose in foetal rat liver

1965 ◽  
Vol 97 (2) ◽  
pp. 365-370 ◽  
Author(s):  
FJ Ballard
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
High Specific Activity ◽  
Direct Conversion ◽  
Seminal Vesicles ◽  
Adult Liver ◽  
Adult Rats ◽  
Liver Slices ◽  
Foetal Rat ◽  
Glucose Formation

1. Glucose, formed from [1-(14)C]fructose or [6-(14)C]fructose in rat-liver slices, has been isolated as gluconate and degraded to give the radioactivity in C-1, C-2-5 and C-6. 2. By using this method it has been shown that, in liver from foetal rats younger than 20 days, glucose is formed from fructose without splitting of the molecule by the aldolase reaction. The rate of glucose formation from fructose in liver from these foetuses is approximately half of the rate in adult liver. 3. The direct conversion of fructose into glucose in foetal rat liver is not via sorbitol as in seminal vesicles, as this pathway cannot be detected. 4. When liver slices are incubated with [U-(14)C]fructose of high specific activity, the labelled intermediates are similar whether from liver from 18-day foetal, newborn or adult rats. 5. These findings are discussed with reference to the changing pathways of fructose metabolism during perinatal development of the liver in the rat.

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