On the Current-Voltage Relationships of Energy-Transducing Membranes: Phosphorylating Membrane Vesicles from Paracoccus denitrificans

1978 ◽  
Vol 6 (6) ◽  
pp. 1292-1295 ◽  
Author(s):  
DOUGLAS B. KELL ◽  
PHILIP JOHN ◽  
STUART J. FERGUSON
Keyword(s):  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Current Voltage

Competition between electron acceptors in membrane vesicles of Paracoccus denitrificans

1981 ◽  
Vol 9 (4) ◽  
pp. 324-326 ◽  
Author(s):  
ROBERT JONES ◽  
JULIAN O. D. COLEMAN ◽  
F. R. WHATLEY
Keyword(s):  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Electron Acceptors

Respiration-driven accumulation of C4 dicarboxylic acids by isolated membrane vesicles of Paracoccus denitrificans

1979 ◽  
Vol 57 (5) ◽  
pp. 436-443 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Sole Carbon Source ◽  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Dicarboxylic Acids ◽  
Energy Coupling ◽  
Carbon Limitation ◽  
Dependent Manner ◽  
Dicarboxylate Transport ◽  
Osmotic Lysis ◽  
Isolated Membrane Vesicles

Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.

scholarly journals Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

Open Biology ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 170206 ◽  
Author(s):  
Febin Varghese ◽  
James N. Blaza ◽  
Andrew J. Y. Jones ◽  
Owen D. Jarman ◽  
Judy Hirst
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Inhibitor Protein ◽  
Wild Type ◽  
Efficient Energy ◽  
Inverted Membrane Vesicles ◽  
Atp Synthases

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans

1980 ◽  
Vol 58 (10) ◽  
pp. 996-1003 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Intact Cell ◽  
Respiratory Control ◽  
Membrane Vesicles ◽  
Bee Venom ◽  
Respiratory Enzymes ◽  
Protein Ratio ◽  
Membrane Topography ◽  
Considerable Controversy

There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria. It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans. The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD: ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of non-permeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control. Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.

scholarly journals The reversibility of active sulphate transport in membrane vesicles of Paracoccus denitrificans

1975 ◽  
Vol 150 (3) ◽  
pp. 527-536 ◽  
Author(s):  
J N Burnell ◽  
P John ◽  
F R Whatley
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Paracoccus Denitrificans ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Thiol Group ◽  
Whole Cells ◽  
Sulphate Transport ◽  
Proton Symport ◽  
Inside Out

An uncoupler-sensitive active transport of sulphate into membrane vesicles prepared from the plasma membrane of Paracoccus denitrificans (previously Micrococcus denitrificans) can be driven by respiration or by a trans-membrane pH gradient (alkaline inside) generated by the addition either of KCL (in the presence of nigericin) or of NH4CL. Valinomycin does not substitute for nigericin. Respiration-driven transport is observed in right-side-out vesicles but not in inside-out vesicles, whereas transport driven by the addition of KCL (in the presence of nigericin) or of NH4CL is observed in both types of membrane vesicle. The active transport of sulphate into these vesicles is shown to be carrier-mediated by its sensitivity to thiol-group reagents. It is proposed that the sulphate carrier in the plasma membrane of P. denitrificans operates by a mechanism of electroneutral proton symport, and is capable of actively transporting sulphate in either direction across the plasma membrane, but that in whole cells respiration-driven proton expulsion drives the accumulative uptake of sulphate.

scholarly journals Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Jianshu Hu ◽  
Elisa Venturi ◽  
Charalampos Sigalas ◽  
Takashi Murayama ◽  
Miyuki Nishi ◽  
...  
Keyword(s):  
Single Channel ◽  
Ryanodine Receptors ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Hek293 Cells ◽  
Cation Channels ◽  
Gating Current ◽  
Nuclear Membranes ◽  
Current Voltage ◽  
Reversal Potentials

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of −13.67 ± 9.02 (n = 7), −2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl−. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

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