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)

Site-specific mutagenesis was used to investigate the functional roles of amino acids in the relatively hydrophobic sequence Ile-Thr-Thr-Cys-Leu-Ala-320, located at the M4S4 boundary of the sarcomplasmic reticulum Ca(2+)-ATPase. Each of the residues was replaced with either a less hydrophogic, a polar, or a charged residue. Mutants Ile-315----Arg and Leu-319----Arg were devoid of any Ca2+ transport function or ATPase activity, while the mutant Thr-317----Asp retained about 5 and 7% of the wild-type Ca2+ transport and ATPase activities, respectively. These three mutants were able to form the ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP, but this intermediate decayed very slowly to the ADP-insensitive phosphoenzyme intermediate (E2P). In the mutants Ile-315----Arg and Leu-319----Arg, the level of E2P formed in the backward reaction with inorganic phosphate was extremely low, but hydrolysis of E2P occurred at a normal rate. These mutants, in addition, displayed a higher apparent affinity for Ca2+ than the wild-type enzyme. In the mutants Ile-315----Ser and Ile-315----Asp, the Ca2+ transport and ATPase activities were moderately reduced to 30-40% of the wild-type activities, but normal affinities for Ca2+, Pi, and ATP were retained, as was the low affinity modulatory effect of ATP. Mutation of Thr-316 to Asp, Thr-317 to Ala, Cys-318 to Ala and Ala-320 to Arg had little or no effect on Ca2+ transport or ATPase activities. Introduction of two negative and one positive charge by triple mutation of the Ile-Thr-Thr-317 sequence created a mutant enzyme that, although completely inactive, was inserted into the membrane, consistent with a location of these residues on the cytoplasmic side of the M4S4 interface. Our findings suggest that the amphipathic character of the S4 helix and/or the distribution of charges in S4 is important for the stability of the E2P intermediate.
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PMID:Functional consequences of alterations to hydrophobic amino acids located at the M4S4 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum. 183

We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea star Pisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA(glu) and tRNA(thr) are 3' to 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.
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PMID:Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea star Pisaster ochraceus. 197 16

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.
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PMID:Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8). 213 47

The synthetic heptapeptide, Ile-Arg-Ile-Cys-Arg-Lsy-Gly-ethoxy, an analog of one of the actin binding sites on myosin head (S-site) (Suzuki, R., Nishi, N., Tokura, S., and Morita, F. (1987) J. Biol. Chem. 262, 11410-11412) was found to completely inhibit the acto-S-1 (myosin subfragment 1) ATPase activity. The effect of the heptapeptide on the binding ability of S-1 for F-actin was determined by an ultracentrifugal separation. Results indicated that the heptapeptide scarcely dissociated the acto-S-1 complex during the ATPase reaction. Consistent results were obtained from the acto-S-1 ATPase activities determined as a function of S-1 concentrations in the absence or presence of the heptapeptide at a fixed F-actin concentration. The heptapeptide reduced the maximum acto-S-1 ATPase activity without affecting the apparent dissociation constant of the acto-S-1 complex. The heptapeptide bound by a site on actin complementary to the S-site probably inhibits the activation of S-1 ATPase by F-actin. These results suggest that S-1 ATPase is necessary to rebind transiently with F-actin at the S-site in order to be activated by F-actin. This is consistent with the activation mechanism proposed assuming the two actin-binding sites on S-1 ATPase (Katoh, T., and Morita F. (1984) J. Biochem. (Tokyo) 96, 1223-1230).
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PMID:Inhibition of actomyosin subfragment 1 ATPase activity by analog peptides of the actin-binding site around the Cys(SH1) of myosin heavy chain. 213 23

New antibiotic pumilacidins A, B, C, D, E, F and G were isolated from the culture broth of a strain of Bacillus pumilus. They are cyclic acylheptapeptide composed of a beta-hydroxy fatty acid, two L-leucine, two D-leucine, L-glutamic acid, L-aspartic acid and L-isoleucine (or L-valine). Pumilacidin components were inhibitory to herpes simplex virus type 1 and H+, K(+)-ATPase and demonstrated antiulcer activity in rat.
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PMID:Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. 215 95

The cryopreservation of hepatocytes is of particular interest as a step in the possible treatment of some inborn disorders of metabolism. This study examines the metabolic damage that occurs as a result of the freeze-thaw procedures and during subsequent incubation periods of isolated rat hepatocytes. Even for freshly prepared hepatocytes, the presence of 1.8 M of Me2SO during incubation led to a rapid decline in viability. Optimal recovery after cryopreservation was obtained when incubation was started after the progressive removal of Me2SO. A buffer medium characterized by an intracellular electrolyte composition (Euro-Collins) proved particularly beneficial to the membrane integrity, probably by protecting the (Na+,K+)ATPase pump activity. The interpretation of viability using the trypan blue exclusion test was generally confirmed by the metabolic analysis of protein synthesizing activity and membrane transport function which are regarded as more rigorous tests of functional viability. The incorporation of L-[U-14C]isoleucine into the proteins of fresh hepatocytes during the first hour of incubation progressively leveled off over the next 2 hr. The cryopreserved hepatocytes showed a similar pattern although at a lower level of activity. Even after 3 hr of preincubation, the subsequent addition of labeled isoleucine still indicated a residual protein synthesizing activity. The active transport of alpha-amino[1-14C]isobutyric acid through the cell membranes reached a peak value after 60 min of incubation of fresh hepatocytes, and after 40 min of incubation of cryopreserved cells, followed by a steep decline as expression of rapid membrane deterioration. Again, the membrane transport pattern for the cryopreserved samples occurred at a lower level of activity. After preincubation of fresh and cryopreserved hepatocytes for 180 min, subsequent addition of labeled alpha-aminoisobutyric acid did not show any further significant metabolic activity. Initially the amino acid availability appeared to control protein synthesizing activity while, as membrane transport became seriously damaged, incorporation leveled off with only a low metabolic activity remaining. Although cryopreserved hepatocytes were susceptible to faster deterioration during subsequent incubation, considerable metabolic activity was retained. However, fresh and cryopreserved hepatocytes expressed metabolic functions at significantly different activities. Moreover, the differences between fresh and cryopreserved cells varied with the particular cellular function being examined.
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PMID:The effects of cryopreservation on membrane integrity, membrane transport, and protein synthesis in rat hepatocytes. 215 75

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the alpha chain, a largely intact beta chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the alpha chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect--mediated, presumably, by conformational changes of the protein.
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PMID:A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations. 216 48

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.
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PMID:Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue. 216 43

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.
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PMID:Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis. 218 28

A site-directed mutation in the gene which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of Ala-25 by Tyr, has been constructed and characterized. A plasmid carrying the mutation was used to transform strain AN943 (uncE429). The resulting strain is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield. Membranes prepared from the mutant possess low levels of ATPase activity and are proton-impermeable. The F1-ATPase activity was found to be inhibited by 80% when bound to the membrane. When carried on a plasmid, the mutation is dominant in complementation tests with all mutant unc alleles tested and when transformed into wild-type strain AN346, the mutation results in an uncoupled phenotype. A mutant which overcomes this dominance was isolated and found to possess an 11-amino-acid deletion extending from Ile-55 to Met-65 within the c-subunit. These results are discussed in relation to the previously isolated Ala-25 to Thr mutant (Fimmel, A.L., Jans, D.A., Hatch, L., James, L.B., Gibson, F. and Cox, G.B. (1985) Biochim. Biophys. Acta 808, 252-258) and in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).
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PMID:The F1F0-ATPase of Escherichia coli. The substitution of alanine by tyrosine at position 25 in the c-subunit affects function but not assembly. 252 60


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