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

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

Adaptation to low-K+ media increases H(+)-K(+)-ATPase but not H(+)-ATPase-mediated pHi recovery in OMCD1 cells

1997 ◽  
Vol 273 (2) ◽  
pp. C558-C571 ◽  
Author(s):  
J. Guntupalli ◽  
M. Onuigbo ◽  
S. Wall ◽  
R. J. Alpern ◽  
T. D. DuBose
Keyword(s):  
Atpase Activity ◽  
Inhibitory Concentration ◽  
Collecting Duct ◽  
Low Ph ◽  
Bafilomycin A1 ◽  
Inhibitory Effects ◽  
Medullary Collecting Duct ◽  
Low K ◽  
Phi Regulation ◽  
In Cells

Studies in rat and rabbit outer medullary collecting duct of inner stripe origin (OMCDis) suggest that both H(+)-ATPase and H(+)-K(+)-ATPase participate in H+ secretion. However, the relative contributions of these transporters, and, in particular, that of H(+)-K(+)-ATPase to K+ absorption have not been defined precisely. The present study was designed to delineate more clearly the response of these two transporters to hypokalemia and acidosis in a newly developed mouse OMCD1 cell line. In cells grown in normal K+ (5 mM) media, intracellular pH (pHi) recovery was similar either in the presence or absence of K+ in the perfusate (delta pHi/min = 0.014 +/- 0.001 vs. 0.017 +/- 0.003, not significant). The inhibitory effects of Sch-28080 (10 microM) and bafilomycin A1 (10 nM) on pHi recovery were evident only in the presence and absence of K+ in the perfusate, respectively. In cells grown in low-K+ (2.5 mM) media to simulate chronic hypokalemia, pHi recovery was significantly faster than in cells grown in normal K+ media (delta pHi/min = 0.045 +/- 0.01 vs. 0.014 +/- 0.001, P < 0.01) and was inhibited specifically by Sch-28080, not by bafilomycin A1. In contrast, in cells preconditioned to low pH (7.0) to simulate chronic acidosis, the enhanced pHi recovery was abolished by bafilomycin A1 but not by Sch-28080. 86Rb+ uptake, when used as a K+ congener, was inhibited by Sch-28080. The K(m) for 86Rb+ uptake (H(+)-K(+)-ATPase activity) and the 50% inhibitory concentration for Sch-28080 were 270 and 5.0 microM, respectively. These studies provide evidence that, in morphologically homogeneous OMCD1 cells, 1) both H(+)-K(+)-ATPase and H(+)-ATPase participate in pHi regulation, 2) the H(+)-K(+)-ATPase is selectively upregulated by preconditioning in low-K+ media, and 3) conversely, preconditioning in low-pH media stimulates only the H(+)-ATPase. Thus, in OMCDis, the H(+)-K(+)-ATPase and H(+)-ATPase respond selectively and independently to chronic hypokalemia and acidosis, respectively.

scholarly journals A New Saponin with Anti-HIV-1 Protease Activity from Acacia pennata

2018 ◽  
Vol 13 (4) ◽  
pp. 1934578X1801300
Author(s):  
Van-Dung Nguyen ◽  
Hong-Loan Thi Nguyen ◽  
Linh-Chi Do ◽  
Vu Van Tuan ◽  
Phuong Thien Thuong ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Inhibitory Concentration ◽  
Ethanol Extract ◽  
Half Maximum ◽  
Human Embryonic Kidney ◽  
Inhibitory Effects ◽  
Immunodeficiency Virus ◽  
Anti Hiv ◽  
Hiv 1

We investigated the phytochemicals from an ethanol extract of Acacia pennata (L.) Willd stems, a Vietnam medicinal plant, which led to the isolation of a new saponin termed 21β- O-[(2 E)-6-hydroxyl-2,6-dimethyl-2,7-octadienoyl] pitheduloside G (1), as well as pitheduloside G (2), a known saponin. The structures of compounds 1 and 2 were elucidated via spectroscopy and compared with those reported in the literature. The isolates (1 and 2) were tested for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) protease (PR). We found that the new compound, 21β- O-[(2 E)-6-hydroxyl-2,6-dimethyl-2,7-octadienoyl] pitheduloside G (1), possessed potent anti-HIV-1 PR activity, with a half-maximum inhibitory concentration (IC50) value of 2.0 ± 0.2 μM. In contrast, pitheduloside G (2) exhibited much less inhibition, with an IC50 value of 18 ± 0.5 μM. Both compounds were nontoxic in human embryonic kidney 293T cells at concentrations effective against HIV-1 PR. This is the first report regarding the isolation of an anti-HIV-1 PR compound (1) from an Acacia plant species.

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 In Vitro α-Glucosidase and α-Amylase Inhibitory Activities of Free and Bound Phenolic Extracts from the Bran and Kernel Fractions of Five Sorghum Grain Genotypes

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1301
Author(s):  
Yun Xiong ◽  
Ken Ng ◽  
Pangzhen Zhang ◽  
Robyn Dorothy Warner ◽  
Shuibao Shen ◽  
...  
Keyword(s):  
Digestive System ◽  
Inhibitory Concentration ◽  
Inhibitory Effect ◽  
Inhibitory Effects ◽  
Phenolic Contents ◽  
Inhibitory Activities ◽  
Sorghum Grain ◽  
Global Health Challenge ◽  
Phenolic Extracts

Diabetes is a global health challenge. Currently, an effective treatment for diabetes is to reduce the postprandial hyperglycaemia by inhibiting the carbohydrate hydrolysing enzymes in the digestive system. In this study, we investigated the in vitro α-glucosidase and α-amylase inhibitory effects of free and bound phenolic extracts, from the bran and kernel fractions of five sorghum grain genotypes. The results showed that the inhibitory effect of sorghum phenolic extracts depended on the phenolic concentration and composition. Sorghum with higher phenolic contents generally had higher inhibitory activity. Among the tested extracts, the brown sorghum (IS131C)-bran-free extract (BR-bran-free, half-maximal inhibitory concentration (IC50) = 18 ± 11 mg sorghum/mL) showed the strongest inhibition against α-glucosidase which was comparable to that of acarbose (IC50 = 1.39 ± 0.23 mg acarbose/mL). The red sorghum (Mr-Buster)-kernel-bound extract (RM-kernel-bound, IC50 = 160 ± 12 mg sorghum/mL) was the most potent in inhibiting α-amylase but was much weaker compared to acarbose (IC50 = 0.50 ± 0.03 mg acarbose/mL).

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 Two Novel Quassinoid Glycosides with Antiviral Activity from the Samara of Ailanthus altissima

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5679
Author(s):  
Qing-Wei Tan ◽  
Jian-Cheng Ni ◽  
Jian-Ting Shi ◽  
Jian-Xuan Zhu ◽  
Qi-Jian Chen
Keyword(s):  
Data Analysis ◽  
Herbal Medicine ◽  
Inhibitory Concentration ◽  
Biological Activities ◽  
Virus Multiplication ◽  
Ailanthus Altissima ◽  
Inhibitory Effects ◽  
Traditional Herbal Medicine ◽  
Virus Activity ◽  
Ic50 Values

Phytochemistry investigations on Ailanthus altissima (Mill.) Swingle, a Simaroubaceae plant that is recognized as a traditional herbal medicine, have afforded various natural products, among which C20 quassinoid is the most attractive for their significant and diverse pharmacological and biological activities. Our continuous study has led to the isolation of two novel quassinoid glycosides, named chuglycosides J and K, together with fourteen known lignans from the samara of A. altissima. The new structures were elucidated based on comprehensive spectra data analysis. All of the compounds were evaluated for their anti-tobacco mosaic virus activity, among which chuglycosides J and K exhibited inhibitory effects against the virus multiplication with half maximal inhibitory concentration (IC50) values of 56.21 ± 1.86 and 137.74 ± 3.57 μM, respectively.

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 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.

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