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Protein glycation is a spontaneous reaction of reducing sugars with amino groups of proteins, leading to the formation of ketoamines (fructosamines or ribulosamines when the initial reacting sugar a glucose or ribose). Recently, protein-repair enzymes able to remove the accessible ketoamines from proteins have been described. Two enzymes, fructosamine-3-kinase (FN3K) and fructosamine-3-kinase related protein (FN3K-RP) are responsible for protein deglycation. The osamine-3-phosphates formed in this way are unstable and spontaneously decompose with recovery of the initial amine. Two other enzymes involved in earlier steps of deglycation are MDP-1 (magnesium dependent phosphatase-1) and LMW-PTP (low-molecular-weight protein tyrosine phosphatase). Dephosphorylation of proteins glycated with glucose-6-phosphate by MDP-1 converts fructosamine-6-phosphates into substrates for FN3K. LMW-PTP converts ribulosamine-5-phosphates and erythrulosamine-4-phosphates, the glycation products derived from ribose-5-phosphate and erythrose-4-phosphate, to substrates for FN3K-RP. The extent of glycation depends on the reactivity of the sugar. The main factor determining the reactivity of a sugar is its proportion present under open-chain conformation, i.e., with a free carbonyl group. Thus, sugar-phosphates such as ribose-5-phosphate or erythrose-4-phosphate are powerful glycating agents. The physiological concentration of ribose-5-phosphate and erythrose-4-phosphate being very low, the glycation of lysine residues by these phosphate esters is extremely low, unless it takes place in a site (such a catalytic site) with a high affinity for ribose-5-phosphate or erythrose-4-phosphate. To establish the hypothetical role of glycation in the loss of enzymatic activity induced by sugar-phosphates, we studied the kinetic properties of four enzymes known to have (or likely to have) a high affinity for ribose-5-phosphate or erythrose-4-phosphate: • Phosphoglucose isomerase from yeast • Deoxyribose-5-phosphate aldolase from Thermus thermophilus and from Staphylococcus aureus • Phosphoglucomutase 2 (human), known for its phophopentomutase activity First, we tested for (reversible) inhibition of enzymes by ribose-5-phosphate and erythrose-4-phosphate and, in some cases arabinose-5-phosphate (epimer of ribose-5-phosphate) to see whether these compounds bind directly to the enzyme active site. Secondly, prolonged incubations of enzymes in the presence of sugar-phosphates were carried out to see whether this led to progressive inactivation of the enzyme (an irreversible process) possibly due to protein glycation. We next tried to restore impaired enzyme activity by using the known protein-repair enzymes, LMW-PTP and FN3K-RP. We observed an important inactivation of deoxyribose-5-phosphate aldolase from T. thermophilus incubated in the presence of arabinose-5-phosphate. Several experiments led us to conclude that this loss of activity was not related to the formation of ketoamines, but to tight binding of glycolaldehyde in the catalytic site. Erythrose-4-phosphate caused inactivation of phosphoglucomutase 2. The incubation of the previously inactivated enzyme in the presence of LMW-PTP, FN3K-RP and ATP failed to restore the enzymatic activity. La glycation est une réaction spontanée entre les sucres réducteurs et les groupements amines des protéines menant à la formation quasi irréversible de cétosamines (fructosamines ou ribulosamines selon que le sucre réagissant est le glucose ou le ribose). On sait depuis peu que les cellules possèdent des enzymes réparatrices capables d'enlever des cétosamines de la surface des protéines. Le processus de déglycation est amorcé par deux enzymes, la FN3K (fructosamine-3-kinase) et la FN3K-RP (fructosamine-3-kinase related protein). Les cétosamines-3-phosphates ainsi formées sont instables et se décomposent spontanément en régénérant l'amine initiale. Deux autres enzymes interviennent en amont dans le processus de déglycation : la MDP-1 (magnesium dependent phosphatase-1) et la LMW-PTP (low-molecular-weight protein-tyrosine phosphatase). La MDP-1 est capable de déphosphoryler les fructosamines-6-phosphates, produits de la glycation par le glucose-6-phosphate, et la LMW-PTP déphosphoryle spécifiquement les ribulosamines-5-phosphates et les érythrulosamines-4-phosphates, produits de la glycation par le ribose-5-phosphate et l'érythrose-4-phosphate. Les fructosamines, les ribulosamines et les érythrulosamines formées au cours de cette réaction sont à leur tour substrats de la FN3K pour les premières et de la FN3K-RP pour les autres. Tous les sucres n'ont pas le même pouvoir glyquant. La réactivité dépend dans une large mesure de la proportion dans laquelle le sucre se trouve sous forme linéaire, c'est-à-dire avec un groupement carbonyle libre, capable de réagir avec un groupement amine. Ainsi les esters phosphoriques de sucres tels que le ribose-5-phosphate ou l'érythrose-4-phosphate sont de puissants agents glyquants. La concentration physiologique du ribose-5-phosphate et d'érythrose-4-phosphate étant très faible, on s'attend à atteindre tout au plus une stoechiométrie de glycation de l'ordre de 1/1000 pour les lysines quelconques d'une protéine. Cependant, si ce processus de glycation a lieu au niveau de certains sites qui servent normalement à fixer le ribose-5-phosphate ou l'érythrose-4-phosphate, on peut s'attendre à observer des stoechiométries de glycation beaucoup plus élevées et, corollairement, une perte importante d'activité enzymatique. Afin de déterminer de rôle de la glycation dans la perte d'activité enzymatique induite par les sucres phosphorylés, nous avons étudié quatre enzymes connues pour avoir (ou susceptibles d'avoir) une haute affinité pour le ribose-5-phosphate ou l'érythrose-4-phosphate : • la phosphoglucose isomérase de levure, • les désoxyribose-5-phosphate aldolases de Thermus thermophilus et de Staphylococcus aureus, • la phosphoglucomutase 2 humaine (qui est, en fait, une phosphopentomutase). Dans chaque cas, nous avons investigué l'effet du ribose-5-phosphate et de l'érythrose-4-phosphate sur la cinétique, afin de voir si ces composés se lient à l'enzyme. Dans certains cas nous avons testé également l'arabinose-5-phosphate (un épimère du ribose-5-phosphate). Nous avons ensuite réalisé des incubations prolongées des différentes enzymes avec ces esters phosphoriques afin de voir si l'on observait une inactivation de l'enzyme, cette inactivation pouvant être le reflet d'une glycation progressive. On a également tenté de remédier à l'effet d'inactivation en utilisant des enzymes réparatrices connues, telles que la LMW-PTP (low-molecular-weight protein-tyrosine phosphatase) et la FN3K-RP (fructosamine-3-kinase-related protein). Une inactivation importante a été observée dans le cas de la désoxyribose-5-phosphate aldolase de Thermus thermophilus lorsqu'elle est inactivée avec de l'arabinose-5-phosphate. Les études que nous avons faites indiquent que cette perte d'activité n'est pas liée à la formation d'une cétosamine, mais bien à la fixation d'une molécule de glycolaldéhyde au site catalytique. Une inactivation par l'érythrose-4-phosphate a été également observée dans le cas de la PGM2. L'incubation de l'enzyme inactivée en présence de la LMW-PTP, de la FN3K-RP et d'ATP n'a pas permis de restaurer l'activité enzymatique initiale.

FN3KRP protein, human --- Glucose-6-Phosphate Isomerase --- Phosphoglucomutase --- Sugar Phospates --- Fructosamine

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Within-field variability of the plant-available nutrients often results in different fertiliser requirements across a field. Variable rate (VR) fertilisation is one of the advanced techniques in precision agriculture. The VR strategy for P (VRP) is based on standard soil test for P in sampling grid of one hectare for large agricultural fields. The study of spatial variability of soil P within a field revealed that, VRP should be implemented over small areas (perhaps down to few square meters). This can be achieved with an on-the-go P fertilisation system. For on-the-go P fertilisation, three important elements are required: a soil sensor to gather soil information while travelling across a field, a model to derive the actual soil P and a recommendation model to predict corresponding application rate. A VIS-NIR soil sensor for on-the-go gathering of soil spectra (Mouazen et al, 2005) was installed at the front of a planter-applicator for on-the-go gathering of soil spectra. The optical sensor unit to measure soil reflectance spectra was attached to a mobile, fibre-type, VIS-NIR spectrophotometer (CORONA FIBRE VISNIR 1.7, Carl Zeiss) with a measurement range of 305 - 1711 nm. Two visible (VIS) and near infrared (NIR) models were developed for prediction of soil P using wet soils based on soil samples that were collected from Belgium and Northern France. These models were calibrated based on two different measures of soil available P: Olsen P and ammonium lactate extractable P (Pal). The performance of the models was validated by different methodologies. The accuracy of the model was verified using several independent field samples. Since in Belgium the phosphate is recommended based on Pal test, the model developed (Mouazen et al, 2007) based on the Pal was used as basis for VR fertilisation system developed for this dissertation. Another challenge to on-the-go P application is fertiliser recommendation. Generally, fertiliser recommendation is based on many factors rather than just measured soil P. These include soil type, pH, other soil nutrients and soil moisture content. These soil parameters must be simultaneously measured in addition to soil P for actual fertiliser recommendation. Since, for the present research these factors were not available in on-the-go measurements, a recommendation model was established solely based on soil Pal measure. This simplified model was derived from the information provided by Soil Service of Belgium rather than based on the Expert System (Vandendriessche et al., 1993 & 1995) for precise fertiliser recommendation that uses many of the above mentioned parameters. The fertiliser recommendation based on this simplified model could sometimes fail due to ignoring other factors. But this could be remedied in future. A programme (LabVIEW, National Instrument) was developed to record soil spectra, predict soil Pal during on-the-go measurement and calculate required P fertiliser amount. This algorithm provided the signal to the VR fertiliser applicator (ED302, AMAZONE) to change the application rate accordingly. In addition, the coordinates of the sampling points were recorded using a DGPS (Trimble® AgDGPS 132) that was included to document the soil and fertiliser maps, although, for a sensor-based VR application system this positioning system is not required. In an experimental field alternative plots were selected so that every other plot was used for VR application as for uniform rate (UR) treatment. Maize was planted over the entire field. During the planting, only the plots assigned for VR treatment received the rates recommended by on-the-go measurement and the rest received a uniform dose of 30 kg P2O5/ha based on the standard Pal measurement and recommendation by the Soil Service of Belgium. The location of the collected soil P spectra and the corresponding phosphate application rate was recorded using a DGPS. The number of plant leaves and grain yield were considered as growth indices, which might be influenced by P deficiency. The maize was harvested using a combine harvester (CR 960 New Holland) instrumented with a continuous grain flow sensor (New Holland) and a DGPS, which allowed positioning the grain yield while travelling across the field. The plant leaves and crop yield showed lower variation in plots assigned for VR treatment compared to those of UR treatment (coefficient of variation of 25 and 31%, respectively). This may be due to the proper distribution of the fertiliser according to soil requirement. No significant difference was found for the number of plant leaves between VR and UR plots. However, the yield of the plots with the VR treatment was significantly higher than for the UR treatment. Yield increase of 336 kg grain/ha was observed in the VR plots. However, the experimental layout of the plots could not be properly designed using a statistical randomisation method because of the limitation imposed by the machine operation. Therefore, this result needs further investigation to be confirmed. Average application rate of phosphate on VR plots was 28.75 kg P2O5/ha which is 1.25 kg P2O5/ha lower than the recommended UR (30 kg P2O5/ha). This implies that the benefit of using VR strategy is minor in agricultural fields with a high level of soil Pal which is the case for most of the Belgian fields.

Academic collection --- 631.333 --- 631.85 --- 631.416.2 --- Fertilizer distributors. Manure spreaders. Machines and equipment for fertilizer preparation and distribution --- Phosphate fertilizers --- Phosphoric acid --- Theses --- 631.416.2 Phosphoric acid --- 631.85 Phosphate fertilizers --- 631.333 Fertilizer distributors. Manure spreaders. Machines and equipment for fertilizer preparation and distribution