scholarly journals Identity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirin-metabolizing carboxylesterase from rat liver microsomal fractions. A comparative study with enzymes purified in different laboratories

1985 ◽  
Vol 232 (2) ◽  
pp. 479-483 ◽  
Author(s):  
R Mentlein ◽  
R K Berge ◽  
E Heymann
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Monoacylglycerol Lipase ◽  
Enzyme Preparations ◽  
Nitrophenyl Phosphate ◽  
Liver Microsomal Fraction ◽  
Coa Hydrolase ◽  
Rat Liver Cytosol

Two purified carboxylesterases that were isolated from a rat liver microsomal fraction in a Norwegian and a German laboratory were compared. The Norwegian enzyme preparation was classified as palmitoyl-CoA hydrolase (EC 3.1.2.2) in many earlier papers, whereas the German preparation was termed monoacylglycerol lipase (EC 3.1.1.23) or esterase pI 6.2/6.4 (non-specific carboxylesterase, EC 3.1.1.1). Antisera against the two purified enzyme preparations were cross-reactive. The two proteins co-migrate in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Both enzymes exhibit identical inhibition characteristics with Mg2+, Ca2+ and bis-(4-nitrophenyl) phosphate if assayed with the two substrates palmitoyl-CoA and phenyl butyrate. It is concluded that the two esterase preparations are identical. However, immunoprecipitation and inhibition experiments confirm that this microsomal lipase differs from the palmitoyl-CoA hydrolases of rat liver cytosol and mitochondria.

scholarly journals Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

1982 ◽  
Vol 207 (1) ◽  
pp. 133-138 ◽  
Author(s):  
M G Battelli ◽  
E Lorenzoni
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Enzymic Activity ◽  
Oxidized Glutathione ◽  
Adult Rats ◽  
Purification And Properties ◽  
Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

scholarly journals Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning

1980 ◽  
Vol 192 (2) ◽  
pp. 507-516 ◽  
Author(s):  
T J Hopkirk ◽  
D P Bloxham
Keyword(s):  
Pyruvate Kinase ◽  
Rat Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Concentration ◽  
Liver Cytosol ◽  
Maximum Value ◽  
Rat Liver Cytosol ◽  
Slow Turnover

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24–48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.

scholarly journals Purification of phospholipid methyltransferase from rat liver microsomal fraction

1986 ◽  
Vol 237 (3) ◽  
pp. 699-705 ◽  
Author(s):  
M A Pajares ◽  
M Villalba ◽  
J M Mato
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Molecular Mass ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Liver Microsomal Fraction

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

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.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Identification and characterization of cDNA clones encoding two homologous proteins that are part of the asialoglycoprotein receptor

1987 ◽  
Vol 7 (5) ◽  
pp. 1841-1847
Author(s):  
M McPhaul ◽  
P Berg
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Asialoglycoprotein Receptor ◽  
Cdna Clones ◽  
Differential Splicing ◽  
Homologous Proteins ◽  
Different Types ◽  
Identification And Characterization

The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.

scholarly journals Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver

1972 ◽  
Vol 129 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Ragnar Flengsrud ◽  
Bjarne Østerud ◽  
Hans Prydz
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Kinetic Studies ◽  
Disc Electrophoresis ◽  
Nitrobenzoic Acid

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin–fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1μg of protein/ml. 3. The inhibitor had a pI of 3.50–3.75, a molecular weight (from sodium dodecyl sulphate–polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin–fibrinogen complex.

scholarly journals Selective depletion of small basic non-glycosylated proteins in diabetes

1981 ◽  
Vol 198 (1) ◽  
pp. 149-157 ◽  
Author(s):  
F C Samaniego ◽  
F Berry ◽  
J F Dice
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Cytosol ◽  
Physiological Importance ◽  
Subunit Molecular Weight ◽  
Uncontrolled Diabetes ◽  
Streptozotocin Induced Diabetes ◽  
Protein Classes ◽  
Rat Liver Cytosol ◽  
Glycosylated Proteins

Degradative rates of small basic non-glycosylated proteins are preferentially enhanced in rat liver cytosol during severe streptozotocin-induced diabetes. Synthetic rates of these classes of proteins are not selectively enhanced in diabetes, so small basic non-glycosylated proteins should be depleted from liver cytosol as a consequence of this disease. To test this hypothesis, proteins were analysed from normal animals, from diabetic animals receiving insulin and from diabetic animals after insulin withdrawal for 3 days. The proteins were separated according to subunit molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, according to isoelectric point by isoelectric focusing and according to carbohydrate content by affinity chromatography with concanavalin A linked to agarose. Severe uncontrolled diabetes is associated with the predicted depletion of small basic non-glycosylated proteins from liver cytosol. The preferential degradation and loss of these protein classes may be of considerable physiological importance to the animal.

Characterization of cytosolic female rat liver receptors of the lactogenic type

1990 ◽  
Vol 123 (2) ◽  
pp. 231-237
Author(s):  
M. Emtner ◽  
P. Roos
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Human Growth ◽  
Structural Features ◽  
Female Rat ◽  
Membrane Bound

Abstract. Some properties of cytosolic receptors of the lactogenic type from female rat liver were studied and compared with those of membrane-bound (microsomal) receptors. The association constant between the cytosolic receptors and human growth hormone was 2.2 l/nmol, which was not significantly different from the value obtained for the microsomal receptors (3.6 l/nmol). Since unlabelled hGH and human prolactin, but not bovine growth hormone, displaced [125I]hGH bound to receptors from both sources, the cytosolic receptors, like the microsomal receptors, must be lactogenic. Furthermore, the cytosolic receptors were recognized by a monoclonal antibody raised against microsomal receptors from female rat liver. However, covalent cross-linking of cytosolic receptors to [125I]hGH and subsequent sodium dodecyl sulphate electrophoresis gave a single band corresponding to a molecular weight of 42 200 (after subtraction of the molecular weight of hGH), which differs significantly (p<0.01) from the values determined for the two distinct bands given by the microsomal fraction. Moreover, upon molecular sieve chromatography the receptor activity in the two fractions appeared at significantly (p<0.05) different elution volumes. These results show that the cytosolic and microsomal receptors have some structural features in common but are definitely not identical.

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