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 Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

Ca2+ transport in myocardial sarcolemma from rainbow trout

1990 ◽  
Vol 259 (3) ◽  
pp. R453-R460 ◽  
Author(s):  
G. F. Tibbits ◽  
H. Kashihara ◽  
M. J. Thomas ◽  
J. E. Keen ◽  
A. P. Farrell
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Velocity ◽  
Contractile Element ◽  
Phospholipid Content ◽  
Equilibrium Binding ◽  
Voltage Dependent

Sacrolemmal vesicles were isolated from trout ventricles with a yield of 0.51 mg protein/g wet wt of a fraction enriched approximately 15-fold over the crude homogenate as estimated by K(+)-stimulated p-nitrophenylphosphatase (K(+)-pNPPase) activity. Although the K(+)-pNPPase specific activity compared favorably with that of the rat heart, there were some striking differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and specific phospholipid content (mumol/mg protein) of the sacrolemmal fractions between the two species. Two major sarcolemmal Ca2(+)-transport proteins were investigated, the Na(+)-Ca2+ exchanger and the dihydropyridine (DHP) receptor, a component of the voltage-dependent L-type Ca2+ channel. From the initial rates of Na(+)-dependent Ca2+ uptake, it was determined that the exchanger has an apparent Km for Ca2+ of 14 +/- 1 microM and a maximum velocity of 7.7 +/- 1.1 nmol.mg protein-1.s-1 at 21 degrees C. Experiments using the DHP ligand [3H] (+) PN 200-110 to characterize the equilibrium binding to the DHP receptor in the sarcolemmal fraction yielded a Kd of 0.08 nM and maximum binding sites of 3.06 +/- 0.49 pmol/mg protein. Given the smaller dimensions of the trout myocyte and the resultant higher sarcolemmal surface to cytosolic volume compared with the mammalian myocyte, these in vitro findings are consistent with the notion that Ca2+ transport across sarcolemma is a quantitatively important contributor of Ca2+ delivery to and removal from the contractile element.

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.

Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum

1991 ◽  
Vol 260 (6) ◽  
pp. R1168-R1175
Author(s):  
L. Bosca ◽  
K. B. Storey
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Busycon Canaliculatum ◽  
Native Molecular Mass ◽  
Dependent Protein

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

scholarly journals Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria

1988 ◽  
Vol 251 (3) ◽  
pp. 881-885 ◽  
Author(s):  
J B Allred ◽  
C R Roman-Lopez
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Outer Mitochondrial Membrane ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.

scholarly journals α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Williams ◽  
H Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

Site of synthesis of metallothionein in rat liver

1981 ◽  
Vol 59 (4) ◽  
pp. 301-306 ◽  
Author(s):  
M. George Cherian ◽  
Sharon Yu ◽  
Colvin M. Redman
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Adult Rat ◽  
Cadmium Treatment ◽  
Reticulocyte Lysate

Free and membrane-attached polysomes were isolated from the livers of normal and cadmium-treated rats, and were translated using L-[35S]cysteine and a nuclease-treated reticulocyte lysate system. The translation products were analyzed for radioactive metallothionein by immunoprecipitation with antibodies to rat cadmium metallothionein followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In both normal and cadmium-treated rats, radioactive metallothionein was produced by free polysomes but not by membrane-attached polysomes. Cadmium treatment did not increase the in vitro ability of polysomes to synthesize metallothionein. As a control, the translation products of these two classes of polysomes were also analyzed for radioactive albumin and it was confirmed that membrane-attached polysomes produce albumin but do not synthesize metallothionein. The cell-free synthesis of metallothionein by free polysomes was also demonstrated by isolation of nascent metallothionein by Sephadex gel filtration and DEAE-cellulose chromatography. In adult rat liver there are two forms of metallothionein and both were produced in vitro by free polysomes.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

Abstract The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Investigation of larvae digestive β-glucosidase and proteases of the tomato pest Tuta absoluta for inhibiting the insect development

2016 ◽  
Vol 106 (3) ◽  
pp. 406-414 ◽  
Author(s):  
S. Sellami ◽  
K. Jamoussi
Keyword(s):  
Serine Proteases ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tuta Absoluta ◽  
Leaf Miner ◽  
Protease Inhibition ◽  
Tomato Leaf Miner ◽  
Proteolytic Activities

AbstractThe tomato leaf miner Tuta absoluta is one of the most devastating pests for tomato crops. Digestive proteases and β-glucosidase enzymes were investigated using general and specific substrates and inhibitors. Maximal β-glucosidase and proteolytic activities occurred at temperature and pH optima of 30 and 40°C, 5 and 10–11 unit of pH, respectively. Zymogram analysis showed the presence of distinguished β-glucosidase exhibiting a specific activity of about 183 ± 15 µmol min−1 mg−1. In vitro inhibition experiments suggested that serine proteases were the primary gut proteases. Gel based protease inhibition assays demonstrated that the 28 and 73 kDa proteases might be trypsin-like and chymotrypsin-like enzymes, respectively. Overall gut trypsin-like and chymotrypsin-like activities were evaluated to be about 27.2 ± 0.84 and 1.68 ± 0.03 µmol min−1 mg−1, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that T. absoluta gut serine proteases are responsible for Bacillus thuringiensis Cry insecticidal proteins proteolysis. Additionally, bioassays showed that T. absoluta larvae development was more affected by the β-glucosidases inhibitor (D-glucono-δ-lactone) than the serine proteases inhibitor (soybean trypsin inhibitor). These results are of basic interest since they present interesting data of β-glucosidases and gut serine proteases of T. absoluta larvae.

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