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Biomedical subjects

H G Pontis

Publications and source records attributed to H G Pontis.

At least 19 recordsLinked to original sources

Protein kinase and phosphatase activities are involved in fructan synthesis initiation mediated by sugars.

The induction of fructosylsucrose-synthesizing activity (FSS) by sugars was tested using detached primary leaf blades of several wheat (Triticum aestivum L.) cultivars, immersed in different sugars solutions for 24 h in the dark. The highest induction was brought about by sucrose, while glucose, fructose and maltose also caused significant induction. 5-Ketofructose, 3-methylglucose and 6-deoxyglucose, which cannot be metabolized by plants, produced no induction at all. The fact that mannose also failed to induce FSS and that mannoheptulose did not inhibit the induction by sucrose suggests that the hexokinase-sensing system may not be involved. The protein phosphatase inhibitor okadaic acid and the calmodulin-dependent protein kinase antagonist W7 inhibited FSS induction while some types of protein kinase inhibitors, such as staurosporine and genistein, had less or no effect, respectively. Cycloheximide and cordycepin completely inhibited the induction response, indicating that transcription and translation are necessary for the FSS induction. Northern blot experiments using a sucrose:fructan-6-fructosyl transferase probe gave a clear indication that the mRNA for this enzyme, which is almost absent in control leaves, is dramatically increased after a 24-h treatment with 500 mM sucrose, and confirmed the inhibition produced by protein kinase and protein phosphatase inhibitors. Our data indicate that protein kinase and protein phosphatase activities take part in the chain of events that intervenes in the induction of fructan synthesis by sugars.

Calcium-Calmodulin-Dependent Protein Kinases↗

Studies on sucrose-phosphate synthase from rice leaves.

Sucrose-phosphate synthase (SPS, EC 2.4.1.14) biochemical properties and peptide composition have been analyzed in rice leaf seedlings. SPS was purified using DEAE-Sephacel chromatography, gel filtration on Sepharose 6B and anion exchange chromatography on Mono Q. At this stage two enzyme forms (SPS-I and -II) were separated. SPS-II was purified 90-fold; however, SPS-I presented a lower specific activity regarding the previous purification step and an unstable activity. Both enzyme forms had similar apparent Km values for Fru-6P but the SPS-I Km for UDP-Glc was ca. 10-fold higher than the SPS-II one. In addition, they differentiate in the capacity of being modulated by Glc-6-P and Pi: while SPS-II activity was inhibited by Pi and activated by Glc-6-P, SPS-I was not affected by either effectors. A native molecular mass of ca. 420 kDa was found by gel filtration. In SPS expression analysis using leaf rice and wheat germ SPS antibodies, a 116 kDa polypeptide was revealed in rice leaf extracts and no polypeptide was immunoactive in rice roots.

Glucosyltransferases↗

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↗

Sucrose Synthase Expression during Cold Acclimation in Wheat.

When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4 degrees C) above 0 degrees C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23 degrees C to 4 degrees C. The level of SS diminished when plants were moved back to 23 degrees C. Northern blots of poly(A)(+) RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress.

Journal Article↗

Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock.

Chlorella vulgaris cultures have been submitted to a chilling shock, bringing down the growing temperature from to 24 degrees C to 4 degrees C. Growth was stopped immediately, and concomitantly there was an accumulation of sucrose and a decrease in the starch content. The enzymes involved in sucrose metabolism were differentially affected by the chilling shock. Sucrose phosphate synthase activity increased while sucrose synthase was not affected. Simultaneously with the chilling shock, raffinose began to accumulate. When algal cultures were returned at 24 degrees C, raffinose disappeared. The presence of raffinose in algal cells has not been reported before.

Journal Article↗

Reversible unidirectional inhibition of sucrose synthase activity by disulfides.

Sucrose synthase (UDPglucose:D-fructose 2-alpha-D-glucosyltransferase, EC2.4.1.13), which catalyzes the synthesis and cleavage of sucrose, exhibits differences in some properties between the two reactions. When enzyme previously incubated with oxidized glutathione or oxidized thioredoxin was used, sucrose cleavage was inhibited whereas sucrose synthesis proceeded at a normal rate. Sucrose cleavage activity could be restored by incubation with dithiothreitol or reduced glutathione. The thioredoxin effect was influenced by the presence of cleavage reaction substrates--i.e., sucrose and UDP. Thioredoxin action was rather slow compared with the catalytic reaction. These findings may have important implications for understanding the metabolic role of sucrose synthase and oxidized thioredoxin. Theoretically, the fact that an enzyme catalyzing a reversible reaction is inhibited in one direction only suggests that a modification in the enzyme affinities for its substrates must have occurred.

Ascorbic Acid↗

Fructose metabolism in plants. Isolation and properties of pea seed frucktokinase.

Fructokinase from pea (Pisum sativum L.) seed has purified 100-fold. The enzyme required reduced sulfhydryl groups for activity. It also exhibits an absolute requirement for potassium ions (Km = 3 mM) and is unstable when not stored with a high concentration of potassium ions. The isoelectric point of the enzyme is 4.7 and it has a molecular weight of 44 000 +/- 700 daltons as determined by molecular sieve chromatography and sedimentation velocity techniques. A Hill plot of the potassium ion data suggests that two potassium sites are present on the enzyme. The MgATP saturation curve was non-Michaelis-Menten with a slight positive cooperativity. Pea seed fructokinase is highly specific for fructose and ATP. A comparison of pea seed fructokinase properties and those of liver and bacterial origin is presented.

Fabaceae↗