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PubMed1997

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase.

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.

Aldehyde Oxidoreductases↗
PubMed1992

Effects of insulin and glucose on renal phosphate reabsorption: interactions with dietary phosphate.

Both insulin deficiency and glycosuria are known to inhibit the tubular reabsorption of phosphate. This inhibition has previously been evaluated either in the fasted state or on a normal phosphate diet. The goal of this study was to evaluate how dietary phosphate depletion affected the relative effects of insulin deficiency and glycosuria on the tubular reabsorption of phosphate. Rats were maintained on either a low- (0.03%) or normal (0.8%) phosphate diet. After 5 days, one half of the animals in each group received streptozotocin to induce short-term insulin deficiency, whereas the other half received vehicle alone. Two days later, sodium-dependent phosphate uptake by renal brush border membrane vesicles (BBMV) was evaluated in each of the four experimental groups. The effect of glucose on phosphate uptake was determined by the addition of varying concentrations of glucose (between 0 and 32 mmol/L) to the extravesicular transport fluid. BBMV phosphate uptake was about threefold higher in the nondiabetic rats fed a low-phosphate diet as compared with the nondiabetic animals maintained on a normal phosphate diet. In rats maintained on a low-phosphate diet, streptozotocin treatment prevented the increase in BBMV phosphate transport; in contrast, in animals fed a normal phosphate diet, streptozotocin treatment had no effect on BBMV phosphate transport. Extravesicular glucose significantly inhibited phosphate transport in a dose-related manner, regardless of dietary phosphate or insulin status. Because fasting mimics the catabolic state associated with insulin deficiency, BBMV phosphate transport was also measured in rats fasted for 48 h after the administration of streptozotocin or vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Adaptive regulation of Na(+)-dependent phosphate transport in the bovine renal epithelial cell line NBL-1. Identification of the phosphate transporter as a 55-kDa glycoprotein.

1. Na(+)-dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein. 2. The bovine kidney epithelial cell line NBL-1 expresses a Na(+)-dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low-phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34 +/- 0.18 and 2.95 +/- 0.1 nmol.mg-1.3 min-1 and Km values of 17.1 +/- 3.0 and 36.3 +/- 7.14 microM for phosphate transport, respectively. Thus there is a significant (P less than 0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low-phosphate medium. 3. Adaptation of phosphate transport in NBL-1 cells to low-phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein. 4. Cells were adapted to low-phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high-phosphate medium. 5. On the basis of these observations, it is proposed that the phosphate transporter of NBL-1 cells is a 55-kDa glycoprotein. Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na(+)-dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1-3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.

Adaptation, Physiological↗
PubMed1983

Regulation of plasma 1,25-(OH)2-D3 by phosphate: evidence against a role for total or acid-soluble renal phosphate content.

In order to evaluate a possible role for tissue phosphate or phosphorylated compounds in mediating the increase in plasma 1,25-(OH)2-D3 levels during dietary phosphate deprivation, measurements of total and acid-soluble renal cortical phosphate content have been made in both intact and hypophysectomized (hypox) rats eating a normal diet and also after four days of dietary phosphate deprivation. Similar measurements were also made in phosphate-deprived hypophysectomized rats replaced with growth hormone (GH). Total and acid-soluble renal cortical phosphate content averaged 81 +/- 8 mumol/g and 4.1 +/- 0.6 mumol/g, respectively, in intact rats eating the normal diet and were not significantly altered after phosphate deprivation despite a fall in plasma phosphate of about 40% and a fourfold increase in plasma 1,25-(OH)2-D3 levels. Total and acid-soluble renal cortical phosphate content levels were higher in hypox rats, averaging 92 +/- 8 mumol/g and 4.9 +/- 0.7 mumol/g, respectively, but also did not change after phosphate deprivation. Replacement of phosphate-deprived hypox rats with GH resulted in a further fall in plasma phosphate and a significant increase in plasma 1,25-(OH)2-D3 levels, but there was no change in either total or acid-soluble renal cortical phosphate content. The distribution of organophosphorus compounds in the acid-soluble phosphate fraction in these experiments was also evaluated using 31P NMR spectrometry. Although there appeared to be an increase in the total concentration of organophosphorus compounds after phosphate deprivation, this effect was not altered by hypophysectomy or by replacement of phosphate-deprived hypox rats with GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

How dietary phosphate, renal failure and calcitriol administration affect the serum calcium-phosphate relationship in the rat.

BACKGROUND: The effect of hyperphosphataemia on serum calcium regulation in renal failure has not been well studied in a setting in which hypercalcaemia is not parathyroid hormone (PTH) mediated. In azotemic rats with a normal serum calcium concentration, an increased dietary phosphate burden affects serum calcium regulation because of its effects on skeletal resistance to PTH, calcitriol production, and possibly intestinal calcium absorption. Our goal was to determine how hyperphosphataemia affected the development of hypercalcaemia during calcitriol-induced hypercalcaemia and PTH suppression in azotemic rats with established hyperparathyroidism. METHODS: Rats underwent a two-stage 5/6 nephrectomy or corresponding sham operations. After surgery, rats were given a high phosphate diet (P 1.2%) for 4 weeks to exacerbate hyperparathyroidism and were then changed to a normal diet (P 0.6%) for 2 weeks to normalize serum calcium values in the azotemic rats. At week 7, rats were divided into five groups and sacrificed after receiving three intraperitoneal doses of calcitriol (CTR, 500 pmol/100 g) or vehicle at 24 h intervals. The five groups and dietary phosphate content were: group 1, normal renal function (NRF)+0.6% P+vehicle; group 2, NRF+0.6% P+CTR; group 3, renal failure (RF)+0.6% P+vehicle; group 4, RF+1.2% P+CTR; and group 5, RF+0.6% P+CTR. Both the 0.6% and 1.2% phosphate diets contained 0.6% calcium. RESULTS: Serum creatinine values were increased (P<0.05) in 5/6 nephrectomized rats (groups 3, 4 and 5), as were serum calcium values (P<0.05) in CTR-treated rats (groups 2, 4 and 5) and serum phosphate values (P<0.05) in CTR-treated azotemic rats (groups 4 and 5). Serum PTH values were suppressed (P<0.05) in CTR-treated hypercalcemic rats (groups 2, 4 and 5) and increased (P<0.05) in azotemic rats not given CTR (group 3). In the azotemic groups (groups 3, 4 and 5), an inverse correlation was present between serum calcium and phosphate in each group, despite a wide variation in serum calcium values. The slope of the inverse relationship between serum calcium and phosphate was steeper in CTR-treated azotemic rats on a 1.2% phosphate (group 4) diet than on a 0.6% phosphate (group 5) diet (P=0.02). Thus, for a similar increase in the serum phosphate concentration, serum calcium values decreased more in group 4 than in group 5. The independent effect of dietary phosphate on serum calcium values was also confirmed by analysis of covariance. Finally, the serum calcium concentration was shown to be greater for any given serum phosphate value in CTR-treated rats than in those not on CTR. CONCLUSIONS: In azotemic rats with calcitriol-induced hypercalcaemia, the magnitude of hypercalcaemia is affected by: (i) the serum phosphate concentration; and (ii) differences in dietary phosphate content. Calcitriol administration also acts to shift upwards the relationship between serum calcium and phosphate so that a higher serum calcium concentration can be maintained for any given serum phosphate value.

Animals↗
PubMed1978

Effect of phosphate deprivation on renal phosphate transport in the dog.

In order to examine the role of dietary intake of phosphate in regulating renal phosphate transport, clearance and micropuncture studies were performed in 25 dogs with various duration of phosphate deprivation induced by low phosphate diet and aluminum hydroxide gel. In phosphate deprivation of 17-41 days duration, the phosphaturic response to extracellular volume expansion (ECVE) was blunted in the intact group and was virtually abolished in the acutely thyroparathyroidectomized (TPTX) group. With longer phosphate deprivation of 53-110 days, no phosphaturia occurred after ECVE or administration of parathyroid hormone (PTH), even with intact parathyroids. These alterations in phosphaturic responses did not correlate with the plasma phosphate but rather with the duration of phosphate deprivation. Fractional proximal tubule phosphate reabsorption was enhanced in phosphate deprivation and the proximal tubule fluid-to-ultrafilterable phosphate ratio appeared to be a good index for the degree of phosphate deprivation. Acute infusion of phosphate to raise plasma phosphate slightly above normal did not completely restore the responsiveness to ECVE. It is concluded that the adaptive response to phosphate deprivation occurs in both proximal and distal nephron segments and that factors other than plasma phosphate are primarily responsible for such an adaptation.

Animals↗
PubMed1981

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves.

Aldose-6-phosphate reductase (alditol 6-phosphate:NADP 1-oxidoreductase) was isolated and characterized from mature apple leaves (Malus domestica cv. Starkrimson). The enzyme was purified 79-fold. The enzyme catalyzed the following reversible reaction: d-glucose 6-phosphate + NADPH + H(+) right arrow over left arrow d-sorbitol 6-phosphate + NADP(+). No activity was detected when NAD(+) was substituted for NADP(+) or when NADH was substituted for NADPH. The enzyme reduced d-galactose 6-phosphate at a higher rate than d-glucose 6-phosphate. d-Mannose 6-phosphate and 2-deoxy-d-glucose 6-phosphate were reduced at low rates. d-Glucose 1-phosphate, d-fructose 6-phosphate, d-ribose 5-phosphate, d-glucose, and sorbitol did not serve as substrates. The pH optimum for both d-sorbitol 6-phosphate oxidation and d-glucose 6-phosphate reduction was 9.5. The K(m) values for d-sorbitol 6-phosphate oxidation and d-glucose 6-phosphate reduction were 3.9 and 20 millimolar, respectively. AgNO(3) (0.1 millimolar) and p-chloromercuribenzoate (1.0 millimolar) completely inhibited the enzyme.Aldose-6-phosphate reductase activity was also detected in mature leaves from Golden Delicious and Antonovka apples (Malus domestica), Conference and Bartlett pears (Pyrus communis), Redhaven peach (Prunus persica), and Perfection apricot (Prunus armeniaca). This suggests that the enzyme has a wide distribution and plays an important role in sorbitol synthesis.

Journal Article↗
PubMed1992

Derivatives of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate that mimic the actions of adenosine 3',5'-phosphate (cAMP) and guanosine 3',5'-phosphate (cGMP).

A series of new analogues of 1-beta-D-ribofuranosylbenzimidazole 3',5'-phosphate (cBIMP) has been designed according to the properties predicted by the MNDO method, and synthesised from substituted benzimidazoles. Dipole vectors and HOMO and LUMO energies for each benzimidazole base were calculated by the MNDO method and the lipophilicities of the cBIMP derivatives were determined. In general, the cBIMP derivatives activate cAMP-dependent protein kinases I and II and preferentially bind to site B, especially for the type II kinase, with 2-trifluoromethyl-cBIMP and 5,6-difluoro-cBIMP exhibiting the highest site selectivity. Each cBIMP derivative can stimulate cGMP-stimulated cyclic phosphodiesterase (cGS-PDE), with 5,6-dimethyl-cBIMP being as potent as cGMP, and also inhibit cGMP-inhibited phosphodiesterase (cGI-PDE). Only the 2-trifluoromethyl-cBIMP and the Rp-phosphorothioates (cBIMPS) (equatorial P = S) were resistant to hydrolysis by cPDE. The Sp-phosphorothioates were hydrolysed slowly, if at all. In addition to exhibiting a high lipophilicity, the most active compounds for the induction of apoptosis and inhibition of proliferation were also resistant to cPDE (Sp-5,6-dichloro-cBIMPS) and/or were potent activators of cAMP-dependent protein kinase (5,6-dichloro-cBIMP).

Animals↗
PubMed1995

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.

Journal Article↗
PubMed2003

Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae.

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.

Adenosine Triphosphate↗
PubMed1999

Metabolic aspects of phosphate replacement therapy for hypophosphatemia after renal transplantation: impact on muscular phosphate content, mineral metabolism, and acid/base homeostasis.

Hypophosphatemia caused by renal phosphate loss occurs frequently after kidney transplantation. In assumption of systemic phosphorus depletion, the presumed deficit commonly is replaced by oral phosphate supplements. However, such treatment is debatable, because intracellular phosphorus stores have not been assessed in this setting and may not be accurately reflected by serum phosphate concentrations. Moreover, disturbances in mineral metabolism from chronic renal failure, such as hypocalcemia and hyperparathyroidism, may be prolonged with oral phosphate supplements. Conversely, a neutral phosphate salt might improve renal acid excretion and systemic acid/base homeostasis for its properties as a urinary buffer and a poorly reabsorbable anion. Twenty-eight patients with mild early posttransplantation hypophosphatemia (0.3-0.75 mmol/L) were randomly assigned to receive either neutral sodium phosphate (Na(2)HPO(4)) or sodium chloride (NaCl) for 12 weeks and examined with regard to (1) correction of serum phosphate concentration and urinary phosphate handling; (2) muscular phosphate content; (3) serum calcium and parathyroid hormone (PTH); and, (4) renal acid handling and systemic acid/base homeostasis. Mean serum phosphate concentrations were similar and normal in both groups after 12 weeks of treatment; however, more patients in the NaCl group remained hypophosphatemic (93% versus 67%). Total muscular phosphorus content did not correlate with serum phosphate concentrations and was 25% below normophosphatemic controls but was completely restored after 12 weeks with and without phosphate supplementation. However, the percentage of the energy-rich phosphorus compound adenosine triphosphate (ATP) was significantly higher in the Na(2)HPO(4) group, as was the relative content of phosphodiesters. Also, compensated metabolic acidosis (hypobicarbonatemia with respiratory stimulation) was detected in most patients, which was significantly improved by neutral phosphate supplements through increased urinary titratable acidity. These benefits of added phosphate intake were not associated with any adverse effects on serum calcium and PTH concentrations. In conclusion, oral supplementation with a neutral phosphate salt effectively corrects posttransplantation hypophosphatemia, increases muscular ATP and phosphodiester content without affecting mineral metabolism, and improves renal acid excretion and systemic acid/base status.

Acid-Base Equilibrium↗
PubMed2000

Relationship between nitrite reduction and active phosphate uptake in the phosphate-accumulating denitrifier Pseudomonas sp. strain JR 12.

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H(+)-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.

Biological Transport, Active↗
PubMedDate not supplied

Phosphate balance and distribution during total parenteral nutrition: effect of calcium and phosphate additives.

Hypophosphatemia is occasionally observed during total parenteral nutrition (TPN). The phenomenon was recognized since the introduction of TPN and was attributed to preexisting phosphate deficits and inadequate phosphate supplements. Because of the close relationship between phosphate and calcium metabolisms, we speculated that calcium additives may also influence phosphate balance and distribution during TPN. We tested this hypothesis in previously fasted animals receiving TPN with variable amounts of calcium and maintenance or no phosphate. Fasting resulted in considerable losses of phosphate in the urine. Refeeding (with TPN) after fasting produced hypophosphatemia but only in animals receiving calcium additives and no maintenance phosphate in the solution. Addition of moderate or large amounts of calcium decreased phosphate in the muscle in groups not receiving maintenance phosphate. There were no significant changes in bone phosphate. Increasing calcium intake was accompanied by significant and progressive reductions in urine phosphate in animals receiving maintenance phosphate, thus increasing net phosphate retention in those groups. In other respects, refeeding with TPN after fasting displayed features compatible with those of the phosphate depletion syndrome, including hypophosphatemia, hypophosphaturia, hypercalcemia, and hypercalciuria. The magnitude of change in these parameters seemed to be dependent upon the amount of calcium added and the availability of phosphate in the solution.

Animals↗
PubMed1992

Renal phosphate transport and vitamin D metabolism in X-linked hypophosphatemic Gy mice: responses to phosphate deprivation.

Two closely linked, nonallelic genes, Gy and Hyp, result in X-linked hypophosphatemia in mice. The present studies in Gy mice were undertaken to determine whether renal brush-border membrane Na(+)-phosphate cotransport kinetics and adaptive responses of renal phosphate transport and vitamin D metabolism to phosphate deprivation are comparable in the two mutant strains. Transport studies in purified brush-border membrane vesicles over a phosphate concentration range of 10-500 microM demonstrated that the apparent maximum velocity of the high affinity transport system is significantly decreased in Gy mice (420 +/- 110 vs. 710 +/- 100 pmol/mg protein.6 sec, Gy vs. normal; mean +/- SE; P less than 0.05), whereas the affinity of the cotransporter for phosphate is unchanged (apparent Km, 25 +/- 3 vs. 27 +/- 2 microM; NS). Feeding a low phosphate diet results in a significant fall in plasma phosphate and an increase in brush-border membrane Na(+)-phosphate cotransport in both normal (568 +/- 40 to 1416 +/- 139 pmol/mg protein.6 sec; P less than 0.01) and Gy mice (407 +/- 27 to 1236 +/- 132 pmol/mg protein.6 sec; P less than 0.01). While the low phosphate diet elicited a rise in plasma 1,25-dihydroxyvitamin D in normal mice (51 +/- 12 to 158 +/- 12 pM; P less than 0.01), a fall in plasma hormone levels was evident in phosphate-deprived Gy mice (90 +/- 22 to 23 +/- 11 pM; P less than 0.01). Phosphate deprivation decreased 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase), the first enzyme in the renal vitamin D catabolic pathway, in normal mice (117 +/- 21 to 69 +/- 8 fmol/mg protein.min), but increased enzyme activity in Gy mice (172 +/- 14 to 240 +/- 18 fmol/mg protein.min; P less than 0.05). Moreover, under both dietary conditions, 24-hydroxylase activity was significantly elevated in Gy mice. The present results demonstrate that hypophosphatemia in Gy mice can be attributed to a decrease in the maximum velocity of the high affinity Na(+)-phosphate cotransport process in renal brush-border membranes. Our results also show that while renal brush-border membrane phosphate transport is appropriately modulated by phosphate in Gy mice, phosphate regulation of vitamin D metabolism is apparently impaired in the mutant strain. The present findings provide evidence for phenotypic similarities between murine Gy and Hyp mutations.

Animals↗
PubMed1983

Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum.

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

Animals↗