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.

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 The Activity of Heparin Preparations Studied by Amidolytic and Clotting Methods

1977 ◽  
Author(s):  
A.N. Teien ◽  
U. Abildgaard ◽  
M. Höök ◽  
U. Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
Factor Xa ◽  
Test System ◽  
Assay Method ◽  
Thrombin Time ◽  
Assay Methods ◽  
Specific Activities ◽  
The Difference ◽  
Heparin Preparation

Two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparins and two heparin preparations separated by affinity chromatography (“High affinity heparin”=HAH and “Low affinity heparin”=LAH) were assayed by the activated partial thromboplastin time method (APTT), the calcium thrombin time method (CaTT) and two amidolytic methods (measuring the accelerating effect of heparin on the inactivation of thrombin or factor Xa by antithrombin III), with and without plasma in the test system. The specific activities of the various heparins were expressed relative to that of the 3rd. Int. Standard (=100). Found specific activities ranged 3 - 198 (LAH and HAH, respectively). In all assay systems HAH had the highest specific activity, followed by one of the commercial preparations and the 3rd Int. Standard. LAH and human heparin had very low specific activities, except in the APTT test system, an assay method which in addition mirrors other anticoagulant effects of heparin than the acceleration of antithrombin III. Apart from the higher effect of LAH and human heparin on the APTT, the difference in specific activities found for each individual heparin preparation with these various assay methods was slight.In view of the reproducibility and simplicity of the amidolytic methods, it is suggested that they be adapted for heparin standardization.

EVIDENCE FOR THE INDUCTION OF URIDINE AND DEOXY-URIDINE PHOSPHORYLASES OF REGENERATING RAT LIVER IN VITRO

1965 ◽  
Vol 43 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Esther W. Yamada
Keyword(s):  
Amino Acids ◽  
Tissue Culture ◽  
Rat Liver ◽  
Culture Medium ◽  
Specific Activity ◽  
Tissue Culture Medium ◽  
Uridine Phosphorylase ◽  
Regenerating Rat Liver ◽  
Specific Activities

Increases in the specific activities of undine and deoxyuridine phosphorylases of slices of regenerating rat liver were found 4 hours after incubation in tissue-culture medium containing uridine or 6-azauridine. These increases were not found when the tissue-culture medium contained either 8-azaguanine or puromycin, or when it lacked amino acids. Although both uridine and 6-azauridine were more effective in increasing the specific activity of uridine phosphorylase than that of deoxyuridine phosphorylase, azauridine was more effective than uridine in increasing the specific activities of both enzymes.In time studies, in which slices of regenerating rat liver were incubated in tissue-culture medium containing optimal concentrations of uridine, the specific activities of the two enzymes reached maximum levels at 3–4 hours. Puromycin prevented these increases.

scholarly journals Dual-digitonin-pulse perfusion. Concurrent sampling of periportal and perivenous cytosol of rat liver for determination of metabolites and enzyme activities

1987 ◽  
Vol 243 (1) ◽  
pp. 87-95 ◽  
Author(s):  
B Quistorff ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Glutamine Synthetase ◽  
Rat Liver ◽  
High Purity ◽  
Alanine Aminotransferase ◽  
Specific Activity ◽  
Perfusion Technique ◽  
Short Intervals ◽  
Specific Activities

A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed ‘dual-digitonin-pulse perfusion’. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.

scholarly journals Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme

1994 ◽  
Vol 300 (1) ◽  
pp. 15-19 ◽  
Author(s):  
E Figueiredo ◽  
M C Duque-Magalhães
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Metal Affinity ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
The Matrix ◽  
Carboxypeptidase Inhibitor ◽  
Matrix Fraction

A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.

scholarly journals Studies on Fractionated Heparin

1977 ◽  
Author(s):  
P. A. Lane ◽  
I. Macgregor ◽  
R. Michalski ◽  
V. V. Kakkar
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
Factor Xa ◽  
Assay Method ◽  
Clotting Time ◽  
Reactive Material ◽  
Heparin Fractions ◽  
Specific Activities ◽  
Low Activity

Commercial porcine heparin has been divided into five different molecular weight components following gel filtration upon a polyacrylamide-agarose gel matrix. Rechromatography has shown that the two extreme fractions, which contain very low and very high molecular weight material, were totally separable upon gel filtration, while the intermediate fractions contained material in common with other fractions. Four of the five fractions contained almost equivalent specific activity when measured by an anti-Factor Xa clotting assay (l) and yet had very different specific activities if assayed by either thrombin clotting time or kaolin-cephalin clotting time methods. The highest MW fraction had low specific activity in all of the clotting assays, suggesting that it contained the greatest percentage of a non-reactive material demonstrated by Rosenberg et al, (2) while the lowest MW fraction had low activity only in thrombin clotting time and KCCT assays. The lowest MW fraction produced a smaller acceleration of purified fibrin monomer polymerisation rate and also smaller inhibition of thrombin induced platelet aggregation in plasma. The demonstration of different results for specific activities of the heparin fractions, depending upon the assay method used is in agreement with a recent study (3) and has important implications in regard to the pharmacopeal assays for heparin. It also suggests that heparins with higher specific anti-Factor Xa (or antithrombotic) effect can be prepared from commercial heparins, although these may have a relatively low ability to neutralise thrombin in plasma.

Structural and Metabolic Interrelationships Among Glycerophosphatides of Rat Liver In Vivo

1971 ◽  
Vol 49 (12) ◽  
pp. 1347-1356 ◽  
Author(s):  
B. J. Holub ◽  
A. Kuksis
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Specific Activity ◽  
Acyl Transfer ◽  
De Novo Synthesis ◽  
Individual Molecular Species ◽  
Relative Specific Activity ◽  
Proportional Contribution ◽  
Specific Activities

The specific activities of individual molecular species of rat liver diacylglycerylphosphorocholine (PC), diacylglycerylphosphoroethanolamine (PE), and diacylglycerophosphorylinositol (MPI) were determined and compared following intravenous injection of glycerol-14C. PC, PE, and MPI contained 41, 51, and 83%, respectively, tetraenoic species, and 40,17, and 9% combined mono-, di-, and trienoic species. The rest of the phosphatide mass of PC, PE, and MPI was contributed by 18, 32, and 8% penta- and hexaenoic species, respectively. The proportions of chemical classes of the glycerophosphatides differed by 1.1- to 18-fold while the fatty acid associations within the unsaturation classes common to these phosphatides varied 2.2- to 17-fold. After 5 min exposure to radioactive glycerol, the mono-, di-, and trienoic species of the PC, PE, and MPI possessed 13–18, 15–50, and 6–42 times, respectively, the specific activity of the tetraenes of the corresponding phosphatide classes. While the pentaenoic and hexaenoic species of PC and MPI had specific activities three to five times those of the respective tetraenes, the higher polyenes of PE were considerably more radioactive and approached the specific activity of the dienoic species of this phosphatide. With progressing time up to 60 min, the tetraenoic species of PC, PE, and MPI showed increases in relative specific activity of 50, 64, and 109%, respectively, in the three phosphatides. These results are consistent with an effective de novo synthesis of the oligoenoic species and a transacylation of the tetraenoic species of all liver glycerophosphatides tested. The proportional contribution of de novo synthesis in comparison to acyl transfer is apparently greater to the formation of PC and PE than to that of MPI.

Purification and characterization of D-β-hydroxybutyrate dehydrogenase expressed in Escherichia coli

1993 ◽  
Vol 71 (7-8) ◽  
pp. 406-410
Author(s):  
Les Jones ◽  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Functional Properties ◽  
Rat Liver ◽  
Specific Activity ◽  
Purification And Characterization ◽  
E Coli ◽  
Specific Activities ◽  
Viable Method

D-β-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 μmol/(min∙mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression, lipid requiring.

scholarly journals Purification and characterization of paraoxon hydrolase from rat liver

1997 ◽  
Vol 321 (3) ◽  
pp. 595-601 ◽  
Author(s):  
Lourdes RODRIGO ◽  
Fernando GIL ◽  
Antonio F. HERNANDEZ ◽  
Anabel MARINA ◽  
Jesus VAZQUEZ ◽  
...  
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Amino Acid Sequences ◽  
Rabbit Serum ◽  
Heat Inactivation ◽  
Ph Profile ◽  
Apparent Homogeneity ◽  
Nitrophenyl Phosphate ◽  
Cibacron Blue 3Ga ◽  
Serum Paraoxonase

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.

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.

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