Sucrose-phosphatase

After glucose synthesis in photosynthesis, the disaccharide sucrose (a-D-Glc(l —> 2)(3-D-Fru) is used as a readily transportable sugar. Sucrose synthesis successively involves the following UDP-glucose + fructose-6-phosphate —> sucrose-6-phosphate + UDP [via sucrose phosphate synthase] sucrose-6-phosphate + H20 —> sucrose + P [via sucrose-6-phosphatase]. 

Though a phosphate of fructose is involved in this reaction it is the 6-phosphate, and so the phosphate ester group remains intact and does not play a direct part in the glycosyl transfer. The second step is to remove this phosphate, by way of a specific sucrose phosphatase, thereby generating free sucrose, at the expense of a certain amount of Gibbs free energy. 

The phosphate group is split off from sucrose-6-phosphate by sucrose phosphatase. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

Figure 5 Model of phosphorus (P) deficiency-induced physiological changes associated with the release of P-mobilizing root exudates in cluster roots of white lupin. Solid lines indicate stimulation and dotted lines inhibition of biochemical reaction sequences or mclaholic pathways in response to P deliciency. For a detailed description see Sec. 4.1. Abbreviations SS = sucrose synthase FK = fructokinase PGM = phosphoglueomutase PEP = phosphoenol pyruvate PE PC = PEP-carboxylase MDH = malate dehydrogenase ME = malic enzyme CS = citrate synthase PDC = pyruvate decarboxylase ALDH — alcohol dehydrogenase E-4-P = erythrosc-4-phosphate DAMP = dihydraxyaceConephos-phate APase = acid phosphatase.

Numerous modifications of in vitro culture systems have been developed for the estimation of BBB transfer [52]. Culture systems in use are either primary cultures of brain microvessel endothelial cells (BMEC) or immortalized endothelial cell hues. BMEC may be grown in co-culture with astrocytes or in astrocyte-conditioned medium. Astrocyte-derived factors increase the tightness of the barrier as measured by transendothelial electrical resistance (TEER) and by the permeability of hydrophUic markers such as sucrose. They also up-regulate the expression of BBB-enriched enzymes such as y-glutamyl transpeptidase (y-GTP) and alkaline phosphatase. A setup of the in vitro technique in a transwell system for transport studies is depicted in Figure 2.5. 

H. Luoma, Phosphoms translocation between enamel and Streptococcus mutans in the presence of sucrose and fluoride with observations on the acid phosphatase of S. mutans. Caries Res. 14 (1980) 248-257. 

In another approach subcellular fractionation of human PMNs, stimulated in vitro with phorbol myristate acetate or opsonized zymsan, was performed by rate-zonal centrifugation in sucrose gradients By varying the centrifugal force the NADPH oxidase could be separated from the activities of enzymes in the lysosomal granules but was similar to the distribution of markers for the plasma membrane, aryl phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. 

FIGURE 20-27 Regulation of sucrose phosphate synthase by phosphorylation. A protein kinase (SPS kinase) specific for sucrose phosphate synthase (SPS) phosphorylates a Ser residue in SPS, inactivating it a specific phosphatase (SPS phosphatase) reverses this inhibition. The kinase is inhibited allosterically by glucose 6-phosphate, which also activates SPS allosterically. The phosphatase is inhibited by Pi, which also inhibits SPS directly. Thus when the concentration of glucose 6-phosphate is high as a result of active photosynthesis, SPS is activated and produces sucrose phosphate. A high P, concentration, which occurs when photosynthetic conversion of ADP to ATP is slow, inhibits sucrose phosphate synthesis. 

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

Later, it was found that alkaline phosphatase was also released by osmotic shock E. coli were exposed to 0.5 M sucrose containing dilute tris-HCl buffer and EDTA, and then the centrifuged cells were rapidly dispersed in the shock medium of cold water or cold 5 X 10 4 M MgCl2. Although the cells were 80% viable with the latter case, almost all of the enzyme was released (9, 10). Other evidence indicates that the only important structural effect of EDTA is to increase the permeability of the cell wall (11, 12). Escherichia coli grow normally in the... 

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