Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Schäli, C; Vaughn, Duke A.; Fanestil, Darrell D.

doi:10.1016/0005-2736(84)90307-9

Cited by 18 publications

(7 citation statements)

References 19 publications

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“…Recently it has been shown that enzymatic removal of alkaline phosphatase from the brush-border membrane with phosphatidylinositol-specific phospholipase C does not af fect the Pj uptake into these membrane vesi cles [9], Thus, our results as well as observations of other investigators convincingly show that non-specific alkaline phosphatase hydrolyzes at a high rate phosphate esters present in the mucosal solution, providing the possibility to absorb Pj from organic sources. Moreover, the hydrolytic action of the enzyme increases the concentration of Pj at the transport site and stimulates the phosphate uptake by ab sorptive cells.…”

Section: Discussionsupporting

confidence: 71%

Absence of Alkaline Phosphatase-Linked Inorganic Phosphate Transport in the Chick Jejunum

Valinietse¹,

Bauman²,

Galvanovsky³

1985

Digestion

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The role of non-specific alkaline phosphatase-linked inorganic phosphate (Pi) transport in the chick jejunum was investigated by using the everted gut sac technique. The highest intensity of transport was observed for free P_i. The maximal transport rate was considerably reduced when β-glycerophosphate was used as a single source of phosphorus. These results suggested absence of the alkaline phosphatase-linked transport system for inorganic phosphate in the epithelium of the chick jejunum. Studies of Mg effect on P_i absorption as well as examination of temporal changes in P_i transport rate and β-glycerophosphate hydrolysis rate by intestinal brush border alkaline phosphatase gave further backing to this suggestion.

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Section: Discussionsupporting

confidence: 71%

Absence of Alkaline Phosphatase-Linked Inorganic Phosphate Transport in the Chick Jejunum

Valinietse¹,

Bauman²,

Galvanovsky³

1985

Digestion

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“…Na+-dependent phosphate-transport activities were reported previously (Kinne & Faust, 1977;Lin et al, 1984), but the characteristics of these reconstituted activities were not described in detail, and those studies were apparently not pursued further. The more detailed studies reported by Schali & Fanestil (1985) and by Schali et al (1986) showed a Na+-dependence for phosphate uptake which was small in comparison with the total uptake. Our procedure should therefore prove useful for the purification and identification of the phosphate carrier, a step which is essential for the characterization of this transport activity at the molecular level and for our understanding of the mechanisms involved in the renal re-absorption of phosphate.…”

Section: Discussionmentioning

confidence: 75%

“…Yet a full understanding of the mechanisms involved awaits the identification and isolation of the carrier molecule. The main problem associated with this approach stems from the fact that reconstitution of the fully functional phosphate carrier has proved difficult to achieve, despite several worthy attempts (Kinne & Faust, 1977;Lin et al, 1984;Schali & Fanestil, 1985;Schali et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter

Vachon

Delisle

Laprade

et al. 1991

Biochemical Journal

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A simple and rapid procedure was developed for the reconstitution of Na(+)-dependent phosphate-transport activity from bovine kidney brush-border membranes. The phosphate transporter appears to be particularly sensitive to extraction conditions. To prevent its inactivation, the phosphate carrier was solubilized in a buffer containing its substrates, Na+ and phosphate, CHAPS, dithiothreitol, brush-border membrane lipids and glycerol. The uptake of phosphate by reconstituted vesicles was strongly stimulated by the presence of a transmembrane Na+ gradient. This stimulation was abolished when the Na+ gradient was dissipated by monensin. The affinity of the carrier for phosphate was similar in proteoliposomes and in brush-border membrane vesicles (apparent Kt = 40 microM). The transporter was also stimulated by the presence of a high concentration of phosphate on the trans side of the membrane. The reconstituted transport activity was inhibited by arsenate, a known inhibitor of phosphate transport. However, the bovine phosphate carrier, intact or reconstituted, was much less sensitive to inhibition by phosphonoformic and phosphonoacetic acids than were those of other species studied so far. SDS/PAGE revealed that only a small number of brush-border membrane proteins were incorporated into the proteoliposomes. This reconstitution procedure should be useful for the purification and identification of the carrier protein.

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“…Purification was achieved by extraction of BBM with chloroform/methanol, followed by hydroxyapatite chromatography. Previous attempts to restore Na+-P1 co-transporter activity into artificial liposomes were not successful, and only poor Na+-dependent Pi uptake was measured after reconstitution of the separated protein fractions [12,13]. We believe that generation of functional proteoliposomes was possible for three reasons.…”

Section: Discussionmentioning

confidence: 95%

Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes

Dębiec

Lorenc

Ronco

1992

Biochemical Journal

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A protein with Na+/Pi co-transporter activity has been extracted from rabbit brush-border membranes with chloroform/methanol and purified by hydroxyapatite chromatography. The protein has been incorporated by the dilution method into liposomes formed from different types and ratios of lipids. The greatest reconstitution has been achieved into liposomes prepared from cholesterol (20%), phosphatidylcholine (20%), phosphatidylethanolamine (30%) and phosphatidylserine (30%) (CH/PC/PE/PS). Pi uptake by these proteoliposomes had the following characteristics: (i) the initial rate was markedly greater in the presence of an inwardly directed Na+ gradient (600 pmol/10 s per mg) than with a K+ gradient (65 pmol/10 s per mg); (ii) maximal uptake was increased 8-fold above the equilibrium value ('overshoot') when a Na+ gradient was applied; (iii) Pi was not merely bound to proteoliposomes but was transported intravesicularly; and (iv) Na(+)-dependent Pi uptake was sensitive to the known phosphate transport inhibitors. This first successful attempt of reconstitution of Na+/Pi transport activity into proteoliposomes led us to isolate and characterize physico-chemically the protein responsible. Its isoelectric point was about 5.8, and urea/SDS gel electrophoresis revealed a broad band of molecular mass ranging from 63 to 66 kDa under both reducing and non-reducing conditions. In the native form, the molecular mass analysed by gel filtration was estimated to be 170 +/- 10 kDa, suggesting that the protein is a polymer, probably stabilized by hydrophobic bonds. Endoglycosidase F treatment decreased the molecular mass to approx. 50 kDa. It is postulated that this acidic glycoprotein might represent a subunit of the intact Na+/Pi co-transporter from rabbit kidney brush-border membranes.

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Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Cited by 18 publications

References 19 publications

Absence of Alkaline Phosphatase-Linked Inorganic Phosphate Transport in the Chick Jejunum

Absence of Alkaline Phosphatase-Linked Inorganic Phosphate Transport in the Chick Jejunum

Reconstitution of the renal brush-border membrane sodium/phosphate co-transporter

Reconstitution and characterization of a Na+/Pi co-transporter protein from rabbit kidney brush-border membranes

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