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E Echeverria

Publications and source records attributed to E Echeverria.

10 recordsLinked to original sources
PubMed1998

Citrate uptake into tonoplast vesicles from acid lime (Citrus aurantifolia) juice cells.

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.

Adenosine Triphosphate↗
PubMed1998

Fru 2,6-P2 and citrate: intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.

Grapefruit leaf PFP was studied for its activation by fructose 2,6-bisphosphate (Fru 2,6-P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0 +/- 0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 nM) with a Ka(citrate) of 4.5 +/- 0.09, 6.9 +/- 0.05 and 38.2 +/- 1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6-P2, but non-linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6-P2 was further investigated in citrus leaves and juice cells. Fru 2,6-P2 was only found in the cytosol of juice cells. Fru 2,6-P2 was detected under both conditions with higher concentrations found under aerobiosis.

Citric Acid↗
PubMed1996

Activation of sucrose-phosphate synthase by a protein factor/sucrose-phosphate phosphatase.

The possible presence of a sucrose-phosphate synthase (SPS) activating/stabilizing factor (SAF) presumably lost during SPS purification was investigated. Rice leaf protein extracts were chromatographed in a DEAE-Sephacel column. SPS activity of previously purified rice enzyme was enhanced to different extent by aliquots of fractions from such column. The activating capacity could not be replaced by albumin, but was nullified by EDTA. When the fractions were boiled or treated with TCA, the activating capacity disappeared suggesting its proteinaceous nature. The presence of 10 microM okadaic acid had no effect on the stimulatory action of SAF on SPS denying the possibility to SAF to be a SPS-phosphatase. Although it overlaps somehow with sucrose synthase (SS) in DEAE-Sephacel fractions, the activating protein factor and SS eluted separately during Sephadex G-200 chromatography. The activating ability was saturable at a fixed SPS concentration and was able to enhance SPS activity from other plant sources. Simultaneous studies on the activities of SPS and sucrose-phosphate phosphatase (SPP), closely linked to SPS, allowed us to suggest that SAF could be SPP. The presence of SAF/SPP did not alter the affinity of SPS for its substrates but helped to reverse the Pi inhibition at low Fru-6-P concentrations. We conclude that SPS may possibly interact with SPP, contributing to a more effective sucrose synthesis.

Chromatography, Gel↗
PubMed1994

Increased affinity of histamine H1 binding to membranes of human myometrium at the end of pregnancy.

1. The characterization of H1 binding sites in membrane preparations of human myometrium obtained from pregnant and non-pregnant women was performed by using 3H-mepyramine as the radioactive ligand. 2. Saturation curve analysis revealed that 3H-mepyramine is bound to a single class of binding sites. Changes in the H1 site binding parameters were observed at the end of pregnancy, resulting in an increased affinity relative to non-pregnant tissue (Kd: 131.0 +/- 8.8 (non-pregnant) and 72.5 +/- 7.5 (pregnant) nM, n = 6, P < 0.01). 3. A reduction in receptor concentration at the end of pregnancy was also observed, [Bmax: 565.2 +/- 43.7 (non-pregnant) and 309.6 +/- 25.9 (pregnant) fmol/mg prot, n = 6, P < 0.01]. It is possible that this reduction in Bmax could be attributed to a dilution factor due to the increase in membraneous proteins that occurs during gestation.

Female↗
PubMed1991

Sucrose Phosphate Is Not Transported into Vacuoles or Tonoplast Vesicles from Red Beet (Beta vulgaris) Hypocotyl.

Tonoplast vesicles and vacuoles isolated from red beet (Beta vulgaris L.) hypocotyl accumulated externally supplied [(14)C]sucrose but not [(14)C]sucrose phosphate despite the occurrence of sucrose phosphate phosphohydrolytic activity in the vacuole. The activities of sucrose synthase and sucrose phosphate synthase in whole cell extracts were 960 and 30 nanomoles per milligram protein per minute, respectively; whereas, no sucrose synthesizing activity was measured in tonoplast preparations. The results obtained in this investigation are incompatible with the involvement of sucrose phosphate synthase in the process of sucrose synthesis and accumulation in the storage cells of red beet.

Journal Article↗
PubMed1990

Developmental Transition from Enzymatic to Acid Hydrolysis of Sucrose in Acid Limes (Citrus aurantifolia).

The sucrose breakdown mechanisms in juice sacs of acid lime (Citrus aurantifolia [Christm.] Swing.) were investigated throughout fruit development. All three enzymes of sucrose catabolism (sucrose synthase, acid, and alkaline invertase) are present during the initial stages. The activities of these enzymes declined rapidly and disappeared by stage 5 (80% development) but not before vacuolar pH had decreased to approximately 2.5. At this stage, sucrose breakdown occurs by acid hydrolysis. By attaining a vacuolar pH of 2.5 prior to enzyme disappearance, the cell maintains a continuous ability to break down sucrose throughout ontogeny. Thus, acid limes possess a unique and coordinated system for sucrose breakdown that involves both enzymatic and nonenzymatic pathways.

Journal Article↗
PubMed1989

Vacuolar Acid hydrolysis as a physiological mechanism for sucrose breakdown.

Sucrose breakdown in mature acidic ;Persian' limes (Citrus aurantifolia [Christm.] Swing.) occurred at a rate of 30.6 picomoles per milliliter per day during 9 weeks storage at 15 degrees C. Neither enzyme of sucrose catabolism (sucrose synthase or acid/alkaline invertase) was present in extracts of mature storage tissue. The average vacuolar pH, estimated by direct measurement of sap from isolated vacuoles and by the methylamine method, was about 2.0 to 2.2. In vitro acid hydrolysis of sucrose at physiological concentrations in a buffered solution (pH 2.2) occurred at identical rates as in matured limes. The results indicate that sucrose breakdown in stored mature acidic limes occurs by acid hydrolysis.

Journal Article↗
PubMed1988

Enzyme activities associated with maize kernel amyloplasts.

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.

Journal Article↗
PubMed1985

Isolation of amyloplasts from developing maize endosperm.

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4 degrees C or 5 hours at 30 degrees C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl(2). After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.

Journal Article↗