Na+-K+-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution

1991 ◽  
Vol 261 (4) ◽  
pp. 87-91
Author(s):  
Mikhail P. Danilenko ◽  
Vera C. Turmukhambetova ◽  
Oleg V. Yesirev ◽  
Vsevolod A. Tkachuk ◽  
Mikhail P. Panchenko
Keyword(s):  
Atpase Activity ◽  
Pertussis Toxin ◽  
Gel Filtration ◽  
Protein S ◽  
Half Maximum ◽  
Inhibitory Effects ◽  
Cardiac Sarcolemma ◽  
Dependent Inhibition ◽  
Gi Protein ◽  
Zwitterionic Detergent

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 μM) decrease in the Na+-K+-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[-thio]triphosphate (0.1 μM GTPS) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 μM) and is abolished in sarcolemma treated with pertussis toxin (20 μg/ml) in the presence of 100 μM NAD. GTPS alone reduces Na+–K+-ATPaseactivity by 45% (half-maximum inhibitory = 1 μM). The apparent affinity of the enzyme for GTPS is increased 10-fold in the presence of 1 μM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTPS-dependent inhibition of the Na+-K+-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL–6B column resulted in a separation of Na+-K+-ATPase and pertussis toxin-sensitive Gi activities. Na+-K+-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20–30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTPS, or Gpp(CH2)p] at micromolar concentrations were restored when the Na+-K+-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5'-[β-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na+-K+-ATPase activity in the reconstitution system. These results suggest that a pertussis toxin-sensitive Gi protein may act as a transducer of the inhibitory hormonal signals on Na+-K+-ATPase in the sarcolemma. cardiac sarcolemma

Na+-K+-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution

1991 ◽  
Vol 261 (4) ◽  
pp. L87-L91
Author(s):  
Mikhail P. Danilenko ◽  
Vera C. Turmukhambetova ◽  
Oleg V. Yesirev ◽  
Vsevolod A. Tkachuk ◽  
Mikhail P. Panchenko
Keyword(s):  
Atpase Activity ◽  
Pertussis Toxin ◽  
Inhibitory Concentration ◽  
Gel Filtration ◽  
Protein S ◽  
Half Maximum ◽  
Inhibitory Effects ◽  
Cardiac Sarcolemma ◽  
Dependent Inhibition ◽  
Zwitterionic Detergent

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 μM) decrease in the Na+-K+-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[ggr-thio]triphosphate (0.1 μM GTPggrS) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 μM) and is abolished in sarcolemma treated with pertussis toxin (20 μg/ml) in the presence of 100 μM NAD. GTPggrS alone reduces Na+-K+-ATPase activity by 45% (half-maximum inhibitory = 1 μM). The apparent affinity of the enzyme for GTPgγS is increased ≈10-fold in the presence of 1 μM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTPgγS-dependent inhibition of the Na+-K+-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na+-K+-ATPase and pertussis toxin-sensitive Gi activities. Na+-K+-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20–30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTPggrS, or Gpp(CH2)p] at micromolar concentrations were restored when the Na+-K+-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5' -[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na+-K+-ATPase activity in the reconstitution system. These results suggest that a pertussis toxin-sensitive Gi protein may act as a transducer of the inhibitory hormonal signals on Na+-K+-ATPase in the sarcolemma. cardiac sarcolemma

scholarly journals Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

1992 ◽  
Vol 287 (2) ◽  
pp. 443-446 ◽  
Author(s):  
O A Coso ◽  
A Díaz Añel ◽  
H Martinetto ◽  
J P Muschietti ◽  
M Kazanietz ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Pertussis Toxin ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
Binding Activity ◽  
Gi Protein ◽  
Liver Membranes ◽  
Blocking Capacity ◽  
Polypeptide Band

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

scholarly journals Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways

2005 ◽  
Vol 34 (2) ◽  
pp. 459-472 ◽  
Author(s):  
M Zhang ◽  
Y Tao ◽  
B Zhou ◽  
H Xie ◽  
F Wang ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Oocyte Maturation ◽  
Protein S ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Inhibitory Effects ◽  
Cumulus Expansion ◽  
Gi Protein ◽  
Oocyte Meiotic Maturation ◽  
Pig Oocyte

Atrial natriuretic peptide (ANP) as well as its receptors is found in mammalian ovary and follicular cells and its function in oocyte meiotic maturation has also been reported in Xenopus, hamster and rat. But the results are controversial and the physiological mechanism of ANP on oocyte maturation is not clear, especially the relationship between gonadotrophin and ANP as well as the signal transduction, and these need further study. The present study conducted experiments to examine these questions by using drug treatment and Western blot analysis and focused on pig oocyte meiotic maturation and cumulus expansion in vitro. The results revealed that ANP could inhibited FSH-induced pig oocyte maturation and cumulus expansion and prevent the full phosphorylation of mitogen-activated protein kinase in both oocytes and cumulus cells, and that these inhibitory effects could be mimicked by 8-Br-cyclic guanosine 5′-monophosphate (8-Br-cGMP), but blocked by a protein kinase G (PKG) inhibitor KT5823. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, could enhance the inhibitory effect of ANP on oocyte maturation. A specific analogue of ANP, C-ANP-(4–23), which binds to the natriuretic peptide receptor-C (NPRC), had no effect in either FSH-induced or spontaneous oocyte maturation. Treatment with forskolin, a stimulator of adenylate cyclase, had a biphasic effect; 44 h treatment induced cumulus expansion but inhibited oocyte maturation while 2 h treatment induced maturation of cumulus-enclosed oocytes (CEOs). Both ANP and C-ANP-(4–23) could inhibit the effect of forskolin on CEO maturation, and these inhibitory effects of ANP/C-ANP-(4–23) could be blocked by preincubation with pertussis toxin (PT), consistent with mediation by a Gi protein(s) in the cumulus cells. All these results suggest that ANP is a multifunctional regulator of FSH and forskolin on pig CEO maturation by two signalling mechanisms: one is via a cGMP/PKG pathway, the other is via NPRC receptors in cumulus cells and the activation of the PT-sensitive Gi protein(s).

Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors

1979 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Amundsen ◽  
M. Larsbraaten ◽  
K. Lyngaas ◽  
E. Fareid
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Plasma Kallikrein ◽  
Peptide Substrate ◽  
Total Inhibition ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plus Fraction ◽  
Time Dependent Inhibition ◽  
Esterase Inhibitor

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.

scholarly journals Further properties and possible mechanism of action of adenosine 5′-triphosphate–d-glucose 6-phosphotransferase from rat liver

1967 ◽  
Vol 105 (2) ◽  
pp. 473-482 ◽  
Author(s):  
M. J. Parry ◽  
D G Walker
Keyword(s):  
Catalytic Properties ◽  
Gel Filtration ◽  
Thiol Group ◽  
Acceptor Molecule ◽  
Magnesium Ions ◽  
Purification Procedure ◽  
Inhibitory Effects ◽  
Bivalent Ions ◽  
Low Glucose ◽  
Yeast Hexokinase

1. Magnesium ions are the most effective bivalent ions in the glucokinase reaction. 2. The molecular weight of rat hepatic glucokinase is 48000–49000 as assessed by gel filtration on Sephadex G-100. 3. Anomalous kinetic behaviour at low glucose concentrations appears to be due to the formation during the purification procedure of fragments possessing modified catalytic properties, but is unlikely to be of physiological significance. 4. Extension of previous studies (Parry & Walker, 1966) suggests that glucokinase catalyses a reaction of the random Bi Bi type similar to that of yeast hexokinase. 5. The inhibitory effects of various thiol reagents suggest that a thiol group may be involved at or near the binding site of the acceptor molecule.

Zinc- and copper-binding proteins in the plasma of winter flounder (Pseudopleuronectes americanus)

1980 ◽  
Vol 58 (4) ◽  
pp. 609-613 ◽  
Author(s):  
P. E. Fletcher ◽  
G. L. Fletcher
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Protein S ◽  
Egg Yolk ◽  
Winter Flounder ◽  
Zinc Binding ◽  
Copper Binding ◽  
Copper Binding Proteins

Zinc- and copper-binding proteins were isolated from the plasma of winter flounder using gel filtration chromatography. A single copper-binding protein fraction of molecular weight 170 000 was isolated from the plasma of both sexes.In male and female flounder over 95% of the plasma zinc was associated with a zinc-binding protein(s) with a molecular weight of 76 000. In male flounder the remaining zinc appeared to be bound to a protein(s) of molecular weight 186 000. In female flounder the remaining 5% of the zinc was associated with two zinc-binding fractions with apparent molecular weights of 186 000 and 340 000 – 370 000.Extracts of plasma vitellogenin and egg yolk proteins revealed significant quantities of zinc and copper. It is hypothesized that the female specific zinc-binding protein (340 000 – 370 000) was vitellogenin.

scholarly journals Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2,I2 and F2alpha on hormone-induced cAMP accumulation in cultured hepatocytes

1988 ◽  
Vol 172 (2) ◽  
pp. 293-297 ◽  
Author(s):  
Oyvind MELIEN ◽  
Randi WINSNES ◽  
Magne REFSNES ◽  
Ivar P. GLADHAUG ◽  
Thoralf CHRISTOFFERSEN
Keyword(s):  
Pertussis Toxin ◽  
Camp Accumulation ◽  
Cultured Hepatocytes ◽  
Inhibitory Effects

scholarly journals Chemical aspect of the influence of cobalt ions on atpase activity

2000 ◽  
Vol 65 (7) ◽  
pp. 507-515 ◽  
Author(s):  
Ljubica Vujisic ◽  
Danijela Krstic ◽  
Jovan Vucetic
Keyword(s):  
Experimental Data ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Synaptic Plasma Membrane ◽  
Cobalt Ions ◽  
Dependent Inhibition ◽  
Ic50 Values ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

The influence of Co 2+ ions on the activities of Na+/K+-ATPase and Mg2+ -ATPase, enzymes from rat brain synaptic plasma membrane, was studied. The aim of this study was to investigate the inhibition of both ATPases activities byexposure tocobalt ions as a function of experimentally added CoSO4. The "free" Co2+ concentrations in the reaction mixturewere also calculated and discussed. CoSO4 induced a dose-dependent inhibition of both enzymes. The IC50 values of Co 2+, as calculated from the experimental curves, were 168 mM for Na+/K+-ATPase and 262 mMfor Mg 2+-ATPase, and for the recalculated free Co 2+ concentration 75.4 mM for Na+/K+-ATPase and 136 mM for Mg 2+-ATPase. The obtained linear Dixon's plot for Na+/K+-ATPase implies equilibium binding of cobalt with inhibitory sites on the enzyme. The kinetic parameters for both enzymes in presence and absence of CoSO4 were calculated from the experimental data. The results of the kinetic analysis show that inhibition of Na+/K+-ATPase induced by CoSO4 is non-competitive, and for Mg 2+-ATPase that there are two sites of different sensitivities or two different enzymes.

scholarly journals Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

2020 ◽  
Author(s):  
Jialei Sun
Keyword(s):  
Rna Polymerase ◽  
Polymerase Activity ◽  
Inhibitory Effects ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Dependent Inhibition ◽  
Catalytic Metal ◽  
Dose Dependent Inhibition ◽  
Dose Dependent ◽  
Low Activity

AbstractThe outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+ or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+ promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.

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