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Advances in experimental medicine and biology 02/1986; 208:543-8. · 1.09 Impact Factor
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ABSTRACT: Human placental brush border membranes were obtained from full-term normal vaginal deliveries. A sodium dependent transport system for phosphate was identified in these membranes. In the presence of a sodium gradient, at pH 7, T degree 35 degrees C, the apparent Km and Vmax were 86.5 +/- 24 microM and 1.9 +/- 0.18 nmol mg-1 30 s-1. The apparent Km was pH dependent, increasing to 500 +/- 55 microM when the pH was elevated to 8.5. At low substrate concentration, the optimal uptake was obtained at pH 7.0. Both monovalent and divalent forms of phosphate were transported so that alteration in the transport by pH is believed to be the result of a direct action on the transport system. Finally, sodium levels in the incubation medium dramatically influenced phosphate uptake by increasing the maximal velocity without modifying the affinity for the substrate. This active transport through the placental brush border membranes is believed to be at the origin of the maternal-fetal gradient of phosphate observed during pregnancy.
Pediatric Research 12/1985; 19(11):1179-82. · 2.70 Impact Factor
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ABSTRACT: It is known that parathyroidectomy, administration of parathyroid hormone (PTH), and dietary phosphate depletion or excess result in variations in phosphaturia and in phosphate transport through brush border membrane vesicles isolated from the kidneys of various animals. Parathyroid hormone has been shown to ultimately phosphorylate some brush border membrane proteins and it has been postulated that the resulting phosphaturia is related to this phosphorylation. However, it is not known whether the regulation of phosphate transport by the diet is affected through similar pathways. Our experiments were designed to study the phosphorylation of brush border membrane with [gamma-32P]ATP using the intrinsic protein kinase of the membranes. Five groups of rats were used: normal, phosphate loaded, phosphate depleted, and thyroparathyroidectomized and acutely loaded with parathyroid hormone. In each series of animals, the proteins whose phosphorylation was cAMP dependent were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their phosphorylation with various concentrations of ATP, in the presence or absence of cAMP in the incubation medium, was quantified. In the normal rat, 17 proteins were phosphorylated, the phosphorylation of two of them (Mr, 71 000 and 84 000) being cAMP dependent. Maximal response to cAMP for these two proteins was obtained with 10 microM cAMP. The peaks of phosphorylation were observed at pH 7 for protein 71 000 and pH 10 for protein 84 000. When brush border membranes from normal rats were incubated with 10-100 microM ATP, cAMP-dependent phosphorylation increased to reach a maximal phosphorylation of 4.44 +/- 0.90 pmol/mg protein for protein 71 000 and 1.32 +/- 0.15 pmol/mg protein for protein 84 000.(ABSTRACT TRUNCATED AT 250 WORDS)
Canadian Journal of Physiology and Pharmacology 12/1985; 63(11):1362-9. · 1.95 Impact Factor
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ABSTRACT: Phosphate uptake by brush-border membrane (BBM) vesicles prepared from hypophosphatemic mice (Hyp) is reduced by half relative to BBM vesicles from normal mice. To investigate this abnormality, we studied the protein composition of BBM, their capacity to bind inorganic phosphate, and their protein kinase activity with and without the addition of exogenous cAMP, in normal and Hyp mice. Gradient polyacrylamide gel electrophoresis of BBM proteins showed 27 bands which were identical in normal and Hyp mice. Incubation of the membranes with ortho[32P]phosphate at 0 degrees C revealed a phosphate binding protein with an apparent molecular weight (Mr) of 79000, which has been previously identified in rats as the monomer of alkaline phosphatase. In normal mice, the Scatchard plot of phosphate binding was not linear, suggesting heterogeneity of the binding sites with two major components. At high substrate concentrations, the affinity (K) was 1.42 mM and maximal binding (Bmax) was 83 pmol/mg protein. At low substrate concentrations, these values were 0.07 mM and 10.9 pmol/mg, respectively. In Hyp mice BBM, only one binding system was found with K and Bmax values of 0.38 mM and 53.8 pmol/mg. Incubation of the membranes with 25 microM[gamma-32P]ATP resulted in the phosphorylation of 11 proteins. The major band (Mr: 79000) corresponded to the inorganic phosphate binding protein, i.e., to the alkaline-phosphatase monomer. The 11 proteins showed maximal phosphorylation at pH 10. The protein of 79000 Mr showed a second peak of phosphorylation at pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)
Canadian Journal of Physiology and Pharmacology 12/1984; 62(11):1394-400. · 1.95 Impact Factor
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ABSTRACT: The effect of phenylglyoxylation on brush-border-membrane functions was studied with membrane vesicles from rat kidney cortex. Na+-gradient-dependent uptake of phosphate, glucose and alanine was inhibited by 65, 88 and 70% by pre-incubation of vesicles with 50 mM-phenylglyoxal for 2 min. The inhibition showed a dependency for alkaline pH. Borate co-operativity in butanedione inactivation was used to prove that inhibition was caused by arginine modification. Intravesicular volumes, alkaline phosphatase, aminopeptidase M and Na+-H+ exchange were not affected by phenylglyoxal treatment. Inhibition of phosphate uptake was studied in more detail and showed that the chemical modification introduced by phenylglyoxal inhibited the overshoot of phosphate uptake caused by the Na+ gradient, and decreased the apparent maximal velocity of the phosphate-transport system in its interaction with Na+. Phosphate uptake measured in the absence of Na+ was not affected by phenylglyoxal. Shunting of the transmembrane electrical potential with K+ and valinomycin had no effect on phenylglyoxal inhibition, proving that the alteration of transmembrane electrical potential could not be responsible for this effect. Phenylglyoxal had no ionophoric effect on the Na+ gradients studied (1-100 mM). Na+ efflux was also unaffected by phenylglyoxal treatment. Na+, harmaline and amiloride were ineffective in protecting against phenylglyoxal inhibition, suggesting that the site modified was not an Na+-binding site. These results indicate the involvement of highly reactive arginine residues in phosphate, glucose and alanine uptake.
Biochemical Journal 12/1984; 223(3):793-802. · 4.90 Impact Factor
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ABSTRACT: Na+ uptake was measured in brush border membrane isolated from rat kidney cortex. In the presence of 100 mM NaCl gradient, Na+ equilibrated across the membranes within 30 minutes, but uptake was linear only for the first 10 seconds. Total uptake could be analysed in terms of a diffusion component and a saturable component (Kt = 5 mM, Jmax = 0.62 pmol/microgram prot/s). Uptake at 1 mM NaCl was found to be inhibited at 60% by amiloride; stimulation of uptake by pH gradient (inside greater than outside) supports the functioning of a Na+-H+ antiport. Uptake at 100 mM NaCl was insensitive to amiloride in the absence of pH gradient. The influence of anions on Na+ uptake was in agreement with known permeabilities for generation of electrical potentials (Thiocyanate greater than chloride greater than gluconate) and was observed at 100 mM Na+ but not at 40 microM. These results suggest that rat renal brush border membrane vesicles are leaky towards Na+ and present a permeability coefficient for Na+ much higher than what is expected from the in vivo conditions.
Biochemical and Biophysical Research Communications 10/1984; 123(2):562-8. · 2.48 Impact Factor
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ABSTRACT: Dietary phosphate restriction and acute parathyroidectomy in rat are known to be associated with a selective increase in phosphate uptake by renal cortical brush border membranes (BBM). Conversely, phosphate loading and parathyroid hormone (PTH) administration result in a decrease of this uptake. In the present study, we investigated whether the response of the membrane to these various stimuli implies similar or different modifications of the kinetic properties of this membrane, whether these modifications affect one or both of the two systems of phosphate transport previously described, whether both superficial and deep nephron populations are involved, and whether the two stimuli: dietary phosphate, and parathyroid activity, are additive or not. Kinetic studies of phosphate (PO4) uptake by BBM vesicles were performed in seven groups of rats: control (N), acutely thyroparathyroidectomized (TPTX), PTH loaded (PTH), phosphate loaded (P+), phosphate depleted (P-), phosphate depleted with acute thyroparathyroidectomy (P-TPTX), and phosphate depleted-PTH loaded (P-PTH). In each of these experimental conditions, superficial and deep nephrons were investigated. Results indicated that 1. BBM from deep nephrons present a greater capacity for PO4 transport than those from superficial nephrons; 2. Whereas a dual system of PO4 uptake is observed in superficial BBM, deep BBM present only one single system; 3. Phosphate in the diet influences PO4 uptake by BBM to a greater extent in the deep than in the superficial nephrons; 4. PTH status on the contrary, equally influences both populations; 5. TPTX does not significantly enhance PO4 uptake in phosphate depleted rats; however, PTH loading curtails this uptake; 6.(ABSTRACT TRUNCATED AT 250 WORDS)
Pflügers Archiv - European Journal of Physiology 05/1984; 400(4):356-62. · 4.46 Impact Factor
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ABSTRACT: The kinetics of sodium gradient dependent phosphate uptake by the renal brush border membrane vesicles of the rat have ben studied under various conditions of temperature and pH. From 7 to 30 degrees C the Lineweaver-Burk plots are linear, and the apparent Km progressively increases from 54 to 91 microM. Above 30 degrees C, the apparent Km continues to increase to reach 135 microM at 40 degrees C, but a break is observed in the Lineweaver-Burk plots at the substrate concentration of 300 microM. The existence of this break, confirmed by the Eadie-Hofstee plot supports the hypothesis of a dual mechanism of phosphate transport, one for low concentrations of substrate with a Km of 100 microM and the other for high concentrations with a Km of approximately 240 microM. When the two components of the Eadie-Hofstee plot are analyzed according to a nonlinear regression program, these two values of Km become 70 microM and 1.18 mM, respectively. The Vmax continuously increases with temperature. However, the Arrhenius plot (In Vmax vs. 1/TK) shows an abrupt discontinuity at 23 degrees C. pH experiments were performed at 35 degrees C. In the absence of a proton gradient, increasing the pH from 6.5 to 7.5 and 8.5 decreases the apparent Km from 341 to 167 and 94 microM, respectively. When only the divalent form of phosphate is considered as the substrate, the apparent Km does not vary anymore with the pH and remains around the mean value of 105 microM. The uniformity of the apparent Km for the total phosphate uptake, when only the divalent phosphate is considered as being the substrate, suggests that this divalent form is the only one which is transported. Whatever the substrate considered, total phosphate or divalent phosphate, the highest Vmax is obtained at pH 7.5 which probably approximates the optimum pH inside the vesicles for the phosphate uptake.
Canadian Journal of Physiology and Pharmacology 03/1984; 62(2):229-34. · 1.95 Impact Factor
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ABSTRACT: Brush border membranes (BBM) have been prepared from fresh samples of normal human kidney cortex and compared to that from mouse, rat, and rabbit. Human BBM presents a sodium dodecyl sulfate gel electrophoresis protein pattern similar to that of the animal species with 22 proteins having the same molecular weight (MW). Incubation with inorganic 32P reveals a phosphate-binding protein (MW 78,000) common to the animal species. However, the binding capacity is lower in man: 4.3 +/- 2.2 pmol Pi/mg protein compared to 9.9 +/- 2.1, 29.7 +/- 4.3, and 31.1 +/- 5.2 in rabbit, mouse, and rat, respectively. The MW of the binding protein corresponds to that of the monomer of alkaline phosphatase. Alkaline phosphatase activity follows the same increasing order in the four species. The Na gradient-dependent Pi uptake by human BBM vesicles is low: Vmax is 0.90 +/- 0.05 nmol/mg/20 s compared to 1.3 +/- 0.1, 1.5 +/- 0.2, and 5.2 +/- 0.2 in rabbit, mouse, and rat, respectively. However, the Km values are within the same range for the four species.
Renal physiology 02/1984; 7(2):65-71.
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Progress in clinical and biological research 02/1984; 168:319-24.
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Advances in experimental medicine and biology 02/1984; 178:29-32. · 1.09 Impact Factor
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Advances in experimental medicine and biology 02/1984; 178:37-40. · 1.09 Impact Factor
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ABSTRACT: Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by gamma-32P ATP at 0 degree C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1 +/- 5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein. Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparent Km: one at 40 microM for low and one at 390 microM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.
Pflügers Archiv - European Journal of Physiology 09/1983; 398(3):227-32. · 4.46 Impact Factor
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ABSTRACT: Brush border membranes (BBM) have been prepared from fresh samples of normal human kidney cortex and compared to that from mouse, rat, and rabbit. Human BBM presents a sodium dodecyl sulfate gel electrophoresis protein pattern similar to that of the animal species with 22 proteins having the same molecular weight (MW). Incubation with inorganic 32P reveals a phosphate-binding protein (MW 78,000) common to the animal species. However, the binding capacity is lower in man: 4.3 ± 2.2 pmol Pi/mg protein compared to 9.9 ± 2.1, 29.7 ± 4.3, and 31.1 + 5.2 in rabbit, mouse, and rat, respectively. The MW of the binding protein corresponds to that of the monomer of alkaline phosphatase. Alkaline phosphatase activity follows the same increasing order in the four species. The Na gradient-dependent Pi uptake by human BBM vesicles is low: Vmax is 0.90 + 0.05 nmol/mg/ 20 s compared to 1.3 + 0.1, 1.5 + 0.2, and 5.2 + 0.2 in rabbit, mouse, and rat, respectively. However, the Km values are within the same range for the four species.
Kidney and Blood Pressure Research 08/1970; 7(2):65-71. · 1.46 Impact Factor
