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N Nelson

Publications and source records attributed to N Nelson.

At least 145 records · Page 8Linked to original sources
PubMed1992

The vacuolar H(+)-ATPase--one of the most fundamental ion pumps in nature.

An electrochemical gradient of protons (PMF) is a universal high-energy intermediate in biological systems. Two related families of proton pumps, denoted F- and V-ATPases, are among the principal generators of a PMF from ATP and can form ATP at the expense of a PMF. The enzymes of these two families share a similar structure and subunit composition; some subunits in the two families evolved from common ancestors. Other subunits having no common ancestry were added independently to the various enzymes and defined the two separate families. The general mechanism for the proton pumping activity is similar in the two families. However, whereas F-ATPases can act in both proton pumping and ATP formation, the V-ATPases of eukaryotes function exclusively as ATP-dependent proton pumps. The catalytic and membrane sectors of F-ATPases and archaebacterial V-ATPases can separately catalyze their specific partial activities of ATPase and proton conduction. The catalytic and membrane sectors of the eukaryotic V-ATPases cannot act separately. This property is correlated with the presence of a large proteolipid that traverses the membrane four times. The gene duplication of the smaller proteolipid in the formation of the large proteolipid was one of the most important events in the evolution of the V-ATPases of eukaryotic cells.

Adenosine Triphosphatases↗
PubMed1992

Mutations in the yeast vacuolar ATPase result in the mislocalization of vacuolar proteins.

The vacuolar ATPase of the yeast Saccharomyces cerevisiae acidifies the vacuolar lumen and generates an electrochemical gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of genes (delta vat) encoding the A (69 x 10(3) M(r)), B (57 x 10(3) M(r)) or c (16 x 10(3) M(r)) subunits of the vacuolar ATPase accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. A kinetic analysis suggests that these precursor proteins accumulate in, and are secreted from, the Golgi complex or post-Golgi vesicles. In addition, subcellular fractionation shows that vacuolar hydrolase-invertase hybrid proteins are inefficiently localized to the vacuole in delta vat strains. This result suggests that the vat mutations cause a steady-state defect in vacuolar protein sorting. The vat mutations also affect the sorting of vacuolar membrane proteins. Precursor forms of alkaline phosphatase are accumulated in vat mutant cells, but to a lesser extent than is seen for the soluble vacuolar hydrolases. This finding, coupled with the insensitivity of alkaline phosphatase to the ATPase inhibitor bafilomycin A1, suggests that vacuolar membrane protein sorting is less sensitive to changes in lumenal pH when compared with the targeting of soluble vacuolar proteins. These results indicate that acidification of the vacuolar system is important for efficient sorting of soluble proteins to the vacuole.

Adenosine Triphosphatases↗
PubMed1992

The membrane sector of vacuolar H(+)-ATPase by itself is impermeable to protons.

The sensitivity for vanadate of proton uptake activities of isolated chromaffin granules and yeast vacuoles was investigated. About 0.5 mM vanadate caused 50% inhibition of ATP-dependent proton uptake activity in both membranes. The proton conductivity across chromaffin granule membranes and yeast vacuoles was assayed in the presence and absence of the catalytic sectors of their respective V-ATPases. Removal of the catalytic sectors by cold treatment in the presence of MgATP did not change the proton conductivity of the membranes. Similar results were obtained with yeast vacuoles of mutants that did not assemble the catalytic sector of the enzyme. These results are in contrast to the effect of removing the catalytic sectors of F-ATPases from various sources. The mechanistic and biological significance of the difference in the behavior of the two families of proton pumps is discussed.

Animals↗
PubMed1991

Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is one of the two multisubunit pigment-protein complexes in the thylakoid membranes of cyanobacteria. Subunit III of photosystem I complex was isolated from a mutant of the cyanonbacterium Synechocystis sp PCC 6803, which lacks subunit II. The sequence of its NH2-terminal residues was determined and corresponding oligonucleotide probes were used to isolate the gene encoding this subunit. The gene, designated as psaF, codes for a mature protein of 15705 Da that is synthesized with a 23-amino acid extension. The deduced amino acid sequence is homologous to subunit III from spinach and Chlamydomonas reinhardtii. The presequence of subunit III shows characteristics typical of bacterial presequences and exhibits remarkable amino acid identity around the proteolytic processing site when compared to corresponding regions from the precursors of eukaryotic subunit III. There are two conserved hydrophobic regions in the mature subunit III which may cross or interact with thylakoid membrane. The gene psaF exists as a single copy in the genome and is expressed as a monocistronic RNA. A stable mutant strain in which the gene psaF was replaced by a gene conferring resistance to kanamycin was generated by targeted mutagenesis. Photoautotrophic growth of the mutant strain was comparable with that of the wild type suggesting that function of subunit III is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Addition of more MgSO4 to BG11 medium enhanced growth of the mutant strain but not of the wild type cells.

Amino Acid Sequence↗
PubMed1991

ADP/ATP translocator is essential only for anaerobic growth of yeast Saccharomyces cerevisiae.

All three genes (AAC1, AAC2 and AAC3) encoding the mitochondrial ADP/ATP translocator, were inactivated in a haploid yeast strain by a gene disruption technique. The triple mutant was still able to grow on fermentable carbon sources but only in the presence of oxygen. Under aerobic conditions neither translocator-protein nor carrier-mediated transport was detected in all mutants in which the AAC2 gene was disrupted. It was further shown that a functional AAC genes product is essential only for anaerobic growth of Saccharomyces cerevisiae but not for growth under derepressed conditions. Under anaerobic conditions a non-detectable amount of AAC3 gene product is sufficient to ensure the cell growth and multiplication.

Adenosine Diphosphate↗
PubMed1991

Mutational analysis of yeast vacuolar H(+)-ATPase.

Yeast mutants in which genes encoding subunits of the vacuolar H(+)-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-[14C]dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of the genes encoding two chaperone proteins of the cyanobacterium Synechocystis sp. PCC 6803.

Molecular chaperones help other proteins in their correct folding and assembly. We have cloned the genes, cpn60 and dnaK, which encode proteins belonging to the chaperonin-60 and the 70-kDa heat shock protein families from the transformable cyanobacterium Synechocystis sp. PCC 6803. These genes are present in single copies in the genome, and the major transcripts for each gene are monocistronic. Comparison of deduced amino acid sequences reveals that cyanobacterial chaperonin-60 is equally homologous to bacterial and plant chaperonin-60 proteins while the product of dnaK is more similar to its bacterial homologues than to its eukaryotic counterparts. The DNA fragments sequenced in these studies also contain five other open reading frames. One of them, ORF60-5, encodes a protein whose deduced amino acid sequence shows remarkable similarity to those of a family of peripheral membrane proteins involved in metabolite transport in bacteria. The transcript levels of dnaK and cpn60 of Synechocystis sp. PCC 6803 increase in response to stress conditions such as heat shock, ultraviolet exposure, and oxidative stress. This is one of the first examples of cyanobacterial gene expression being regulated by environmental stresses.

Amino Acid Sequence↗
PubMed1991

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803.

The two operons atp1 and atp2, encoding the subunits of the F0F1 ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F0F1 ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5' end of atp1, coding for a putative gene product similar to uncI in Escherichia coli. A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.

Amino Acid Sequence↗
PubMed1991

Vanadate-sensitive ATPase from chromaffin granule membranes formed a phosphoenzyme intermediate and was activated by phosphatidylserine.

Vanadate-sensitive ATPase (115 kDa molecular weight) in adrenal chromaffin granules is an intrinsic membrane enzyme with its catalytic site located at the outer surface of the granules. Upon incubation with [gamma-32P]ATP, the purified ATPase formed an alkaline-labile phosphoenzyme intermediate, which was inhibited by vanadate but not by Na+ or K+. Ratio of ATPase or phosphatase activity and formation of phosphoenzyme intermediate was constant during purification after the first glycerol density gradient centrifugation. Phosphatidylserine specifically activated the enzyme about three-fold by increasing the Vmax value without changing the Km for ATP. Other phospholipids, including phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, as well as lysophospholipids and detergents, had no effect. These results indicated that the vanadate-sensitive ATPase belongs to the P-type ATPases, which differ from known cation-translocating P-type ATPases.

Adenosine Triphosphatases↗
PubMed1991

Structure and pharmacology of the proton-ATPases.

There are three main classes of proton-ATPases named P-, V- and F-ATPases. Their function is to synthesize ATP from a universal high-energy intermediate (protonmotive force), or to generate the intermediate by hydrolysing ATP. Every cell contains at least one kind of these enzymes and the protonmotive force is used for numerous secondary reactions, including uptake and storage of neurotransmitters in synaptic vesicles. The ubiquitous distribution of proton-ATPases and their central role in energy conversion makes them indispensable components of cell metabolism. One of the functions of V-ATPases is to acidify the interior of organelles inside the cell. As Nathan Nelson discusses here, this property is being used for designing and targeting of drugs.

Adenosine Triphosphatases↗
PubMed1990

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules.

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a rat brain GABA transporter.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

Amino Acid Sequence↗
PubMed1990

Cloning of the human brain GABA transporter.

A cDNA clone encoding a transporter for the neurotransmitter gamma-aminobutyric acid in human brain was cloned and sequenced. The cDNA contains an open reading frame encoding a hydrophobic protein of 599 amino acids with a calculated molecular weight of 67022 Da. Hydropathy analysis revealed twelve potential transmembrane segments. The human protein is highly homologous to the protein from rat brain. Northern hybridization demonstrated a ubiquitous distribution of the transporter in various parts of the brain.

Amino Acid Sequence↗
PubMed1990

A third ADP/ATP translocator gene in yeast.

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.

Amino Acid Sequence↗
PubMed1990

A meta-analysis of stages I and II Hodgkin's disease.

To compare radiotherapy alone to chemotherapy plus radiotherapy in the treatment of early stage Hodgkin's disease, the English language medical literature was searched for reports on randomized clinical trials in Stages I and II Hodgkin's disease from 1975 through 1986. Twenty-three reports with 2999 patients were entered into matched study analysis. Data on extended-field radiotherapy (EF), involved-field (IF), chemotherapy alone, combination chemotherapy and radiotherapy (CM), disease stage, laparotomy staging, and complications were gathered. A proportional hazard rate was used to estimate and compare relapse-free (RFS) and overall survival rates (S). Iteratively reweighted least square analysis was used to estimate survival curves. Twelve-year RFS for CM (889 patients) was significantly superior to EF (1350 patients) (P less than 0.01). Twenty-year RFS in EF was better than IF (760 patients) (P less than 0.01). Twelve-year S for CM was not significantly different than for EF but was better than for IF (P less than 0.05).

Combined Modality Therapy↗
PubMed1990

Response of mouse tissues to neutron and gamma radiation: protection by WR-3689 and WR-77913.

The response of mouse intestine to d(22 MeV) + Be neutrons, p(50.5 MeV) + Be neutrons, or 137Cs gamma-rays was examined using intestinal crypt-cell survival as an endpoint. The RBEs for 50.5 MeV neutrons relative to 137Cs-gamma-rays of response levels of 5 and 50 surviving cells/circumference were 1.80 and 1.86. For d(22 MeV) + Be neutrons, the RBEs at comparable cell survivals were 1.86 and 1.93. For LD50/7 day (gastrointestinal syndrome), the RBE for 50.5 MeV neutrons was 1.56 and for LD50/30 (bone marrow syndrome), the RBE was 1.29. The ability of the phosphorothioates WR-3689 [S-2-(3-methylamino-propylamino) ethylphosphorothioic acid, 450 mg/kg] and WR-77913 [S-2-(3-amino-2-hydroxypropyl) phosphorothioic acid, 800 mg/kg] to protect against p(50.5 MeV) + Be neutrons and gamma-rays also was examined in mice using lethality at seven or 30 days as the endpoints. Results were compared to previous studies with d(22 MeV) + Be neutrons WR-2721 (400 mg/kg), an appropriate comparison given the similar RBEs of the two neutron sources relative to gamma-rays. WR-77913 is a less effective protector than is WR-2721 while in some cases WR-3689 is as effective as WR-2721. The neutron DMFs for LD50/7 were 1.12 for WR-3689 and 0.99 for WR-77913. For the bone marrow syndrome (LD50/30) the neutron DMFs were 1.47 for WR-3689 and 1.21 for WR-77913. For a given type of radiation, WR-3689 is more effective than WR-77913. Both phosphorothioates protect less well against neutron radiation than against photons, as was reported previously for WR-2721.(ABSTRACT TRUNCATED AT 250 WORDS)

Amifostine↗
PubMed1990

Disruption of genes encoding subunits of yeast vacuolar H(+)-ATPase causes conditional lethality.

The main function of vacuolar H(+)-ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membranes of the vacuolar system. The enzyme is composed of a catalytic sector with five subunits (A-E) and a membrane sector containing at least two subunits (a and c). We disrupted two genes of this enzyme, in yeast cells, one encoding a subunit of the membrane sector (subunit c) and another encoding a subunit of the catalytic sector (subunit B). The resulting mutants did not grow in medium with a pH value higher than 6.5 and grew well only within a narrow pH range around 5.5. Transformation of the mutants with plasmids containing the corresponding genes repaired the mutations. Thus failure to lower the pH in the vacuolar system of yeast, and probably other eukaryotic cells, is lethal and the mutants may survive only if a low external pH allows for this acidification by fluid-phase endocytosis.

Cloning, Molecular↗