Phosphate-binding

Calcium salt. Calcium gluconate is the preferred salt in PN because it is has a low dissociation in solution with lesser free calcium available at a given time to bind phosphate (as opposed to calcium chloride, which dissociates rapidly in solution). 

As in Sect. 2, the early works in this field will only be briefly summarized. Highly charged polyazoniamacro(oligo)cycles and guanidinium crowns bind phosphates and nucleotides via electrostatic interactions [1,65], and polyammonium cations, like spermine, are generally known to associate with the phosphate backbone of RNA and DNA strands [66]. 

The pH-rate profile for the action of the enzyme shows a typical pH maximum, with sharply lower rates at either higher or lower pH than the optimum these facts suggest that both an acidic and a basic group are required for activity (Herries, 1960). The two essential histidine residues could serve as these groups if, in the active site, one were protonated and the other present in its basic form. The simultaneous acid-base catalysis would parallel that of the model system (discussed below) of Swain and J. F. Brown. The essential lysine, which binds phosphate, presumably serves to bind a phosphate residue of the ribonucleic acid. These facts led Mathias and coworkers to propose the mechanism for the action of ribonuclease that is shown in (13) (Findlay et al., 1961). 

Most important is that the target protein is soluble, stable and monodisperse in the selected buffer. This will be checked in the limited protein characterization (see below). Phosphate buffer is very convenient since there are no H signals, and is often the first choice for ligand-detected NMR techniques. However, there are several proteins that bind phosphate (notably phosphatases) for which phosphate buffer is not a good choice. Fortunately, there is now a large selection of commercially available buffers in deuterated form. 

EXAFS studies indicate a Zn(II)-Fe(III) distance of 393 pm which decreases to 372 pm on binding phosphate. The change of Zn-edge structure and the appearance of a new peak in the Fourier transform of the EXAFS suggests that the inhibitor also binds to the Zn(II) site irrespective of pH. 

Oligopyrrolic clusters with thiophene spacers 88 have been synthesized and their ability to bind phosphate in the solution phase evaluated <03TL6695>. Tetrathiafulvalene (TTF) analogs consisting of fused thiophenes 89-91 have been synthesized and the mechanism of formation of TTF derivatives from 1,8-diketones has been analyzed <03T8107>. 

For chronic use, e.g. hypoparathyroidism, alfa-calcidol or calcitriol are needed. Dietary calcium is increased by giving calcium gluconate (an effervescent tablet is available) or lactate. Aluminium hydroxide binds phosphate in the gut causing hypo-phosphataemia, which stimulates renal formation of the most active vitamin D metabolite and usefully enhances calcium absorption. 

Monobasic sodium phosphate should not he administered concomitantly with aluminum, calcium, or magnesium salts since they bind phosphate and could impair its ahsorption from the gastrointestinal tract. Interaction between calcium and phosphate, leading to the formation of insoluble calcium phosphate precipitates, is possible in parenteral admix-tures.< >... 

These acyclic Robson systems provided initial evidence for this mode of binding. Recently, a dicopper(II) complex of this type (47) was proven to bind phosphate esters and increase their rate of hydrolytic cleavage (120), which... 

Hypophosphatemia and phosphate depletion may result from inadequate intestinal phosphate absorption. Patients taking aluminum- or magnesium-containing antacids may develop hypophosphatemia, because these antacids bind phosphate in the intestine, rendering it nonabsorbable. The hypophosphatemia observed in patients with malabsorption maybe more closely related to their secondary hyperparathyroidism than to malabsorption of phosphate. Because phosphate is abundant in most foods, dietary deprivation is not usually a cause of phosphate depletion in patients with normal intestinal function and an adequate diet. 

Therapy for hyperphosphatemia is directed toward correcting the cause of the high serum phosphate. In renal failure and in hypoparathyroidism, dietary restriction of phosphate and agents that bind phosphate in the intestine (calcium carbonate and others) are useful in lowering the serum phosphate concentrations. 

There is little motion of the protein, the main movement being closure of a loop over the active site after binding phosphate and substrate analogue. There are indications that when an acceptor maltooligosaccharide and glucose-1-phosphate are both bound, the phosphate is forced down and in an unfavourable torsional angle however there is no indication of any direct covalent interaction between the nucleophilic phosphate and the phosphate attached to the pyridoxal moiety, which had been proposed on the basis on NMR studies on glycogen phosphorylase. There is also no indication of any covalent intermediate. Structurally, the S i mechanism is plausible whereas the doubledisplacement mechanism would create difficulties. 

As we ve just seen, control of the E. coli glnA gene depends on two proteins, NtrC and NtrB. Such two-component regulatory systems control many responses of bacteria to changes in their environment. Another example involves the E. coli proteins PhoR and PhoB, which regulate transcription in response to the concentration of free phosphate. PhoR is a transmembrane protein, located In the Inner (plasma) membrane, whose periplasmic domain binds phosphate with moderate affinity and whose cytosolic domain has protein kinase activity PhoB is a cytosolic protein. 

This mechanism of binding phosphate ions into insoluble forms with aluminium or iron leads to considerable losses of phosphate fertilizers in the soil, since in this case, only a fraction of the phosphorus supplied may be utilized by the plants. 

In Other examples anion binding can cause quenching of the Ru (bpy)3 MLCT emission. In aqueous solution polyaza receptors 80-82 bind phosphate and ATP anions, producing up to a 15% reduction in the emission intensity of max at 605 nm [16]. Similarly, 76 shows up to a 40% reduction in the intensity of the luminescent emission at 630 nm in the presence of dihydrogenphosphate in DMSO solution. 

Polyamines that exist as polycationic species in water under appropriate pH conditions are suitable scaffolds to capture phosphate derivatives by the formation of multiple-hydrogen bonding and electrostatic interaction. It has previously been shown that naturally occurring polyamines such as spermine and spermidine bind phosphates or DNA [11]. Nakai et aL reported that the apparent affinity constants, logICapp for the 1 1 complexation of spermine with AMP, ADP, and ATP were foimd to be 2.6, 3.1 and 4.0, respectively, in 50 mM tris buffer (pH 7.5) [12]. Based on this moderate affinity, several artificial phosphate receptors containing polyamines were developed. Because of the difficulty of captming phosphate anions by non-covalent interaction in aqueous media, however, successful examples of artificial small molecular receptors or sensors for phosphates are still relatively scarce. 

Similar to guanidinium, imidazole, a basic group of histidine side chains, might be used as a binding unit for phosphate in artificial receptors. As far as we know, however, there are no reported examples of such receptors that can successfully bind phosphates in aqueous media. Known examples are hmited to receptors used in organic media. This again indicates the difficulty in the development of sophisticated phosphate receptors usable in aqueous solutions. 

Metal-based receptors have found particularly interesting applications in the recognition of phosphorylated species of biological interest (e.g. phospho-rylated amino acids and peptides). This area is reviewed in depth by Tamaru and Hamachi with particidar emphasis on a series of receptors based on zinc(II) centres which have been shown to bind phosphates with very high binding constants in aqueous media. The applications of this type of receptor for the detection of samples of biological interest are also presented. 

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