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)

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.
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PMID:Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. 217 44

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.
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PMID:Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. 283 17

Vanadate is known to inhibit several phosphatases including Na+, K+-ATPase, alkaline phosphatase, and glyceraldehyde-3-P dehydrogenase. Inhibition presumably results because vanadium adopts a stable structure which resembles the transition state of phosphate during the reactions involving these enzymes. We performed experiments to further examine the effects of vanadate (VO3-4) on erythrocyte (red blood cells (RBC] glycolytic intermediates. RBC obtained from human subjects were centrifuged and washed with lactated Ringer's 5% dextrose. 31P nuclear magnetic resonance analysis of the RBC revealed the characteristic peaks for the 3-phosphate and 2-phosphate of 2,3-diphosphoglycerate (DPG), inorganic phosphate (Pi), and ATP. Incubation of RBC with 10(-6) M VO3-4 led to a disappearance of ATP and 2,3-DPG while the peak for Pi increased. By the end of 4 h over 90% of the VO3-4 had been reduced to VO2+ (vanadyl) in the RBC. The effects of 10(-4) M iodoacetamide and 10(-5) M ethacrynic acid, known inhibitors of glyceraldehyde-3-P dehydrogenase that act by interactions with sulfhydryl groups (-SH) of the enzyme, were similar to those of VO3-4. Incubation with vanadyl did not affect the peaks for Pi, 2-DPG, or 3-DPG. Furthermore, using electron spin resonance we demonstrated that in the presence of glyceraldehyde-3-P dehydrogenase, VO3-4 is reduced to VO2+. The findings demonstrate that VO3-4 inhibits glycolysis at micromolar concentrations and that the ion is reduced to VO2+ in the cell. The similarity of the effect of VO3-4 to those of iodoacetamide and ethacrynic acid suggests that interactions with -SH groups is its mechanism of inhibition. Since under physiological conditions intracellular VO3-4 concentrations are in the micromolar range and may exist in oxidized and/or reduced forms, VO3-4 could regulate the activity of glyceraldehyde-3-P dehydrogenase through changes in the redox state of the enzyme rather than by substituting for the PO3-4 ion.
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PMID:Mechanism of inhibition of glycolysis by vanadate. 303 65

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++-ATPase, Na+,K+-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogenase, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.
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PMID:Analysis of human erythrocyte membrane vesicles produced by shearing. 644 34

Most members of the guanosine triphosphatase (GTPase) superfamily hydrolyze guanosine triphosphate (GTP) quite slowly unless stimulated by a GTPase activating protein or GAP. The alpha subunits (G alpha) of the heterotrimeric G proteins hydrolyze GTP much more rapidly and contain an approximately 120-residue insert not found in other GTPases. Interactions between a G alpha insert domain and a G alpha GTP-binding core domain, both expressed as recombinant proteins, show that the insert acts biochemically as a GAP. The results suggest a general mechanism for GAP-dependent hydrolysis of GTP by other GTPases.
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PMID:Separate GTP binding and GTPase activating domains of a G alpha subunit. 826 82

Na,K-ATPase is an integral membrane protein complex involved in the regulation of intracellular Na+ and K+ ion levels. The number of accessible Na,K-ATPase complexes on the surface of human platelets before and after activation by adenosine 5'-diphosphate (ADP) was quantitated using ouabain, a highly specific inhibitor of this enzyme. Studies with [3H]ouabain revealed an increase in the number of accessible ouabain binding sites from approximately 55 per resting platelet to approximately 625 per ADP-activated platelet. Ultrastructural localization of this ion transport complex was also studied in resting human platelets or following activation by ADP. Localization was performed using a novel colloidal gold affinity probe which employed ouabain (ouabain-GAP) as a ligand specific for the Na,K-ATPase. Qualitatively, the number of surface-associated ouabain-GAP labels per platelet was observed to increase following activation by ADP. Fracture-labeling studies of frozen preparations of resting and freshly activated platelets suggested ouabain binding sites were associated with intracellular platelet structures during the early stages of activation. Together these results suggest an increase in the amount of ouabain binding sites at the surface of human platelets as an event of activation and that at least some of these additional sites may originate from intracellular sites.
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PMID:Appearance of additional ouabain binding sites on platelet surfaces following activation. 839 97

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.
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PMID:Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins. 865 79

The aim of this study was to estimate the anticataract action of vitamin E using an in vitro methylprednisolone (MP)-induced cataract model. The same severity of early cortical cataract was induced in lenses isolated from male Wistar rats aged 6 weeks by incubation with MP (1.5 mg/ml) in TC-199 medium. The cataractous lenses showed slight increases in lipid peroxide (LPO) content and Na+/K+ ratio and slight decreases in reduced glutathione (GSH) content and glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), a sensitive index of oxidative stress, and Na+,K(+)-ATPase activities. When the cataractous lenses were further incubated in TC-199 medium with and without vitamin E (250 micrograms/ml) for 48 h, the progression of cataract was prevented in the vitamin E-treated lenses, but not in the vitamin E-untreated lenses. The vitamin E-untreated lenses showed a decrease in vitamin E content and an increase in water content in addition to further increases in LPO content and Na+/K+ ratio and further decreases in GSH content and GAP-DH and Na+,K(+)-ATPase activities. In contrast, the changes of these components and enzymes except for GSH were attenuated in the vitamin E-treated lenses. From these results, it can be estimated that vitamin E prevents in vitro cataractogenesis in rat lenses treated with MP by protecting the lenses against oxidative damage and loss of membrane function.
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PMID:Anticataract action of vitamin E: its estimation using an in vitro steroid cataract model. 888 85

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.
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PMID:The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. 951 63

We conducted a mutation analysis of the most conserved region of the neurofibromatosis type 1 (NF1) gene, the guanine triphosphatase (GTPase) activating protein (GAP)-related domain (NF1 GRD), to which the function of tumour suppressor is attributed. Sixty primary neuroectodermal tumours were analysed. The rationale for the study was based on the likelihood of finding structural alterations resulting in loss of function of this region in tumours of neuroepithelial tissues, where the activity of neurofibromin seems to be crucial in regulating the mechanisms of signal transduction and cell transformation mediated by p21 ras. Following analysis of the whole NF1 GRD sequence, no mutations were identified in the tumours analysed. We conclude that the loss of NF1 gene tumour suppressor function, that might lead or contribute to the development of malignancies in neuroectodermal tissues, is not due to structural abnormalities of the region of the gene which interacts with p21 ras.
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PMID:The guanine triphosphatase (GTPase) activating protein (GAP)-related domain of the neurofibromatosis type 1 gene is not mutated in neural crest-derived sporadic tumours. 971 12


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