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)

We have characterized the subunit composition of the chloroplast ATP synthase from Chlamydomonas reinhardtii by means of a comparison of the polypeptide deficiencies in a mutant defective in photophosphorylation, with the polypeptide content in purified coupling factor (CF)1 and CF1.CF0 complexes. We could distinguish nine subunits in the enzyme, four of which were CF0 subunits. Further characterization of these subunits was undertaken by immunoblotting experiments, [14C]dicyclohexylcarbodiimide binding and analysis of their site of translation. In particular, we were able to show the presence of an as yet unidentified delta subunit in CF1 from C. reinhardtii. We have identified a 70-kDa peripheral membrane protein in the thylakoid membranes of C. reinhardtii, which is immunologically related to the beta subunit of CF1. We discuss its conceivable ATPase function with respect to the Ca2+-dependent ATPase activity previously reported in the thylakoid membranes from C. reinhardtii.
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PMID:The chloroplast ATP synthase in Chlamydomonas reinhardtii. I. Characterization of its nine constitutive subunits. 252 91

Bispecific antisera were prepared to a mixture of thylakoid membrane polypeptides 4.1 and 4.2. The identity of these polypeptides as the alpha and beta subunits of coupling factor (CF1) was established based on the cross-reactivity of the antisera toward CF1 from peas and by an analysis of the thm-24 mutant of Chlamydomonas which lacks the CF1 ATPase. Photochemical labeling of thylakoid membranes with hydrophobic and hydrophilic fluorescent probes indicated that these polypeptides did not significantly penetrate the membrane bilayer. Immunoprecipitation of the translation products of thylakoid-bound and soluble ribosomes showed the thylakoids to be the major site of synthesis of the polypeptides. Immunoprecipitation of the products of translation of total cellular RNA in a reticulocyte lysate showed no evidence for substantially higher molecular weight precursors. Further analysis of the thylakoid-bound synthesis of alpha and beta revealed that some of the in vitro synthesized polypeptides had been incorporated into the CF0-CF1 complex based on their release from membranes with trypsin and copurification with the CF0-CF1 ATPase.
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PMID:In vitro synthesis and assembly of the peripheral subunits of coupling factor CF1 (alpha and beta) by thylakoid-bound ribosomes. 285 55

The chloroplast (cp)-encoded CF1 ATPase beta-subunit gene (atpB) of Chlamydomonas reinhardtii and its flanking regions have been sequenced. The derived amino acid (aa) sequence is highly homologous to that of the beta-subunit gene in Escherichia coli, bovine heart mitochondria, and higher plant cp. In contrast to all other cp genomes, the CF1 epsilon subunit gene (atpE) does not lie at the 3' end of the atpB gene but maps to a position 92 kb away in the other single-copy region. Northern blots confirm that the beta subunit is not encoded as part of a dicistronic message as it is in higher plants. The region just upstream from the atpB gene in C. reinhardtii contains two small open reading frames (ORFs) and not the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as is found in cp genomes of higher plants. No transcripts for either ORF were detected, but the codon usage in these ORFs as well as in the atpB gene follows the unique pattern of codon usage previously seen in other cp genes in C. reinhardtii.
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PMID:The sequence of the chloroplast atpB gene and its flanking regions in Chlamydomonas reinhardtii. 287 28

A cDNA library from Chlamydomonas reinhardtii, constructed in the phage expression vector lambda gt11, was probed with antiserum directed against the nuclear-encoded gamma subunit of the chloroplast H+-transporting ATP synthase [ATP phosphohydrolase (H+-transporting) or chloroplast coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A cDNA was isolated and transcribed in vitro. The transcript was translated in vitro and immunoprecipitated with anti-gamma-subunit serum to yield a product that coelectrophoresed with the immunoprecipitated product from in vitro-translated polyadenylylated RNA. These proteins were larger than the mature gamma subunit, either immunoprecipitated as chloroplast coupling factor 1 or as the individual subunit. Thus, the gamma subunit is synthesized as a precursor of greater molecular weight in C. reinhardtii. Furthermore, the precursor protein encoded by the cDNA is imported into pea chloroplasts and processed to a lower molecular weight polypeptide that coelectrophoreses with mature C. reinhardtii gamma subunit. The largest cDNA isolated is about the same length as the corresponding mRNA (approximately equal to 1900 bases long) and probably contains the entire coding region. Southern blot analyses revealed restriction fragment length polymorphisms and that the gamma subunit is probably encoded by an intron-containing single-copy gene.
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PMID:Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: import and cleavage of the precursor protein. 289 28

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, label is incorporated primarily into the alpha, beta, and Mr = 78,000 chains; a much smaller amount is incorporated into the Mr = 69,000 chain. The light chains are not labeled. The incorporation is UV-dependent, ATP-sensitive, and blocked by preincubation of the enzyme with vanadate plus low concentrations of ATP or ADP. These results suggest that the alpha heavy chain contains the site of ATP binding and hydrolysis in the alpha subunit. In the beta subunit, the beta heavy chain and one or both intermediate chains may contain ATP-binding sites.
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PMID:Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP. 293 35

When outer-row dynein arms are extracted from Chlamydomonas flagellar axonemes, they dissociate into two ATPase complexes with sedimentation coefficients of 12S and 18S. We immunized mice with 18S dynein and generated a library of monoclonal antibodies against the polypeptides in this complex. Antibodies were selected which specifically recognize the 18S alpha- and beta-heavy chains and the 83,000-dalton and 70,000-dalton intermediate chains. These antibodies were isolated and characterized for their ability to recognize determinants on both denatured antigens and native 18S dynein; 18S dynein was dissociated in stepwise fashion into smaller aggregates with ionic and nonionic detergents and the resulting subcomplexes were isolated by precipitation with specific monoclonal antibodies. The smallest aggregates isolated were heterodimers between the alpha-chain and a 16,000-dalton light chain and between the two intermediate chains. Additional close associations of the beta-heavy chain with an 18,000-dalton light chain and 70,000-dalton intermediate chain, and a weaker interaction between the intermediate chain heterodimer and light chains of 21,000 daltons and 12,500 daltons, were also observed. We present a model of 18S dynein substructure based upon this information.
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PMID:Protein-protein interactions in the 18S ATPase of Chlamydomonas outer dynein arms. 294 99

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.
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PMID:High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits. 295 7

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.
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PMID:MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. 295 82

This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme.
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PMID:Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes. 296 9

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg2+ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.
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PMID:Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii. 300 55


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