scholarly journals Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1973 ◽  
Vol 135 (4) ◽  
pp. 819-826 ◽  
Author(s):  
W. Howard Evans ◽  
Diana O. Hood ◽  
James W. Gurd
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Protein Band ◽  
Phosphodiesterase Activity ◽  
Liver Plasma Membrane ◽  
Purification And Properties ◽  
Nucleotide Pyrophosphatase ◽  
Neutral Sugars

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate–Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose–detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5′-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000–130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD+, UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.

Phosphorylation of liver plasma membrane-bound calmodulin

1988 ◽  
Vol 66 (8) ◽  
pp. 922-927 ◽  
Author(s):  
Shobha Ghosh ◽  
Jon G. Church ◽  
Basil D. Roufogalis ◽  
Antonio Villalobo
Keyword(s):  
Plasma Membrane ◽  
Calcium Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Bovine Brain ◽  
Antibody Treatment ◽  
Liver Plasma Membrane ◽  
Phosphate Donor ◽  
Membrane Bound ◽  
Stimulation Of

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [γ-32P] ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3′:5′ AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.

scholarly journals Purification and properties of bovine liver plasma membrane 5' nucleotidase

1983 ◽  
Vol 137 (1-2) ◽  
pp. 131-138 ◽  
Author(s):  
Jean HARB ◽  
Khaled MEFLAH ◽  
Yves DUFLOS ◽  
Serge BERNARD
Keyword(s):  
Plasma Membrane ◽  
Bovine Liver ◽  
Liver Plasma Membrane ◽  
Purification And Properties

Characterization of Specific Binding Sites for Corticosterone in Mouse Liver Plasma Membrane

1989 ◽  
Vol 8 (4) ◽  
pp. 229-239 ◽  
Author(s):  
Miguel Trueba ◽  
IÑAki Ibarrola ◽  
Ana Isabel Vallejo ◽  
MarÍA José Sancho ◽  
Aida Marino ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Mouse Liver ◽  
Specific Binding ◽  
Liver Plasma Membrane ◽  
Specific Binding Sites

Purification and properties of an acid proteinase from Choanephora cucurbitarum

1986 ◽  
Vol 32 (2) ◽  
pp. 151-155
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Hydrolytic Activity ◽  
Acid Proteinase ◽  
Metallic Ions ◽  
Diisopropyl Fluorophosphate ◽  
Purification And Properties ◽  
Choanephora Cucurbitarum ◽  
Hydrolysis Of

An acid proteinase has been purified from mycelial extracts of Choanephora cucurbitarum by treatment with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The enzyme hydrolysed haemoglobin rapidly compared with casein, bovine albumin, cytochrome c, and hide powder azure, but failed to hydrolyse any of the synthetic peptides tested. The acid proteinase is a glycoprotein with an apparent molecular weight of 12 700. Optimal hydrolysis of haemoglobin by the proteinase was observed at 20 °C, pH 3.0, and has a Km value of 2.8 mg∙mL−1. Heavy metallic ions, such as Hg2+, Fe2+, and Zn2+, inhibited the hydrolytic activity, whereas Ca2+ and Cu2+ enhanced the enzyme activity by two- and four-fold, respectively, as compared with the controls. Benzamidine and phenylmethanesulfonyl fluoride inhibited severely the enzyme activity, while diisopropyl fluorophosphate, antipain, N-α-p-tosyl-L-lysine chloromethyl ketone inhibited moderately. Phenylmethanesulfonyl fluoride inhibition could be reversed by 2-mercaptoethanol. Reducing agents such as cysteine and dithiothreitol did not enhance the enzyme activity. The data presented suggest that the enzyme has the characteristics of serine proteinases.

Cysteine proteinase of Phascolomyces articulosus: purification and properties

1986 ◽  
Vol 64 (11) ◽  
pp. 2441-2445 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iodoacetic Acid ◽  
Phenylmethylsulfonyl Fluoride ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Salting Out ◽  
Purification And Properties

A proteinase from the mycelial extracts of Phascolomyces articulosus has been purified by salting out with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The proteinase rapidly hydrolysed haemoglobin but failed to hydrolyse any of the synthetic peptides tested. The enzyme is a glycoprotein with an apparent molecular weight of 12 800. The carbohydrate content was estimated to be 65%. It has a temperature optimum of 20 °C, pH optimum of 3.0, and has a Km value of 6.6 mg∙mL−1 for denatured haemoglobin. Iodoacetic acid, iodoacetamide, benzamidine, as well as all the heavy metals tested inhibited the enzyme activity. The enzyme activity was not enhanced by reducing agents such as cysteine, ethylenediaminetetra acetic acid, and dithiothreitol, the latter, however, reversed inhibition by phenylmethylsulfonyl fluoride. The inhibitor studies suggest that the enzyme belongs to the group of cysteine proteinases.

scholarly journals Cytosol intermediates in the transport of iron

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 1051-1055 ◽  
Author(s):  
MT Nunez ◽  
ES Cole ◽  
J Glass
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Binding Protein ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iron Binding ◽  
The Third ◽  
Initial Values ◽  
Iron Supply ◽  
Iron Binding Protein

Three 59Fe-labeled nonheme components of the cytosol were identified when rabbit reticuloyctes were incubated with 59Fe-labeled plasma under conditions in which the iron supply was not limiting. Two of these components were identified as ferritin and transferrin. The latter was characterized by gel filtration as having apparent molecular weight higher than transferrin, indicating that the transferrin may be complexed to another moiety. The third component, referred to as iron- binding protein-I (IBP-I), is as yet uncharacterized. When the reticulocytes were incubated with unlabeled plasma after pulse-labeling with 59Fe-labeled plasma, 59Fe radioactivity in these cytosol components decreased; after 15 min of chase, the 59Fe in ferritin, transferrin, and IBP-I fell to 64.6%, 26.5%, and 65.8% of the initial values, respectively. A good correlation existed between the decrease of 59Fe in these three nonheme compartments and the associated increase in 59Fe-heme. The data presented suggest that cytosol ferritin, transferrin, and IBP-I are intermediates in the transport of 59Fe from the plasma membrane to the mitochondria.

scholarly journals The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1972 ◽  
Vol 129 (4) ◽  
pp. 945-952 ◽  
Author(s):  
D. J. Sams ◽  
W. Montague
Keyword(s):  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Cyclic Monophosphate ◽  
Km Value

1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3′:5′-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent Km values for adenosine 3′:5′-cyclic monophosphate (cyclic AMP) of 3μm and 30μm, suggesting the presence of two activities. The pH optimum of the activity with the low Km value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low Km value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

2007 ◽  
Vol 50 (6) ◽  
pp. 731-738 ◽  
Author(s):  
Ping Chen ◽  
LiJun Zhang ◽  
XuanWen Li ◽  
Xie Wang ◽  
Rui Cao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Membrane Proteome ◽  
Liver Plasma Membrane
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