scholarly journals Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0238754
Author(s):  
Melissa Martinez ◽  
Gregory A. Fendley ◽  
Alexandra D. Saxberg ◽  
Maria E. Zoghbi
Keyword(s):  
Oxidative Damage ◽  
Atpase Activity ◽  
Animal Studies ◽  
Atp Hydrolysis ◽  
Aminolevulinic Acid ◽  
Negative Control ◽  
Hydrolysis Rate ◽  
Inner Mitochondrial Membrane ◽  
Experimental Conditions ◽  
Mitochondrial Transporter

Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin’s effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

scholarly journals Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin

2020 ◽  
Author(s):  
Melissa Martinez ◽  
Gregory A. Fendley ◽  
Alexandra D. Saxberg ◽  
Maria E. Zoghbi
Keyword(s):  
Oxidative Damage ◽  
Atpase Activity ◽  
Animal Studies ◽  
Atp Hydrolysis ◽  
Aminolevulinic Acid ◽  
Negative Control ◽  
Hydrolysis Rate ◽  
Inner Mitochondrial Membrane ◽  
Experimental Conditions ◽  
Mitochondrial Transporter

AbstractHeme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, thus intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin’s effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

Tropomyosin-Troponin-Induced Changes in the Partitioning of Free Energy Release of Actomyosin-Catalyzed ATP Hydrolysis as Measured by ATP-Phosphate Exchange

1980 ◽  
Vol 35 (5-6) ◽  
pp. 431-438 ◽  
Author(s):  
Peter Dancker
Keyword(s):  
Free Energy ◽  
Atpase Activity ◽  
Energy Release ◽  
Atp Hydrolysis ◽  
Free Energy Change ◽  
Energy Change ◽  
Experimental Conditions ◽  
Mean Values ◽  
Induced Changes ◽  
Phosphate Exchange

Abstract ATPase activity and ATP-Pi exchange of unregulated (without tropomyosin-troponin) and regulated (with tropomyosin-troponin) acto-HMM were measured in media containing 0.2 mg/ml actin, HMM, and (when present) tropomyosin-troponin, 2 mM MgCl2, 10 m M KCl, 2 mM NaN3, 10 mM Pi(pH 7.0), 3 mM ATP. The following mean values for ATPase activity and for the rate of incorporation of P, into ATP (each per mg HMM and per min) were obtained: unregulated acto-HMM 0.33 nmol Pi and 0.33 nmol Pi, regulated acto-HMM 0.54 nmol Pi and 1.06 nmol P*. The ratio of P4 incorporation rate to ATPase activity was 1.01 × 10-3 for unregulated and 2.02 × 10-3 for regulated acto-HMM. From these ratios and from the overall free energy change of ATP hydrolysis it was calculated that under the prevailing experimental conditions in unregulated acto-HMM 62% and in regulated acto-HMM 66% of the free energy change of ATP hydrolysis occurs after the release of phosphate from actomyosin. It is probably this part of the free energy change that is used by the muscle for the performance of work.

scholarly journals Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity

2005 ◽  
Vol 391 (3) ◽  
pp. 481-490 ◽  
Author(s):  
Odile Ramaen ◽  
Christina Sizun ◽  
Olivier Pamlard ◽  
Eric Jacquet ◽  
Jean-Yves Lallemand
Keyword(s):  
Complex Formation ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Activation Mechanism ◽  
Hydrolysis Rate ◽  
Sequential Assignment ◽  
Atp Binding ◽  
Native Page ◽  
Transient Complex

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642–Ser871) and NBD2 (Ser1286–Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10−5 s−1) and NBD2 (6×10−6 s−1) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 μM. Despite the fact that both monomers bind ATP, no stable NBD1·NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1·NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1·NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1·NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

Oxidative damage to ferritin by 5-aminolevulinic acid

2003 ◽  
Vol 409 (2) ◽  
pp. 349-356 ◽  
Author(s):  
Maria E.M Rocha ◽  
Fernando Dutra ◽  
Brian Bandy ◽  
Regina L Baldini ◽  
Suely L Gomes ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Aminolevulinic Acid

Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

1992 ◽  
Vol 2 (2) ◽  
pp. 105-111 ◽  
Author(s):  
S. Sánchez-Nieto ◽  
R. Rodríguez-Sotres ◽  
P. González-Romo ◽  
I. Bernal-Lugo ◽  
M. Gavilanes-Ruíz
Keyword(s):  
Zea Mays ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Atpase Activity ◽  
Kinetic Analysis ◽  
Atp Hydrolysis ◽  
Membrane Atpases ◽  
Substrate Concentrations ◽  
Tonoplast Atpase ◽  
Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

scholarly journals The Middle Domain of Hsp90 Acts as a Discriminator between Different Types of Client Proteins

2006 ◽  
Vol 26 (22) ◽  
pp. 8385-8395 ◽  
Author(s):  
Patricija Hawle ◽  
Martin Siepmann ◽  
Anja Harst ◽  
Marco Siderius ◽  
H. Peter Reusch ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Fold Increase ◽  
Cellular Protein ◽  
Hydrolysis Rate ◽  
Cellular Activity ◽  
Protein Levels ◽  
Middle Domain ◽  
Client Proteins

ABSTRACT The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.

scholarly journals Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

2000 ◽  
Vol 352 (1) ◽  
pp. 165-173 ◽  
Author(s):  
Sang Yeul HAN ◽  
Dong Yoon PARK ◽  
Sang Dai PARK ◽  
Seung Hwan HONG
Keyword(s):  
Fusion Protein ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Polyclonal Antibodies ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Brain Extract ◽  
Terminal Domain ◽  
Protein Receptor ◽  
And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Hydrogen-rich water-alleviated ultraviolet-B-triggered oxidative damage is partially associated with the manipulation of the metabolism of (iso)flavonoids and antioxidant defence in Medicago sativa

2015 ◽  
Vol 42 (12) ◽  
pp. 1141 ◽  
Author(s):  
Yanjie Xie ◽  
Wei Zhang ◽  
Xingliang Duan ◽  
Chen Dai ◽  
Yihua Zhang ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Medicago Sativa ◽  
H2 Production ◽  
Hydrogen Gas ◽  
Ultraviolet B ◽  
In Vitro Tests ◽  
Antioxidant Defence ◽  
Experimental Conditions ◽  
Uvb Irradiation

External administration of hydrogen gas (H2) benefits plants from multiple environmental stimuli. However, the physiological significance and molecular mechanism of H2 in ultraviolet-B (UVB) irradiation are largely unexplored. Here, the biological function of H2 in the regulation of plant UVB-tolerance was investigated by using hydrogen-rich water (HRW). Results showed that the exposure of alfalfa seedlings to UVB irradiation increased endogenous H2 production. Pretreatment with HRW mimicked the UVB-induced endogenous H2 production. Corresponding UVB-triggered toxic symptoms, in terms of lipid peroxidation and overproduction of reactive oxygen species (ROS), as well as the subsequent growth inhibition, were markedly mitigated. Metabolic profiling analysis by using ultra performance liquid chromatography-mass spectrometric (UPLC-MS), identified 40 (iso)flavonoids in UVB-treated alfalfa plants, with 22 kinds was increased by HRW. These changes resulted in the alternation of (iso)flavonoids profile, with the effective promotion of isoflavone and flavanone subfamilies in particular. These compounds included afromosin, afromosin 7-O-β-D-glucoside-malonate, daidzein, formononetin 7-O-β-D-glucoside-6ʹʹ-O-malonate, garbanzol, matteucin and naringenin. In vitro tests further showed that the HRW-modulated (iso)flavonoids profile upon UVB stress possessed advanced ROS-quenching and antioxidant capacities under our experimental conditions. Meanwhile, UVB-triggered upregulation in the transcription levels of (iso)flavonoids biosynthetic-related genes were substantially strengthened by HRW. The activities and related transcripts of representative antioxidant enzymes were also induced. Taken together, our findings indicate that HRW confers tolerance to UVB-induced oxidative damage partially by the manipulation of (iso)flavonoids metabolism and antioxidant defence in Medicago sativa L.

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