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F Blasco

Publications and source records attributed to F Blasco.

48 records · Page 3Linked to original sources
PubMed1990

Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.

The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the narZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

Amino Acid Sequence↗
PubMed1989

Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer.

The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed. It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively. The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide. In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide. This suggested that the latter is an electron transfer unit of the nitrate reductase complex. Such a view is opposite to the current description of the nitrate reductase. The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction. The NarG polypeptide was found to display a high degree of homology with numerous E. coli molybdoproteins. Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.

Amino Acid Sequence↗
PubMed1984

Identification of a plasma membrane protein involved in Pi transport in the yeast Candida tropicalis.

A plasma membrane protein was found to contain antigenic determinants in common with a Pi-binding protein released by osmotic shock from Candida tropicalis. This plasma membrane protein (M.P. 30) has a molecular mass of 30 kDa as measured by SDS-PAGE and anti-M.P. 30 antibodies inhibit Pi uptake in protoplasts while only the corresponding Fab fragments inhibit Pi transport in whole cells. This plasma membrane protein may be the Pi plasma membrane carrier.

Antibodies↗
PubMed1983

Antigenic relationships between plasma membrane ATPases of two different yeasts, Candida tropicalis and Schizosaccharomyces pombe.

Antibodies raised against purified plasma membrane ATPase from the yeast S. pombe inhibit the plasma membrane-bound ATPase of another yeast, C. tropicalis. The kinetic constants, Vm and Km, of the ATPases are both modified by the antibodies. These results show antigenic relationships between the plasma membrane ATPases belonging to the two yeast genera.

Adenosine Triphosphatases↗
PubMed1982

An investigation into the feasibility of using azide-insensitive ATPase and ConA as yeast plasma membrane markers.

Cytochemical localization of Concanavalin A binding sites in protoplasts of Candida tropicalis, investigated with glycosylated-ferritin and electron microscopy, showed that the lectin was specifically bound to the external protoplast surface. Thus, the plasma membranes have been labelled with 125I-Concanavalin A and followed through the isolation procedure. Relative distribution of 125I-radioactivity and azide-insensitive ATPase activity in the obtained fractions, suggested that this enzyme was an equivocal plasma membrane marker. Despite the presence of internal Concanavalin A binding sites, Concanavalin A could be used unambiguously as an exogenous plasma membrane marker of intact protoplasts.

Adenosine Triphosphatases↗
PubMed1982

The proton-translocating ATPase of Candida tropicalis plasma membrane.

Proton translocation activity of Candida tropicalis plasma membrane ATPase has been demonstrated using a fluorescent delta pH probe (ACMA) and by direct pH measurements. Modifications in fluorescence intensity and H+ transport are highly specific for Mg2+ and ATP, and are sensitive to the well-known inhibitors of the plasma membrane ATPase, vanadate and DCCD. A H+/ATP ratio of 0.54 is found.

Adenosine Triphosphatases↗
PubMed1981

Purification and characterization of the plasma membrane ATPase of Neurospora crassa.

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.

Adenosine Triphosphatases↗
PubMed1981

Characterization of the plasma membrane ATPase of Candida tropicalis.

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.

Adenosine Diphosphate↗
PubMed1977

Cotransport of phosphate and sodium by yeast.

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.

Adenosine Triphosphate↗
PubMed1976

[Demonstration of 2 phosphate transport systems in Candida tropicalis].

Candida tropicalis has two phosphate transport systems, one of which is constitutive and has a low affinity for its substrate (Kmapp 1.2.10(-3) M). The other one characterized by a high affinity for H2PO4- (Kmapp = 4.5.10(-6) M) appears only under phosphate starvation conditions. The regulation of the latter would act on the one hand on the synthesis of binding proteins for P1 (repression-derepression) and on the other on the activation or inactivation of the carriers.

Binding Sites↗