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PubMed1991

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: distinction of the 1-monodeutero-isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy.

The 1H NMR spectrum obtained with the alpha- and beta-anomers of D-[1-2H]fructose 6-phosphate generated from D-glucose 6-phosphate sequentially exposed in D2O to phosphoglucoisomerase, phosphofructokinase and fructose-1,6-diphosphatase differed from that recorded when the deuterated ketohexose phosphate was produced from D-mannose 6-phosphate sequentially exposed in D2O to phosphomannoisomerase, phosphofructokinase and fructose-1,6-diphosphatase. The identification of the 2 isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy provides a new tool to assess the relative extent of interconversion of hexose phosphates in the reactions catalyzed by phosphoglucoisomerase and phosphomannoisomerase, respectively.

Deuterium↗

PubMed2000

Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.

The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.

Adenosine Triphosphate↗

PubMed1995

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

Alcohol Oxidoreductases↗

PubMed2004

Recovery of renal tubule phosphate reabsorption despite reduced levels of sodium-phosphate transporter.

BACKGROUND: The acute effect of parathyroid hormone (PTH) on phosphate transport has been reported to be mediated by rapid downregulation of sodium-phosphate transporter (NaPi-IIa) protein, but the association was observed with pharmacological doses of PTH. OBJECTIVE: To explore the effects of physiological doses of PTH on NaPi-IIa protein and its relationship to phosphate transport. METHODS: Acute clearance studies were performed in parathyroidectomized rats given a bolus i.v. physiological dose (1 microg) of bovine PTH(1-34) and NaPi-IIa protein concentrations were examined at different time intervals. RESULTS: Fractional excretion of phosphate increased from 0.031+/-0.006 (mean+/-S.E.) to 0.238+/-0.059 (P<0.01 compared with baseline and compared with controls) at 40 min and returned to control values by 120 min. Urinary cAMP concentrations were increased at 20 min only. Superficial cortex brush-border membrane (BBM) NaPi-IIa protein was decreased from baseline at both 40 and 120 min (P<0.01) and did not recover at 240 min (P<0.01 compared with baseline and compared with controls). CONCLUSION: These results confirm that PTH, even in physiological dosage, causes a rapid decrease in BBM NaPi-IIa, but subsequent recovery of phosphate reabsorption is poorly correlated with BBM concentrations of NaPi-IIa protein. This suggests that transport mechanisms other than NaPi-IIa are important in renal phosphate reabsorption.

Absorption↗

PubMed1981

Interaction of organic phosphates with bovine hemoglobin. I. Oxylabile and phosphate-labile proton binding.

Adult bovine hemoglobin solutions were studied with respect to the influence of organic phosphates (adenosine-5'-triphosphate, 2,3-diphosphoglycerate, myo-inositolhexaphosphate) on the oxylabile proton binding (fixed-acid Haldane effect). At alkaline pH (less than 7.5) this Haldane effect is increased by organic phosphates, whereas at acid pH (less than 6.5) the effect is decreased; in the neutral pH range no unequivocal trend was found. The influence on the Haldane effect is caused by binding of organic phosphates to both deoxy- and liganded hemoglobin. The binding of 2,3-diphosphoglycerate and myo-inositolhexaphosphate causes a massive proton uptake. In going from deoxy- to liganded hemoglobin in the presence of organic phosphates the proton release due to phosphate release from deoxyhemoglobin and the proton uptake due to phosphate binding by liganded hemoglobin were shown to account for the phosphate-induced part of the fixed-acid Haldane effect.

2,3-Diphosphoglycerate↗

PubMed1997

Crystal structures of ribonuclease A complexes with 5'-diphosphoadenosine 3'-phosphate and 5'-diphosphoadenosine 2'-phosphate at 1.7 A resolution.

High-resolution (1.7 A) crystal structures have been determined for bovine pancreatic ribonuclease A (RNase A) complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) and 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), as well as for a native structure refined to 2.0 A. These nucleotide phosphates are the two most potent inhibitors of RNase A reported so far, with Ki values of 240 and 520 nM, respectively. The binding modes and conformations of ppA-3'-p and ppA-2'-p were found to differ markedly from those anticipated on the basis of earlier structures of RNase A complexes. The key difference is that the 5'-beta-phosphate rather than the 5'-alpha-phosphate of each inhibitor occupies the P1 phosphate binding site. As a consequence, the ribose moieties of the two nucleotides are shifted by approximately 2 A compared to the positions of their counterparts in earlier complexes, and the adenine rings are rotated into unusual syn conformations. Thus, the six-membered and five-membered rings of both adenines are reversed with respect to the others but nonetheless engage in extensive interactions with the residues that form the B2 purine binding site of RNase A. Despite the close structural similarity of the two inhibitors, the puckers of their furanose rings are different: C2'-endo and C3'-endo, respectively. Moreover, their 5'-alpha-phosphates and 3'(2')-monophosphates interact with largely different sets of RNase residues. The results of this crystallographic study emphasize the difficulties inherent in qualitative modeling of protein-inhibitor interactions and the compelling reasons for high-resolution structural studies in which quantitative design of improved inhibitors was enabled. The structures presented here provide a promising starting point for the rational design of tight-binding RNase inhibitors, which may be used as therapeutic agents in restraining the ribonucleolytic activities of RNase homologues such as angiogenin, eosinophil-derived neurotoxin, and eosinophil cationic protein.

Adenosine Diphosphate↗

PubMed1998

CobD, a novel enzyme with L-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2.

The cobD gene of Salmonella typhimurium LT2 has been cloned, sequenced, and overexpressed. The overexpressed protein had a molecular mass of approximately 40 kDa, in agreement with the mass predicted by the deduced amino acid sequence (40.8 kDa). Computer analysis of the deduced amino acid sequence of CobD identified a consensus pyridoxal phosphate-binding motif. The role of CobD in cobalamin biosynthesis in this bacterium has been established. CobD was shown to decarboxylate L-threonine O-3-phosphate to yield (R)-1-amino-2-propanol O-2-phosphate. We propose that the latter is a substrate in the reaction catalyzed by the CbiB enzyme proposed to be responsible for the conversion of adenosylcobyric acid to adenosylcobinamide and that the product of the reaction is adenosylcobinamide phosphate, not adenosylcobinamide as previously thought. The implications of these findings are discussed in light of the demonstrated kinase activity of the CobU enzyme (O'Toole, G. A., and Escalante-Semerena, J. C. (1995) J. Biol. Chem. 270, 23560-23569) responsible for the conversion of adenosylcobinamide to adenosylcobinamide phosphate. These findings shed light on the strategy used by this bacterium for the assimilation of exogenous unphosphorylated cobinamide from its environment. To our knowledge, CobD is the first enzyme reported to have L-threonine-O-3-phosphate decarboxylase activity, and computer analysis of its amino acid sequence suggests that it may be a member of a new class of pyridoxal phosphate-dependent decarboxylases.

Amino Acid Sequence↗

PubMed1989

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides.

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP(+) ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.

Journal Article↗

PubMed1982

Double-blind comparison of the respiratory and sedative effects of codeine phosphate and (+/-)-glaucine phosphate in human volunteers.

1 Two antitussive agents (+/-)-glaucine phosphate and codeine phosphate have been compared with placebo with respect to ventilation, ventilatory response to carbon dioxide, pulse, blood pressure, digit symbol substitution, sedation score and the Zahlen-Verbindung test performance in ten healthy volunteers (22-36 years). The study was double-blind and the two doses of each antitussive agent and the placebo were administered as a syrup. 2 Both codeine phosphate and (+/-)-glaucine phosphate displaced the ventilatory response to carbon dioxide to the right. 3 The effect of codeine phosphate on the ventilatory response to carbon dioxide was not dose dependent: 30 mg produced greater effects than the 60 mg dose. 4 Only the highest dose of (+/-)-glaucine phosphate (60 mg) caused respiratory depression and this was associated with sedation and decreased performance in the digit symbol substitution test. 5 Neither antitussive agent had significant effects upon pulse or blood pressure and codeine phosphate had no detectable sedative activity.

Adult↗

PubMed1979

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.

Acyl Coenzyme A↗

PubMed1985

A simple fluorimetric assay for pyridoxamine phosphate oxidase in erythrocyte haemolysates: effects of riboflavin supplementation and of glucose 6-phosphate dehydrogenase deficiency.

The formation of pyridoxal and its phosphate from pyridoxamine phosphate by red cell haemolysates was measured in a centrifugal analyser by the formation of the fluorescent adduct with semicarbazide. Pyridoxal phosphate was found to react more rapidly than pyridoxal, thus permitting a distinction between the two products, and hence the measurement of phosphatase activity. Activity of the enzyme, pyridoxamine phosphate:oxygen oxidoreductase (deaminating) EC 1.4.3.5 (PPO) was measured in haemolysates from 72 Gambian women with evidence of riboflavin deficiency, and was repeated after 6 weeks of placebo or riboflavin supplementation. Those who received the riboflavin supplement responded with a marked increase in PPO activity, which was matched by a decrease in the activation coefficient (AC) of erythrocyte NAD(P)H2:glutathione oxidoreductase, EC 1.6.4.2 (glutathione reductase, EGR). No difference between the supplemented and unsupplemented groups was observed in the capacity of haemolysates to hydrolyse pyridoxal 5-phosphate, nor in the extent of activation of erythrocyte L-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1. by pyridoxal phosphate. Although the three subjects with low levels of D-glucose 6-phosphate: NADP 1-oxidoreductase EC 1.1.1.49 (G6P-D) had, as expected, correspondingly low AC's of EGR, their unsupplemented activities of PPO were in the same low range as those of the G6P-D-normal subjects, and they responded as G6P-D-normal subjects did to riboflavin supplementation. PPO thus does not appear to resemble EGR in retaining its flavin coenzyme during riboflavin depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed2006

Injectable and rapid-setting calcium phosphate bone cement with dicalcium phosphate dihydrate.

Calcium phosphate cement (CPC) sets in situ with intimate adaptation to the contours of defect surfaces, and forms an implant having a structure and composition similar to hydroxyapatite, the putative mineral in teeth and bones. The objective of the present study was to develop an injectable CPC using dicalcium phosphate dihydrate (DCPD) with a high solubility for rapid setting. Two agents were incorporated to impart injectability and fast-hardening to the cement: a hardening accelerator (sodium phosphate) and a gelling agent (hydroxypropyl methylcellulose, HPMC). The cement with DCPD was designated as CPC(D), and the conventional cement was referred to as CPC(A). Using water without sodium phosphate, CPC(A) had a setting time of 82 +/- 6 min. In contrast, CPC(D) exhibited rapid setting with a time of 17 +/- 1 min. At 0.2 mol/L sodium phosphate, setting time for CPC(D) was 15 +/- 1 min, significantly faster than 40 +/- 2 min for CPC(A) (Tukey's at 0.95). Sodium phosphate decreased the paste injectability (measured as the paste mass extruded from the syringe divided by the original paste mass inside the syringe). However, the addition of HPMC dramatically increased the paste injectability. For CPC(D), the injectability was increased from 65% +/- 12% without HPMC to 98% +/- 1% with 1% HPMC. Injectability of CPC(A) was also doubled to 99% +/- 1%. The injectable and rapid-setting CPC(D) possessed flexural strength and elastic modulus values overlapping the reported values for sintered porous hydroxyapatite implants and cancellous bone. In summary, the rapid setting and relatively high strength and elastic modulus of CPC(D) should help the graft to quickly attain strength and geometrical integrity within a short period of time postoperatively. Furthermore, the injectability of CPC(D) may have potential for procedures involving defects with limited accessibility or narrow cavities, when there is a need for precise placement of the paste, and when using minimally invasive surgical techniques.

Biocompatible Materials↗

PubMed1988

Identification and functional reconstitution of phosphate: sugar phosphate antiport of Staphylococcus aureus.

Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗

PubMed1990

Resolution-enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ions in the early deposits of a solid phase of calcium-phosphate in bone and enamel, and their evolution with age. I: Investigations in the upsilon 4 PO4 domain.

In order to investigate the possible existence in biological and poorly crystalline synthetic apatites of local atomic organizations different from that of apatite, resolution-enhanced, Fourier transform infrared spectroscopy studies were carried out on chicken bone, pig enamel, and poorly crystalline synthetic apatites containing carbonate and HPO4(2-) groups. The spectra obtained were compared to those of synthetic well crystallized apatites (stoichiometric hydroxyapatite, HPO4(2-)-containing apatite, type B carbonate apatite) and nonapatitic calcium phosphates which have been suggested as precursors of the apatitic phase [octacalcium phosphate (OCP), brushite, and beta tricalcium phosphate and whitlockite]. The spectra of bone and enamel, as well as poorly crystalline, synthetic apatite in the upsilon 4 PO4 domain, exhibit, in addition to the three apatitic bands, three absorption bands that were shown to be independent of the organic matrix. Two low-wave number bands at 520-530 and 540-550 cm-1 are assigned to HPO4(2-). Reference to known calcium phosphates shows that bands in this domain also exist in HPO4(2-)-containing apatite, brushite, and OCP. However, the lack of specific absorption bands prevents a clear identification of these HPO4(2-) environments. The third absorption band (610-615 cm-1) is not related to HPO4(2-) or OH- ions. It appears to be due to a labile PO4(3-) environment which could not be identified with any phosphate environment existing in our reference samples, and thus seems specific of poorly crystalline apatites. Correlation of the variations in band intensities show that 610-615 cm-1 band is related to an absorption band at 560 cm-1 superimposed on an apatite band. All the nonapatitic phosphate environments were shown to decrease during aging of enamel, bone, and synthetic apatites. Moreover, EDTA etching show that the labile PO4(3-) environment exhibited a heterogeneous distribution in the insoluble precipitate.

Aging↗

PubMed1988

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport.

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate (p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH2 groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.

Animals↗

PubMed2004

NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59-79) by stabilized amorphous calcium fluoride phosphate nanocomplexes.

The repair of early tooth enamel lesions has been recently demonstrated by tryptic phosphopeptides derived from milk caseins that associate with amorphous calcium phosphate (ACP) forming stable complexes. These casein phosphopeptides (CPP), containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, form calcium phosphate delivery vehicles that retard enamel demineralization and promote remineralization. Recently, we have shown that these peptides also stabilize calcium fluoride phosphate as soluble complexes. These complexes designated CPP-ACFP, have the potential to provide superior clinical efficacy in preventing dental caries and treating and repairing early stages of disease. In an approach to determine the ultrastructure of the casein phosphopeptide-amorphous calcium fluoride phosphate complexes, we have studied the structure of the predominant peptide alpha(S1)-CN(59-79) bound to ACFP using nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. The alpha(S1)-CN(59-79) peptide stabilized calcium fluoride phosphate as amorphous nanocomplexes with a hydrodynamic radius of 2.12+/-0.26 nm. The nanocomplexes exhibited stoichiometry of one peptide to 15 calcium, nine phosphate and three fluoride ions. Sequence-specific resonance assignments were determined for the peptide alpha(S1)-CN(59-79) complexed to the ACFP. The secondary structure of the peptide alpha(S1)-CN(59-79) was characterized by sequential (i, i+1), medium-range (i, i+2) nOes and H alpha chemical shifts. The spectral data were compared with that of the peptide alpha(S1)-CN(59-79) bound to calcium ions, revealing that the structurally significant secondary NH and alpha-chemical shifts were similar.

Amino Acid Motifs↗

PubMed1984

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions.

A higher plant homologue to the high-affinity phosphate transporter gene of yeast (Saccharomyces cerevisiae) PHO84 was isolated from Arabidopsis thaliana. Expression of the Arabidopsis gene PHT1 at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H+ cotransport mechanism of phosphate uptake, and had a Km of 3.1 microM which is within limits characteristic of high-affinity transport mechanisms. These results indicate that PHT1 encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.

Arabidopsis↗