Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.
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PMID:Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. 881 3

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [gamma-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the delta'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.
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PMID:Two subunits of the F0F1-ATPase are phosphorylated in the inner mitochondrial membrane. 950 Sep 82

Apical and basal membrane fractions from Locusta Malpighian tubules were prepared and were characterized by marker enzyme analysis. The apical membranes contained an azide- and orthovanadate-insensitive ATPase activity that was inhibited by bafilomycin A1 (IC50 = 0.44 nM) and NEM (IC50 = 2.15 microM), and thus was characterized as putative V-type ATPase. The enzyme was stimulated by a variety of monovalent cations (Tris > K = Na > choline > Li = Rb) maximal stimulation occurring at 30-40 mM. It was also stimulated by a variety of monovalent anions (maximal activation 30-40 mM), but was strongly inhibited by nitrate and thiocyanate. SDS-PAGE separation of proteins present in the various membrane fractions was carried out. The apical membrane fraction alone contained a 28 kDa protein band that bound a monoclonal antibody specific for a 28 kDa peptide which was a component of the V-type ATPase from midgut of Manduca sexta and, in native gels, possessed ATPase activity which was also sensitive to both bafilomycin and NEM but not to azide or orthovanadate. Binding of the fluorescent monoclonal antibody was located at the apical boundary of the tubule cells. It was concluded that a V-type ATPase is present at the apical surface of Locusta Malpighian tubule cells and that it is involved in their secretory functioning.
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PMID:Characterization of ATPases of apical membrane fractions from Locusta migratoria Malpighian tubules. 968 29

Soluble proteins were isolated from leaves of the common ice plant Mesembryanthemum crystallinum L. in the CAM state of photosynthesis and tested for protease activity using amino acid-beta-naphthylamide (NA)-derivatives in a search for proteolytic activity responsible for cleavage of the V-ATPase subunit B. This cleavage is suggested to occur at the peptide bond between Met192 and Glu193. At neutral pH Met-NA was one of seven derivatives which were cleaved by proteases present in this fraction. Enzymes exhibiting proteolytic activity were separated from other soluble proteins by Superose 12-size exclusion FPLC. Incubation of partially purified protease with tonoplast-enriched membrane vesicle fractions isolated from M. crystallinum in the C3-state of photosynthesis led to a decrease in subunit B (55 kDa) protein amount and to the formation of the polypeptide Di (32 kDa), which has been previously suggested to represent a fragment of subunit B. Cleavage of subunit B and the appearance of Di also occurred during incubation of tonoplast vesicles in the presence of reactive oxygen species. In addition to Di, the polypeptide Ei (28 kDa) appeared after incubation with protease and/or reactive oxygen species. Taken into account that Di and Ei cross-reacted with an affinity purified antiserum directed against subunit B, Di as well as Ei might represent fragments of subunit B. These results open new perspectives with respect to the regulation of V-ATPase modification and turnover.
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PMID:Processing of V-ATPase subunit B of Mesembryanthemum crystallinum L. is mediated in vitro by a protease and/or reactive oxygen species. 1098 65

The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.
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PMID:The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids. 1102 54

Membranes prepared from various members of the genus Halobacterium contained a Triton X-100 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength (< 3 M NaCl) and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90% of total protein. The 60-kDa subunit reacted with dicyclohexylcarbodiimide (DCCD) when inhibition was carried out in an acidic medium. The significance of the two minor components (28 kDa and 12 kDa is not established. The enzyme from H. saccharovorum, which differs from previously described halobacterial ATPases, possesses properties of an F1F0 as well as an E1E2 ATPase.
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PMID:Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum. 1154 91

The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.
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PMID:Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca(2+)-ATPase: probing nucleotide binding by multidimensional NMR. 1180 14

The 14-3-3 protein family is a family of regulatory proteins involved in diverse cellular processes. In a previous study of regulation of individual 14-3-3 isoforms in the germinating barley embryo, we found that a post-translationally modified, 28 kDa form of 14-3-3A was present in specific cell fractions of the germinated embryo. In the present study, we identify the nature of the modification of 14-3-3A, and show that the 28 kDa doublet is the result of cleavage of the C-terminus. The 28 kDa forms of 14-3-3A lack ten or twelve amino acid residues at the non-conserved C-terminus of the protein, respectively. Barley 14-3-3B and 14-3-3C are not modified in a similar way. Like the 30 kDa form, in vitro produced 28 kDa 14-3-3A is still capable of binding AHA2 H+-ATPase in an overlay assay. Our results show a novel isoform-specific post-translational modification of 14-3-3 proteins that is regulated in a tissue-specific and developmental way.
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PMID:Post-translational modification of barley 14-3-3A is isoform-specific and involves removal of the hypervariable C-terminus. 1236 28

Cellular differentiation and programmed cell death are tightly controlled to maintain tissue homeostasis and proper organ function. In a screen for apoptosis specific gene products, we isolated an immediate early apoptosis response gene from myelomonocytic stem cells that appears to play a key regulatory role in a number of cell types and may be of particular importance in cells of the central nervous system. The gene's 28 kDa protein product interacts with the C-terminal ectodomain of the Na+/K+-ATPase (NKA) beta 1 subunit and was therefore named NKIP (NKA Interacting Protein). NKIP is coexpressed with NKA, localizes to lysosomes and the endoplasmic reticulum and is predominantly expressed in excitable tissues including polarized epithelia and the central nervous system. NKIP has been characterized as an endogenous suppressor of the NKA as reduction of NKIP in PC12 cells significantly increases NKA activity. In pluripotent NT2 progenitor cells, NKIP induced rapidly K+-level-dependent cell death. NKIP overexpression induced growth factor-independent neurite outgrowth, which was associated with MEK-independent phosphorylation of the transcription factor ERK1/2. Thus, we have identified NKIP as an important novel protein that interacts to the NKA complex, influencing cellular ion balance, induction of apoptosis and neuronal differentiation.
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PMID:Characterization of NKIP: a novel, Na+/K+-ATPase interacting protein mediates neural differentiation and apoptosis. 1809 56

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.
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PMID:Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion. 2207 17


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