Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.     

Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.

We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.     

Presence of a MgATP/ADP-dependent pp50 phosphatase in bovine brain coated vesicles

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments in which they must be able to undergo repeated membrane fusion and fission. We previously described the presence of cyclic nucleotide- and Ca2+-independent protein kinase activity in bovine brain coated vesicles which specifically phosphorylated a unique Mr = 50,000 coated vesicle integral protein (pp50) on a threonine residue. We describe now the presence in bovine brain coated vesicles of the antagonistic enzymatic activity which dephosphorylates pp50. This phosphoprotein phosphatase occurs under two interconvertible active and inactive forms. The activation process needs the simultaneous presence of Mg2+ and ATP or ADP. Unchelated ATP, but not unchelated ADP, inactivates the pp50 phosphatase. The latter is associated with the vesicular core. MgADP activation of the pp50 phosphatase implicates a different mechanism which does not need a phosphorylated intermediate. Thus, the pp50 phosphatase might belong to a new phosphatase type distinct from the four other classes of well known protein phosphatases.     

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.     

Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.     

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

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Receptor-mediated pinocytosis of IgG and immune complex in rat peritoneal macrophages: an electron microscopic study

The uptake mechanism of homologous IgG and immune complex, and the participation of coated vesicles in this process were studied in rat peritoneal macrophages. Peroxidase-antiperoxidase (PAP) immune complex produced in rat, and purified rat IgG adsorbed to gold particles (IgG-Au) were used as ligands. Freshly collected peritoneal macrophages were preincubated with the ligands at 4 degrees C, washed, warmed up to 37 degrees C, maintained in a serum-free culture medium for 5 sec to 30 min and subsequently fixed for electron microscopy. In the IgG-Au experiments, acid phosphatase reaction was also applied to identify lysosomes, and ruthenium red to trace membranes exposed to the extracellular space. At the end of the preincubation period PAP and IgG were found randomly distributed on the external surface of the plasma membrane. After warming up the cells to 37 degrees C, the ligands bound to the plasma membrane showed a tendency to move towards deep labyrinthic invaginations of the cell surface from where they were internalized via coated pits and coated vesicles. In the initial period, these structures seemed to be the primary carriers of the ligands. In the period between 5 and 10 min, ligands were concentrated in vacuoles (endosomes) located in the deeper cytoplasm, while after 30 min, they were present in large lysosome-like or multivesicular bodies, which were found to be acid phosphatase positive.     

Amphibian intestinal brush border membranes-I. Isolation from Rana catesbeiana tadpole.

1. Intestinal brush border membrane vesicles have been isolated form Rana catesbeiana tadpole. 2. Electron microscopy of brush border membrane vesicles demonstrates a fairly homogenous preparation of vesicles, some of them still containing electron dense material. 3. The dense vesicles probably comprise both microvillus core and membrane. 4. Negative staining of vesicles reveals the presence of knob-like structures (particles) covering the outer surface of the membrane. 5. The membranous fraction is characterized by a high specific activity of alkaline phosphatase, trehalase, glucoamylase, maltase and gamma-glutamyltranspeptidase.     

Comparison of the intracellular pathways of immunoglobulin-G and low density lipoprotein in cultured human term trophoblast cells

Trophoblast cells were cultured on microporous membrane filters. After incubation at different times with gold-conjugated ligands, the cells were processed for electron microscopy. Gold particles indicating the presence of both IgG and LDL appeared in a time-dependent manner in coated pits and coated vesicles. LDL-gold appeared primarily within lysosomes whereas approximately 50% of the internalized IgG-gold appeared within vesicles (diameters ranging from 35 to 80 nm) near the basal regions of the cell. These vesicles may be the protective mechanism which prevents IgG breakdown during transcytosis across trophoblast cells, thus allowing transport of the intact molecule to the fetus.     

Cotranslational secretion of diphtheria toxin and alkaline phosphatase in vitro: involvement of membrane protein(s).

Crude messenger ribonucleic acid fractions isolated from Corynebacterium diphtheriae and Escherichia coli were translated in an E. coli in vitro protein-synthesizing system and yielded precursors of the secreted proteins diphtheria toxin and alkaline phosphatase, respectively. Addition of inverted E. coli inner membrane vesicles to the system during the initial stages of translation resulted in the intravesicular segregation of mature diphtheria toxin and alkaline phosphatase. Outer membrane vesicles or inner membrane vesicles whose cytoplasmic surfaces had been treated with pronase could not mediate transmembrane transfer of diphtheria toxin or alkaline phosphatase. However, inner membrane vesicles isolated from E. coli spheroplasts which had been treated with pronase and inner membrane vesicles complexed with ribosomes during pronase treatment were functional in transmembrane transfer. At temperatures below the phase transition of E. coli membranes, no intravesicular segregation of alkaline phosphatase or diphtheria toxin was observed. The precursor forms of each protein accumulated free from the vesicles. These results suggest that an inner membrane protein, exposed on the cytoplasmic surface, plays an integral role in secretion.     

Ectoplasmic insertion of a glycosylphosphatidylinositol-anchored protein in glycosphingolipid- and cholesterol-containing phosphatidylcholine vesicles.

Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.     

The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose. Vid vesicles then deliver FBPase to the vacuole for degradation. A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole. For this assay, FBPase was fused with a truncated form of alkaline phosphatase. Under in vivo conditions, FBPase-delta60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation. Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-delta60Pho8p. These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system. Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner. The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles. In contrast, the t-SNARE Vam3p was a necessary vacuolar component. Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.     

Protein kinase(s) in bovine brain coated vesicles

Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165, 54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[gamma S] have different effects on 32P incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.     

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of Immunoglobulin-G (IgG) in the human placenta

Summary Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.     

Rat liver canalicular membrane vesicles. Isolation and topological characterization

Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.     

Teratogenic antibody internalization by rat visceral yolk-sac endoderm in vitro: an ultrastructural colloidal gold tracer study

Previous work from our laboratory has demonstrated that specific rabbit immunoglobulins G (IgG) against a glycoprotein antigen of rat kidney proximal tubule or a cross-reacting visceral yolk-sac endodermal cell antigen will induce abnormal embryonic development when they are injected into pregnant rats during the period of organogenesis. It has been proposed that these antibodies may induce embryopathy by interfering with functions of the visceral yolk-sac placenta, an important organ providing nutrients to the embryo at this stage of development. In order to gain some insight into the underlying pathogenic mechanism(s) in which specific teratogenic IgG may interfere with visceral yolk-sac functions, we examined the uptake of these teratogenic IgG by the visceral yolk-sac endodermal cells at the electron microscopic level. The results demonstrated that teratogenic rabbit IgG specifically localized on the fuzzy coat of the external apical cell membrane of the visceral yolk-sac endoderm at the intermicrovillous region. Within 5 min, the IgG were rapidly internalized via coated pits and micropinocytic vesicles. Within 30 min, an increasing proportion of gold particles appeared within uncoated vesicles or vacuoles of various sizes; most of the gold particles were in close proximity to the inner membranous lining of the vesicles. Similar findings were observed after 1- or 2-hr incubation. After 24- to 48-hr culture, however, the gold particles appeared to have dissociated from the inner surface of the vesicle membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.