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)

To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.
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PMID:Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes. 288 19

A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.
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PMID:Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle. 289 70

The nucleotide sequence of the wheat mitochondrial gene for subunit 6 (atp6) of the F1F0 ATPase complex has been determined. Unlike bacterial, chloroplast or animal/fungal mitochondrial atp6 counterparts, which encode proteins of about 230-270 amino acids, the wheat mitochondrial atp6 homologue comprises the latter part of an open reading frame (ORF) of 386 codons. The ATP6 protein may therefore by synthesized with a long N-terminal presequence. This is supported by the finding that the ORF is preceded by a conserved sequence block closely related to ones preceding several other actively transcribed wheat mitochondrial protein-coding genes. The fused upstream ORF is similar in length, but unrelated in sequence, to those preceding the maize and tobacco mitochondrial atp6 genes. In wheat, the atp6 gene is located on a recombinationally active repeated DNA element, whose length of 1.4 kb corresponds approximately to that of the atp6 mRNA. A comparison of the wheat and maize ATP6 sequences reveals unexpectedly high divergence in the region corresponding to the mature N-terminal domain and may reflect mitochondrial DNA rearrangements during atp6 gene evolution in monocotyledonous plants.
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PMID:Sequence analysis of the wheat mitochondrial atp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins. 290 99

The activities of Na+,K+-, and Ca2+-ATPases were determined in plasma membranes obtained from livers of rats treated acutely and chronically with CCl4. Twenty-four hours after a single oral dose of CCl4 the ATPases decreased below 50% of control values. The activity of Ca2+-ATPase returned to normal after 4 days, and Na+,K+-ATPase activity returned to normal values after 12 days. One week after initiation of the chronic intraperitoneal treatment with CCl4, the Na,K+-ATPase decreased to 40% of control values and continued to decrease further until reaching values below 1%. Ca2+-ATPase followed a pattern similar to that obtained with Na+,K+-ATPase, except that the decrease was not as severe. Colchicine treatment prevented the modifications in ATPases when given simultaneously with CCl4 and reverted the alterations in ATPase activities of the CCl4-cirrhotic animals. Because ATPases are known to be modulated by the lipid composition of the membrane, we also determined the cholesterol to phospholipid ratio in all the isolated membranes. The ratios were increased in membranes with low ATPase activity due to an increase in the total concentration of cholesterol. Plasma membranes of cirrhotic rats treated with colchicine showed a low concentration of cholesterol, a decreased cholesterol to phospholipid ratio, and Na+,K+-ATPase activity was almost normal. When plasma membranes of cirrhotic rats were fused with phosphatidyl serine-containing liposomes, the cholesterol to phospholipid ratio decreased and the ATPase activity increased. The ATPase activity of normal plasma membranes decreased below 20% of control values when enriched with cholesterol. Our results suggest that the decrease in the plasma membrane Na+,K+-ATPase activity of the cirrhotic rat is due in part to an increase in its cholesterol concentration and in the cholesterol to phospholipid ratio.
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PMID:Cryptic adenosine triphosphatase activities in plasma membranes of CCl4-cirrhotic rats. Its modulation by changes in cholesterol/phospholipid ratios. 299 43

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.
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PMID:Calcium transport in membrane vesicles of Streptococcus cremoris. 301 12

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.
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PMID:Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. 303 60

bI4 maturase encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. It has been shown previously that a cytoplasmically translated hybrid protein composed of the pre-sequence of subunit 9 of Neurospora ATPase fused to a part of the bI4 maturase can be guided to mitochondria where it could compensate maturase deficiencies. This in vivo complementation of maturase mutants can be easily estimated by restoration of respiration. This work examines the efficiency of different bI4 maturase constructions to restore respiration in different yeast maturase-deficient strains. It is shown that the N-terminal end of the bI4 maturase plays a crucial role in the maturase activity. Moreover, the 12 N-terminal amino acids of the mitochondrial outer membrane protein constitute the most efficient mitochondrial targeting sequence in this system. Surprisingly enough, it was found that the cytoplasmically translated bI4 maturase containing the 254 C-terminal amino acid coded by the intron open reading frame can complement maturase mutations without any added mitochondrial-targeting sequence.
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PMID:Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase. 303 91

A series of recombinant plasmids containing increasing lengths of the 5'-flanking promoter sequences of the chicken conalbumin and ovalbumin genes fused to the sequences coding for the SV40 T-antigen have been constructed. These recombinants were introduced into a variety of established cell lines and primary cultured cells by nuclear microinjection. Promoter activity was estimated by monitoring T-antigen synthesis by indirect immunofluorescence. We show that the microinjected ovalbumin and conalbumin promoter regions do not function in chicken fibroblasts, kidney cells and in a variety of non-chicken cells, irrespective of the presence of steroid hormone receptors. In contrast, these promoter regions are active in primary cultured chicken embryonic hepatocytes and oviduct tubular gland cells, suggesting the presence of cell-specific transcription factors in these cells. Unexpectedly, promoter sequences close to the TATA boxes of both the ovalbumin and conalbumin genes are sufficient to confer cell-specific expression. Most of the controls exerted on the ovalbumin and conalbumin promoters in the whole animal appear to be reproduced in vitro by nuclear microinjection of the chimeric genes into the primary cultured cells. However, the microinjected ovalbumin promoter is active in embryonic hepatocytes and thus escapes the regulation imposed on the corresponding inactive endogenous gene.
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PMID:Cell-specificity of the chicken ovalbumin and conalbumin promoters. 366 76

Steroid hormone regulation of activity of the chicken ovalbumin promoter was studied by microinjection of chimeric genes into the nuclei of primary cultured oviduct tubular gland cells. The chimeric genes contained increasing lengths of ovalbumin gene 5'-flanking sequences fused to the sequence coding for the SV40 T-antigen. Promoter activity was estimated by monitoring synthesis of T-antigen. The activity of the ovalbumin promoter is cell-specifically repressed in these oviduct cells and the repression is relieved upon addition of steroid hormones. The -132 to -425 region of the ovalbumin promoter which is responsible for this negative regulation behaves as an independent functional unit containing the regulatory elements necessary for both repression (in the presence of steroid hormone antagonists) and induced derepression (in the presence of steroid hormones) of linked heterologous promoters.
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PMID:The chicken ovalbumin promoter is under negative control which is relieved by steroid hormones. 366 77

Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.
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PMID:Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7. 435 83


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