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 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.

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 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.

A comparison of acetylation in vitro of microsomal, homogenate, and Golgi fractions of rat liver

1988 ◽  
Vol 66 (10) ◽  
pp. 1152-1161 ◽  
Author(s):  
Hari Sambasivam ◽  
Robert K. Murray
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sialic Acids ◽  
Acetyl Coa ◽  
Acetyltransferase Activity ◽  
Acetylneuraminic Acid

The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.

scholarly journals A simple method for purification of epoxide hydratase from rat liver

1977 ◽  
Vol 163 (2) ◽  
pp. 381-383 ◽  
Author(s):  
R G Knowles ◽  
B Burchell
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Styrene Oxide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Yield ◽  
Simple Method ◽  
Epoxide Hydratase

Rat liver epoxide hydratase was purified 460-fold to homogeneity by detergent solubilization and ion-exchange chromatography. The enzyme obtained in high yield (36%) exhibited a specific activity of 479nmol of styrene glycol formed/min per mg of protein, with styrene oxide as substrate. Only one polypeptide-staining band, mol.wt. 49500, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals Alterations in translatable messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP) in rat liver cytosol during deinduction.

1978 ◽  
Vol 253 (12) ◽  
pp. 4327-4332
Author(s):  
D. Kioussis ◽  
L. Reshef ◽  
H. Cohen ◽  
S.M. Tilghman ◽  
P.B. Iynedjian ◽  
...  
Keyword(s):  
Rat Liver ◽  
Messenger Rna ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Liver Cytosol ◽  
Rat Liver Cytosol
Sign in / Sign up

Export Citation Format

Share Document