Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.
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PMID:S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1). 1213 86

Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.
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PMID:Localization of subunits D, E, and G in the yeast V-ATPase complex using cysteine-mediated cross-linking to subunit B. 1222 Jan 97

Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.
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PMID:Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain. 1460 47

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.
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PMID:Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain. 1595 35

The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and an integral V(0) domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V(0) domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.
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PMID:Structure and regulation of the V-ATPases. 1669 71

1. Secretory pathway Ca(2+) ATPase type 1 (SPCA1) is a newly recognized Ca(2+)/Mn(2+)-transporting pump localized in membranes of the Golgi apparatus. 2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues. 3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains. 4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells. 5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically. 6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, alpha-tubulin or MBP. 7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells. 8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.
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PMID:Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures. 1675 24

Adeno-associated virus (AAV) is a nonpathogenic single-stranded human parvovirus which usually requires the presence of a "helper" virus for strong DNA replication. In addition to adeno- and herpes viruses, human papillomavirus (HPV) can serve as an AAV helper. We recently published that HPV type 16 (HPV-16) E1 protein contributes significantly as an individual helper gene for AAV-2 DNA replication and transcription. As Rep78 and E1 are the corresponding DNA helicase/replication proteins of AAV and HPV, respectively, and Rep78 and E1 have a degree of homology, we assayed whether these two proteins interact physically. The full length proteins were purified from bacteria as GST-E1 and MBP-Rep78 and used in five assays to observe Rep78-E1 interactions. All five assays (pull-down, coimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), chemical cross-linking, and ATPase activity) provided evidence consistent with Rep78-E1 interaction. Most intriguing, an overall decrease in ATPase activity was observed when both proteins were present together. These data strongly suggest that E1 and Rep78 interact and that this interaction modulates at least some of their individual biochemical functions. This study adds to our understanding of AAV-HPV interaction biology, E1's modulation of Rep78 biochemistry, Rep78's modulation of E1 biochemistry and provides initial clues which may lead to the underlying mechanism of HPV E1 helper function for AAV DNA replication.
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PMID:AAV-2 Rep78 and HPV-16 E1 interact in vitro, modulating their ATPase activity. 1809 9

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.
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PMID:A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes. 1849 52

Isoforms of RUSH interact with a RING-finger binding protein (RFBP), which is a splice variant of the Type IV P-type ATPase, ATP11B. Splice arrays and RT-PCR showed that although most splice variants in RUSH and ATP11B are conserved in human and rabbit, the RFBP isoform is specific to rabbit. Interactions between the discontinuous PVITHC-HAKCPL sequence in the RING-domain of RUSH and the KVIRLIKIS sequence in the catalytic loop of RFBP were first identified with pull-down assays. Fine mapping involved probing CLIPS-constrained RING peptides with GST-tagged KVIRLIKIS. When the companion site in RFBP was fine mapped by replacement analysis with MBP-tagged RING, a four-fold increase in binding was noted for the KVIRLDKIS mutant. Direct comparison of splicing events in the RUSH and ATP11B genes between human and rabbit shows high structural stability in these protein interactions sites, which are 100% conserved in all mammalian orthologs.
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PMID:Conservation of inter-protein binding sites in RUSH and RFBP, an ATP11B isoform. 1858 49

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.
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PMID:Poliovirus 2C protein forms homo-oligomeric structures required for ATPase activity. 1952 Aug 52


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