A-Dephosphorylation

Synthesis of glycogen from UDP-glucose is catalyzed by glycogen synthase, which is rate-controlling. As in other tissues, glycogen synthase occurs in both a phosphory-lated form, synthase-b, which depends on Glc-6-P as a positive modulator, and a dephosphorylated, independent synthase-a form, which is much more active. 

Radiolabeled RNA can be generated either by incorporation of a (a32p) nucleoside triphosphate during an in vitro transcription reaction or by the transfer of (y P)-ATP to the 5 terminus of a dephosphorylated RNA molecule (41). The authors prefer the first mentioned method, as it needs only a single enzymatic reaction. 

Fig. 5 (a) Dephosphorylation of Fmoc-Y(p)-OH (8) to hydrogelator Fmoc-Y-OH (9) by alkaline phosphatase, (b) Conversion of the [3-peptide precursor 10 to hydrogelator 11 using acid phosphatase at pH 4.8... 

The detailed mechanism by which ATP hydrolysis is coupled to transport awaits determination of the protein s three-dimensional structure, but a current model (Fig. 11-37) proposes that the ATPase cycles between two forms, a phosphorylated form (designated P-Enzn) with high affinity for K+ and low affinity for Na+, and a dephosphorylated form (Enz ) with high affinity for Na+... 

Although some investigators believe that dihydrofuranone is derived from ribose-5-phosphate through a dephosphorylation-dehydration mechanism, others believe it can be formed by a typical Halliard reaction betveen amines and sugars, in which the Amadori products dehydrate and eliminate to amines. Both routes are likely to occur. 

Several studies have revealed that the D2 receptors modulate the state of phosphorylation of DARPP-32. In mouse striatal slices, the D2 agonist quinpirole decreased the basal phosphorylation of DARPP-32 on Thr-34, and antagonized the phosphorylation produced by the application of D1 agonist, forskolin or 8-bromo-cAMP (Nishi et al., 1997). This D2 effect was calcium-dependent and was blocked by cyclosporine A, an inhibitor of calcineurin, suggesting that it involved an increase in intracellular Ca2+ and a dephosphorylation of DARPP-32 by calcineurin. This study showed that an activation of... 

Another problem of the DHAP-dependent aldolases is that the product is phos-phorylated. The aldol reaction has therefore to be followed by a dephosphorylation step. In the sequences in Schemes 5.26-5.28 this deprotection is performed in situ. Otherwise several enzymes, such as phytase, are available for this purpose and the reaction normally proceeds under mild conditions [40, 43, 46]. 

The activity of pyruvate dehydrogenase is regulated by two mechanisms product inhibition and covalent modification (Section 6.5). The enzyme complex is allosterically activated by NAD+, CoASH, and AMP. It is inhibited by high concentrations of ATP and the reaction products acetyl-CoA and NADH. In vertebrates these molecules also activate a kinase, which converts the active pyruvate dehydrogenase complex to an inactive phosphorylated form. High concentrations of the substrates pyruvate, CoASH, and NAD+ inhibit the activity of the kinase. The pyruvate dehydrogenase complex is reactivated by a dephosphorylation reaction catalyzed by a phosphoprotein phosphatase. The phosphoprotein phosphatase is activated when the mitochondrial ATP concentration is low. 

Lakshmi and Balasubramanian (1980) showed the presence of a new multiple form of arylsulfohydrolase B in human and monkey brain. Arylsulfohydrolase B, can be separated by DEAE-cellulose chromatography (Mathew and Balasubramanian, 1984). The B, form totally binds to Sephadex G-200 and was not eluted with 1.0 M NaCl, 0.5 M glucose, 0.5 M glucose plus 0.5 M NaCl, 0.5 M KSCN, 1 M urea, or 1% Triton X-100. The treatment of arylsulfohydrolase B with Escherichia coli alkaline phosphatase results in the formation of a less acidic form, presumably due to dephosphorylation. The dephosphorylated form does not bind to DEAE-cellulose. Inorganic phosphate and serine phosphate but not mannose 6-phosphate can inhibit this dephosphorylation. The kinetic properties of the phosphorylated and dephosphorylated arylsulfohydrolase are quite similar. The possibility that arylsulfohydrolase B is a dephosphorylated form of B, has been ruled out by the significant differences between substrate concentration and activity curves of these enzymes. 

These second messengers interact with yet another type of protein, the ion channels. They open the gates on the ion channels and allow positively charged ions to flow into the cell, therefore creating an electrical potential between the cell and its environment. This is relieved when the synapse at the other end of the cell, in the olfactory bulb, fires across to the next nerve cell in line and the transduction process is set in motion. The second messengers also set in train a process which deactivates the receptor by phosphorylation, and a dephosphorylation is necessary to make it active again. 

A major effect of insulin is on the adipose tissue, both in regard to glucose uptake and intracellular metabolic effects. The major metabolic conversion in response to insulin is a dephosphorylation of hormonesensitive lipase to convert this enzyme into the inactive form. A second important metabolic aspect is an increase in synthesis and secretion to the blood-vessel surface of lipoprotein lipase. This enzyme is responsible for breaking down circulating triacylglycerols, particularly from VLDL as well as from chylomicrons, into fatty acids and glycerol. 

GC can also be used to separate partial hydrolytic products of certain larger molecules, such as phospholipids. An enzymatic hydrolysis readily yields the products (e.g., monoglycerides, ceramides and diglycerides) that are amenable to GC after a proper derivative formation [345,346]. A dephosphorylation of phospholipids by an enzyme prior to GC was also used by Kuksis et al. [337,347] to assess the effect of certain drugs on hyperlipidemic conditions. 

Glucose breakdown by heart muscle is regulated at several steps. Phos-phofructokinase (PFK), which converts fructose 6-phosphate to fructose 1,6-diphosphate, is a key step. PFK is activated by 5 -AMP and phosphate which lower the of the enzyme for fructose 6-phosphate and is inhibited by ATP, ATP Mg, and citrate which, conversely, raise the of the enzyme for fructose 6-phosphate. Whether PFK is also regulated by a dephosphorylation-phosphorylation process similar to that described above is not decided. Brand and Soling (1975) suggest the presence of such a regulatory mechanism for PFK isolated from liver. 

IXl. In contrast to the suggested stimulation of LHC kinase by a reduced pQ-pool (5) we observe a dephosphorylation (slow phase of oxygen increase) despite of a more reduced pool (fig. 3). 

Cloning of the "140 bp DNA" in M13mp8 vector. DNA from the "140 bp DNA" was subjected to SI nuclease treatment followed by a dephosphorylation using bacterial alkaline phosphatase. The DNA was en treated with T4 polynucleotide kinase and the ends repaired with T4 DNA polymerase. After each enzymatic step, the solutions were extracted with phenol and chloroform and the DNA recovered by ethanol precipitation. The blunt-ended DNA was cloned by blunt-end ligation in the Hindi site of M13mp8 vector. All these steps were achieved according to standard procedures (15). Ml 3 clones (26 total) were obtained. 

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