Characterization of a membrane tyrosine phosphatase in AR42J cells: regulation by somatostatin

1992 ◽  
Vol 262 (6) ◽  
pp. G1007-G1014 ◽  
Author(s):  
N. Tahiri-Jouti ◽  
C. Cambillau ◽  
N. Viguerie ◽  
C. Vidal ◽  
L. Buscail ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Maximum Level ◽  
Somatostatin Analogue ◽  
Maximal Velocity ◽  
Control Activity ◽  
Transient Activation ◽  
High Level ◽  
Ptpase Activity

A phosphotyrosyl protein phosphatase (PTPase) activity has been characterized in the plasma membranes of confluent AR42J pancreatic tumor cells using 32P-labeled poly(Glu, Tyr) as substrate. Membrane PTPase activity exhibited an apparent Michaelis constant of 3 microM and an apparent maximal velocity of 0.9 nmol.min-1.mg-1. It was inhibited by orthovanadate, zinc, poly(Glu,Tyr) and was stimulated by EDTA and dithiothreitol. Gel filtration of solubilized plasma membranes gave a peak of enzyme activity at a relative molecular weight of 70,000. Plasma membrane PTPase activity was changed during AR42J cell growth. At the beginning of culture, the control PTPase activity was minimal. Over the 5 days of culture, PTPase activity increased to reach a maximum (3.5-fold over control activity) preceding confluency by 2 days. Then the high level of PTPase activity was sustained until confluency. Incubation of the cells with the stable somatostatin analogue SMS 201-995 (SMS) resulted in a rapid and transient activation of crude membrane PTPase activity. Activation reached a maximum level within 5 min of addition and return to control levels within 20 min. The effect of SMS was dose dependent with half-maximal and maximal activation occurring at 6 pM and 0.1 nM SMS respectively.

scholarly journals Regulation of an hepatic low-Mr membrane-associated protein-tyrosine phosphatase

1993 ◽  
Vol 292 (1) ◽  
pp. 1-5 ◽  
Author(s):  
P S Tappia ◽  
P G P Atkinson ◽  
R P Sharma ◽  
G J Sale
Keyword(s):  
Tyrosine Phosphatase ◽  
Gel Filtration ◽  
Protein Tyrosine Phosphatases ◽  
Single Species ◽  
Particulate Fraction ◽  
Egf Receptors ◽  
Western Blots ◽  
Protein Tyrosine ◽  
Receptor Signal ◽  
Ptpase Activity

Protein-tyrosine phosphatases (PTPases), active against autophosphorylated insulin and epidermal growth factor (EGF) receptors in rat liver, are predominantly membrane associated. Fasting of rats for 48 h decreased hepatic particulate PTPase activity by 15.0-26.9%. This reduction in particulate PTPase activity was due to a rather specific decrease in activity of > 85% of a single species of PTPase, termed PTPase I. Disappearance of PTPase I activity from the particulate fraction was not accounted for by its translocation to the cytosol. PTPase I displayed the highest activity against autophosphorylated insulin and EGF receptors, relative to activity against a 32P-labelled peptide substrate, of three PTPases resolved from the liver particulate fraction. The M(r) value of PTPase I, as determined by gel filtration on a Superose 12 column was approx. 42,000, indicating that PTPase I belongs to the low-M(r) class of PTPases. An antibody raised against PTPase 1B, the prototype of this class of PTPases, did not react with PTPase I in Western blots. The potential importance of the novel change in activity of PTPase I in the regulation of insulin-receptor signal transduction is discussed.

scholarly journals Characterization of the binding of plasminogen activators to plasma membranes from human liver

1992 ◽  
Vol 287 (3) ◽  
pp. 911-915 ◽  
Author(s):  
G Nguyen ◽  
S J Self ◽  
C Camani ◽  
E K O Kruithof
Keyword(s):  
Human Liver ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Mannose Receptor ◽  
Hepatoma Cells ◽  
Tissue Type ◽  
High Affinity ◽  
Liver Membranes ◽  
Membrane Bound ◽  
Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

scholarly journals Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Y Ikehara ◽  
Y Hayashi ◽  
S Ogata ◽  
A Miki ◽  
T Kominami
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Soluble Form ◽  
Microsomal Membranes ◽  
Hydrophobic Nature ◽  
Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

scholarly journals Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells

1989 ◽  
Vol 262 (2) ◽  
pp. 449-456 ◽  
Author(s):  
C Hanekom ◽  
A Nel ◽  
C Gittinger ◽  
A Rheeder ◽  
G Landreth
Keyword(s):  
T Cells ◽  
Time Course ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Jurkat Cells ◽  
Serine Kinase ◽  
Transient Activation ◽  
Normal Human ◽  
Dose Dependent

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.

Relationship between the membrane potential of the contractile vacuole complex and its osmoregulatory activity inParamecium multimicronucleatum

2002 ◽  
Vol 205 (20) ◽  
pp. 3261-3270 ◽  
Author(s):  
Heidi K. Grønlien ◽  
Christian Stock ◽  
Marilynn S. Aihara ◽  
Richard D. Allen ◽  
Yutaka Naitoh
Keyword(s):  
Reference Electrode ◽  
Maximum Level ◽  
The Other ◽  
Contractile Vacuole ◽  
Hypertonic Solution ◽  
Hypotonic Solution ◽  
Atpase Inhibitor ◽  
Filling Phase ◽  
High Level

SUMMARYThe electric potential of the contractile vacuole (CV) of Paramecium multimicronucleatum was measured in situ using microelectrodes,one placed in the CV and the other (reference electrode) in the cytosol of a living cell. The CV potential in a mechanically compressed cell increased in a stepwise manner to a maximal value (approximately 80 mV) early in the fluid-filling phase. This stepwise change was caused by the consecutive reattachment to the CV of the radial arms, where the electrogenic sites are located. The current generated by a single arm was approximately 1.3×10-10 A. When cells adapted to a hypotonic solution were exposed to a hypertonic solution, the rate of fluid segregation, RCVC, in the contractile vacuole complex (CVC) diminished at the same time as immunological labelling for V-ATPase disappeared from the radial arms. When the cells were re-exposed to the previous hypotonic solution, the CV potential, which had presumably dropped to near zero after the cell's exposure to the hypertonic solution, gradually returned to its maximum level. This increase in the CV potential occurred in parallel with the recovery of immunological labelling for V-ATPase in the radial arm and the resumption of RCVC or fluid segregation. Concanamycin B, a potent V-ATPase inhibitor, brought about significant decreases in both the CV potential and RCVC. We confirm that (i) the electrogenic site of the radial arm is situated in the decorated spongiome, and (ii) the V-ATPase in the decorated spongiome is electrogenic and is necessary for fluid segregation in the CVC. The CV potential remained at a constant high level(approximately 80 mV), whereas RCVC varied between cells depending on the osmolarity of the adaptation solution. Moreover, the CV potential did not change even though RCVC increased when cells adapted to one osmolarity were exposed to a lower osmolarity, implying that RCVC is not directly correlated with the number of functional V-ATPase complexes present in the CVC.

Hybrid Pigments Based on Montmorillonite and Anionic Dyes for Leather Finishing

2021 ◽  
Vol 320 ◽  
pp. 198-203
Author(s):  
Anna Bondaryeva ◽  
Olena Mokrousova ◽  
Olena Okhmat
Keyword(s):  
Maximum Level ◽  
Steric Factor ◽  
Anionic Dyes ◽  
Mineral Particles ◽  
High Level ◽  
Leather Finishing ◽  
Positively Charged ◽  
Sodium Form

The work is focused on obtaining hybrid pigments by adsorption of anionic dyes on positively charged montmorillonite. Modification of the sodium form of montmorillonite by chromium hydroxocomplexes was provided to ensure effective adsorption of anionic dyes on the surface of mineral particles. A high level of adsorption of anionic dyes as a result of steric factor was revealed. It was shown that the adsorption of dyes depended on the pH of the medium and was characterized by a maximum level at pH 4.5 – 6.0. The scheme of obtaining hybrid pigments, which were characterized by good сovering ability, resistance to stratification, especially saturated and intense colour was proposed.

Glucose transport by isolated plasma membranes of the bovine blood-brain barrier

1997 ◽  
Vol 272 (5) ◽  
pp. C1552-C1557 ◽  
Author(s):  
W. J. Lee ◽  
D. R. Peterson ◽  
E. J. Sukowski ◽  
R. A. Hawkins
Keyword(s):  
Glucose Transport ◽  
Blood Brain Barrier ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Extracellular Fluid ◽  
Brain Barrier ◽  
Maximal Velocity ◽  
Plasma Membrane Vesicles ◽  
Cerebral Microvessels ◽  
Blood Brain

Luminal and abluminal endothelial plasma membrane vesicles were isolated from bovine cerebral microvessels, the site of the blood-brain barrier. Glucose transport across each membrane was measured using a rapid-filtration technique. Glucose transport into luminal vesicles occurred by a stereospecific energy-independent transporter [Michaelis-Menten constant (K(m)) = 10.3 +/- 2.8 (SE) mM and maximal velocity (Vmax) = 8.6 +/- 2.0 nmol.mg protein(-1).min-1]. Kinetic analysis of abluminal vesicles also showed a transport system with characteristics similar to the luminal transporter (K(m) = 12.5 +/- 2.3 mM and Vmax = 10.0 +/- 1.0 nmol.mg protein-1.min-1). These functional, facilitative glucose transporters were symmetrically distributed between the luminal and abluminal membrane domains, providing a mechanism for glucose movement between blood and brain. The studies also revealed a Na-dependent transporter on the abluminal membrane with a higher affinity and lower capacity than the facilitative transporters (K(m) = 130 +/- 20 microM and Vmax = 1.59 +/- 0.44 nmol.mg protein-1.min-1. The abluminal Na-dependent glucose transporter is in a position to transport glucose from the brain extracellular fluid into the endothelial cells of the blood-brain barrier. The functional significance of its presence there remains to be determined.

scholarly journals High-Level Expression of Plasmodium vivax Apical Membrane Antigen 1 (AMA-1) in Pichia pastoris: Strong Immunogenicity in Macaca mulatta Immunized with P. vivax AMA-1 and Adjuvant SBAS2

1999 ◽  
Vol 67 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Clemens H. M. Kocken ◽  
Martin A. Dubbeld ◽  
Annemarie Van Der Wel ◽  
Jack T. Pronk ◽  
Andrew P. Waters ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Plasmodium Vivax ◽  
Apical Membrane ◽  
Gel Filtration ◽  
Methylotrophic Yeast ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
High Level

ABSTRACT The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of thePlasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Δglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Δglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Δglyc43–487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant,Pichia-expressed PV66Δglyc43–487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

scholarly journals Analisis Natrium Benzoat pada Saos di Yogyakarta

2015 ◽  
Vol 2 (2) ◽  
pp. 69
Author(s):  
Siti Fatimah ◽  
Dian Wuri Astuti ◽  
Ni Putu Ayu Kurniasih
Keyword(s):  
Research Methods ◽  
Sodium Benzoate ◽  
Research Result ◽  
Maximum Level ◽  
Research Purpose ◽  
Slow Process ◽  
Quantitative Test ◽  
Acid Titration ◽  
Qualitative Test ◽  
High Level

Background: Preservative can block or slow process of fermentation, acidity and analyzes that cause by microbe. Sodium benzoate is one of organic preservative that easy to dissolve and usually additional with various of ingredients like sauce. Sauce is a fluid that can make food tasty. Sauce can be lasting if add by sodium benzoate. Sodium benzoate as preservative can be dangerous for healthy if out maximum level, and that interested research sodium benzoate. The research purpose to know about sodium benzoate and then determine content level existence on samples of sauce bottle on Beringharjo market in Yogyakarta. Research methods: The research describe sodium benzoate exiztence on sauce and than determine sodium benzoate level on saos. The research object sodium benzoate. Statistical variable in this research is one variable that is the existence and level of sodium benzoate on samples. Method that used for analyzed sodium benzoate in this case is base-acid titration in alkalimetry with two test that is qualitative test and quantitative test. This data is set out in table. Result: The research result by samples is 100% contain sodium benzoate and preservative with high level or out of maksimum level that has been certained and did not fulfill the terms of regulation BPOM Nomor 36 Tahun 2013 is 41,70%. Conclusion: there is sodium benzoate preservative on samples of sauce bottle on Beringharjo market in Yogyakarta with level maximum 7.001,61 mg/kg and level minimun 420,175 mg/kg

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