scholarly journals Stability of the cbb3-Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions

2008 ◽  
Vol 190 (16) ◽  
pp. 5576-5586 ◽  
Author(s):  
Annette Peters ◽  
Carmen Kulajta ◽  
Grzegorz Pawlik ◽  
Fevzi Daldal ◽  
Hans-Georg Koch
Keyword(s):  
Oxidase Activity ◽  
Rhodobacter Capsulatus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Conditions ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Membrane Bound ◽  
Binuclear Center ◽  
The Stability ◽  
Copper Oxidase

ABSTRACT Cytochrome cbb 3-type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme b-CuB binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound c-type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome cbb 3-type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of Rhodobacter capsulatus cbb 3-type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a Nout-Cin topology. In the absence of CcoQ, cbb 3-type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the cbb 3-type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in R. capsulatus CcoQ was required for optimal cbb 3-type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa cbb 3-type oxidase complex.

scholarly journals Chasing Particularities of Guanine- and Cytosine-Rich DNA Strands

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 434 ◽  
Author(s):  
Marko Trajkovski ◽  
Janez Plavec
Keyword(s):  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dna Recombination ◽  
Cd Spectroscopy ◽  
G Quadruplex ◽  
Recombination Processes ◽  
Dna Strands ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Myc Gene

By substitution of natural nucleotides by their abasic analogs (i.e., 1′,2′-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson–Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

scholarly journals The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes

1989 ◽  
Vol 262 (1) ◽  
pp. 233-240 ◽  
Author(s):  
D J B Hunter ◽  
K R Brown ◽  
G W Pettigrew
Keyword(s):  
Oxidase Activity ◽  
Sodium Iodide ◽  
Azotobacter Vinelandii ◽  
Pseudomonas Stutzeri ◽  
Selective Removal ◽  
Bacterial Respiration ◽  
Cellular Location ◽  
Diamine Oxidase Activity ◽  
Membrane Bound

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN‘N’-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.

scholarly journals FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

scholarly journals Membrane Sequestration of PII Proteins and Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

2007 ◽  
Vol 189 (16) ◽  
pp. 5850-5859 ◽  
Author(s):  
Pier-Luc Tremblay ◽  
Thomas Drepper ◽  
Bernd Masepohl ◽  
Patrick C. Hallenbeck
Keyword(s):  
Gel Electrophoresis ◽  
Membrane Fraction ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Photosynthetic Bacterium ◽  
Wild Type ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Pii Proteins ◽  
High Level

ABSTRACT Both Rhodobacter capsulatus PII homologs GlnB and GlnK were found to be necessary for the proper regulation of nitrogenase activity and modification in response to an ammonium shock. As previously reported for several other bacteria, ammonium addition triggered the AmtB-dependent association of GlnK with the R. capsulatus membrane. Native polyacrylamide gel electrophoresis analysis indicates that the modification/demodification of one PII homolog is aberrant in the absence of the other. In a glnK mutant, more GlnB was found to be membrane associated under these conditions. In a glnB mutant, GlnK fails to be significantly sequestered by AmtB, even though it appears to be fully deuridylylated. Additionally, the ammonium-induced enhanced sequestration by AmtB of the unmodifiable GlnK variant GlnK-Y51F follows the wild-type GlnK pattern with a high level in the cytoplasm without the addition of ammonium and an increased level in the membrane fraction after ammonium treatment. These results suggest that factors other than PII modification are driving its association with AmtB in the membrane in R. capsulatus.

scholarly journals C7 is expressed on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3640-3648 ◽  
Author(s):  
Fleur Bossi ◽  
Lucia Rizzi ◽  
Roberta Bulla ◽  
Alessandra Debeus ◽  
Claudio Tripodo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blot Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Complement Components ◽  
Albumin Leakage ◽  
Sds Page ◽  
Membrane Bound ◽  
Terminal Complement

Abstract We describe a novel localization of C7 as a membrane-bound molecule on endothelial cells (ECs). Data obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Northern blot analysis, and mass spectrometry revealed that membrane-associated C7 (mC7) was indistinguishable from soluble C7 and was associated with vimentin on the cell surface. mC7 interacted with the other late complement components to form membrane-bound TCC (mTCC). Unlike the soluble SC5b-9, mTCC failed to stimulate ECs to express adhesion molecules, to secrete IL-8, and to induce albumin leakage through a monolayer of ECs, and more importantly protected ECs from the proinflammatory effect of SC5b-9. Our data disclose the possibility of a novel role of mC7 that acts as a trap for the late complement components to control excessive inflammation induced by SC5b-9.

Possible Role of a Membrane Phosphopolypeptide in the Inhibition of Platelet Function by Cyclic Amp

1979 ◽  
Author(s):  
J.E.B. Fox ◽  
A. Smith ◽  
R.J. Haslam
Keyword(s):  
Cyclic Amp ◽  
Platelet Function ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Human Platelets ◽  
Differential Centrifugation ◽  
Phosphorylation Reaction ◽  
Membrane Bound

The inhibition of platelet function by PGE1, which is mediated by cyclic AMP, is associated with an increased phosphorylation of a 24,000 dalton platelet polypeptide (P24) (Haslam et al. (1979) Biochem J. 178, 397). The significance of this phosphorylation reaction has been investigated. Washed human platelets that had been labelled with 32P were I incubated with 2 μM PGE1 for 2min, sonicated and separated into subcellular fractions by differential centrifugation. Analysis of the phosphopolypeptides present in these fractions by SDS-polyacrylamide gel electrophoresis showed that P24 was enriched in a 19,000- 90,000 g fraction that contained both plasma and intracellular membranes and accumulated 45Ca2+ by an ATP-dependent, oxalate-stimulated process. Uptake of 45Ca2+ by membranes from PGE1-treated platelets was significantly greater (approx. 50%) than by membranes from control platelets. When the former membranes were loaded with 45Ca2+ in the presence of oxalate and centrifuged througll a discontinuous sucrose density gradient, thre membrane fractions were obtained. Enrichment of both 45Ca2+ and P24 was greatest in the most dense of these. Since platelet responses to aggregating agents are believed to be mediated by Ca2+ ions, we suggest that PGE1 may inhibit these processes by causing the cyclic AMP-dependent phosphorylation of the membrane-bound P24 which then stimulates the active transport of Ca2+ ions out of the platelet cytosol.

scholarly journals Enzymatic Removal of Nitric Oxide Catalyzed by Cytochrome c′ in Rhodobacter capsulatus

2001 ◽  
Vol 183 (10) ◽  
pp. 3050-3054 ◽  
Author(s):  
Richard Cross ◽  
David Lloyd ◽  
Robert K. Poole ◽  
James W. B. Moir
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Oxidase Activity ◽  
Rhodobacter Capsulatus ◽  
Resistance Mechanism ◽  
Growth Conditions ◽  
Anaerobic Conditions ◽  
No Removal ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

ABSTRACT Cytochrome c′ from Rhodobacter capsulatushas been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome c′ synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome c′ is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N2O), thus showing that cytochromec′ is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome c′ is distinct from the NO reductase pathway that is involved in denitrification. Cytochromec′ is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome c′ synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of R. capsulatus by NO suggests that one role for cytochromec′ is to maintain oxidase activity when both NO and O2 are present.

scholarly journals Electron transport in Paracoccus halodenitrificans and the role of Ubiquinone

1984 ◽  
Vol 30 (5) ◽  
pp. 572-577 ◽  
Author(s):  
Lawrence I. Hochstein ◽  
Sonja E. Cronin
Keyword(s):  
Enzyme Activity ◽  
Electron Transport ◽  
Ultraviolet Irradiation ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Succinate Oxidation ◽  
Partial Restoration ◽  
Membrane Bound ◽  
Nadh Oxidase Activity

The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm). When the membranes were extracted with n-pentane, NADH oxidase activity was lost. Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes. Succinate oxidation was not inhibited by dicoumarol or HQNO, but was affected by ultraviolet irradiation or n-pentane extraction. However, the addition of the ubiquinone fraction to the membranes extracted with n-pentane did not restore enzyme activity. These observations suggested that NADH and succinate were not oxidized through a common ubiquinone pool.

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