Influence of glioma tumour microenvironment on the transport of ANG1005 via low-density lipoprotein receptor-related protein 1.

BACKGROUND: ANG1005 consists of three molecules of paclitaxel conjugated via ester bonds to the 19-amino-acid peptide Angiopep-2. The new chemical agent has been shown to cross the blood-brain barrier (BBB) by receptor-mediated transcytosis via low-density lipoprotein receptor-related protein 1 (LRP1). The experiments here examined the role of LRP1 in the subsequent endocytosis of drug into cancer cells. METHODS: Localisation of ANG1005 and Angiopep-2 was examined by immunohistochemistry and in-vivo near-infrared fluorescence imaging in mice carrying orthotopic glioma tumours. Transport of ANG1005 and Angiopep-2 was examined in U87 glioblastoma cell lines. RESULTS: Systemically administered ANG1005 and Cy5.5Angiopep-2 localised to orthotopic glioma tumours in mice. The glioma transplants correlated with high expression levels of LRP1. Decreasing LRP1 activity, by RNA silencing or LRP1 competitors, decreased uptake of ANG1005 and Angiopep-2 into U87 glioblastoma cells. Conversely, LRP1 expression and endocytosis rates for ANG1005 and Angiopep-2 increased in U87 cells under conditions that mimicked the microenvironment near aggressive tumours, that is, hypoxic and acidic conditions. CONCLUSION: ANG1005 might be a particularly effective chemotherapeutic agent for the wide array of known LRP1-expressing brain and non-brain cancers, in particular those with an aggressive phenotype.

Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2.

BACKGROUND AND PURPOSE: Paclitaxel is highly efficacious in the treatment of breast, head and neck, non-small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P-glycoprotein (P-gp) at the blood-brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel. EXPERIMENTAL APPROACH: Paclitaxel molecules were conjugated to a brain peptide vector, Angiopep-2, to provide a paclitaxel-Angiopep-2 conjugate named ANG1005. We determined the brain uptake capacity, intracellular effects and antitumour properties of ANG1005 in vitro against human tumour cell lines and in vivo in human xenografts. We then determined ANG1005 activity on brain tumours with intracerebral human tumour models in nude mice. KEY RESULTS: We show by in situ brain perfusion that ANG1005 enters the brain to a greater extent than paclitaxel and bypasses the P-gp. ANG1005 has an antineoplastic potency similar to that of paclitaxel against human cancer cell lines. We also demonstrate that ANG1005 caused a more potent inhibition of human tumour xenografts than paclitaxel. Finally, ANG1005 administration led to a significant increase in the survival of mice with intracerebral implantation of U87 MG glioblastoma cells or NCI-H460 lung carcinoma cells. CONCLUSIONS AND IMPLICATIONS: These results demonstrate the antitumour potential of a new drug, ANG1005, and establish that conjugation of anticancer agents with the Angiopep-2 peptide vector could increase their efficacy in the treatment of brain cancer.

Stimulation of tPA-dependent provisional extracellular fibrin matrix degradation by human recombinant soluble melanotransferrin.

Tissue-type plasminogen activator (tPA) and its substrate plasminogen (Plg) are key components in the fibrinolytic system. We have recently demonstrated, that truncated human recombinant soluble melanotransferrin (sMTf) could stimulate the activation of Plg by urokinase plasminogen activator and inhibit angiogenesis. Since various angiogenesis inhibitors were shown to stimulate tPA-mediated plasminogen activation, we examined the effects of sMTf on tPA-dependent fibrinolysis. This study demonstrated that sMTf enhanced tPA-activation of Plg by 6-fold. sMTf also increased the release of [125I]-fibrin fragments by tPA-activated plasmin. Moreover, we observed that the interaction of sMTf with Plg provoked a change in the fibrin clot structure by cleaving the fibrin alpha and beta chains. Overall, the present study shows that sMTf modulates tPA-dependent fibrinolysis by modifying the clot structure. These results also suggest that sMTf properties could involve enhanced dissolution of the provisional extracellular fibrin matrix.

Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.

We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.

Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate.

The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.

Cytosolic proteins of 21-23 kDa are methylated by kidney cortex membrane-associated C-terminal carboxyl methyltransferases.

We have studied the effect of a soluble fraction from kidney cortex on the C-terminal carboxyl methylation of 21-23 kDa proteins catalyzed by membrane-associated methyltransferases. Addition of soluble proteins to isolated luminal, antiluminal and intracellular membranes resulted in a large increase in the methylation of the membrane-associated 21-23 kDa substrates. Fractionation of the soluble extract from the cortex by Q-Sepharose anion exchange chromatography showed the presence of two distinct peaks of proteins presenting stimulating activities, eluting at 0.15 M (peak I) and 0.4 M (peak II) NaCl, respectively. Both peaks eluted as proteins of apparent molecular sizes of 40 kDa upon Superose 6 gel-filtration chromatography. No methylation activity towards N-acetyl-S-trans,trans-farnesylcysteine (AFC), a good substrate for C-terminal carboxyl methyltransferases, was associated with either peaks. In contrast, the increase in methylation induced by these proteins was strongly inhibited by AFC, suggesting that the methylation induced by these factors occurred on C-terminal isoprenylated cysteine residues. Both partially purified proteins competitively inhibited the methylation of AFC by the membrane-associated enzymes, suggesting that they may represent substrates for the methyltransferases. Immunoblotting of these partially purified soluble substrates with a rabbit polyclonal antibody directed against the small G-protein CDC42 showed the presence of this protein in peak I but not in peak II. Taken together, these results suggest the presence in a soluble fraction from the kidney of distinct methyl-accepting proteins, one of these being tentatively identified as the small G-protein CDC42.

High levels of P-glycoprotein detected in isolated brain capillaries.

P-glycoprotein (P-gp) is a highly-conserved membrane protein expressed in various multidrug-resistant cell lines. P-glycoprotein was detected in capillaries isolated from human, beef and rat brains with a Western immunoblotting procedure using the monoclonal antibody C219 (mAb C219) specific for P-gp. The mAb C219 detected a 180 kDa protein in brain capillaries isolated from all three species. The largest amount of antigen was detected in capillaries isolated from human brain. Specific binding was assessed by competitive inhibition of mAb C219 binding by the synthetic epitope VQEALD. The glycoprotein nature of the brain capillary proteins was confirmed by its sensitivity to N-glycanase treatment, which reduced their apparent molecular mass by 5 to 10 kDa. In addition, immunohistochemical studies using the antibodies C219, JSB-1 and C494 were performed. Bovine and rat capillaries showed reactivity only with the mAb C219. Heavy staining of human brain capillaries was observed with both antibodies C219 and JSB-1, while only weak staining was observed with antibody C494. These results clearly show that P-glycoprotein is strongly expressed at the blood-brain barrier (BBB) site and suggest that this protein may play a physiological role in regulating the access of certain molecules to the central nervous system, or in the secretory functions of the BBB.

Functional size of C-terminal protein carboxyl methyltransferase from kidney basolateral plasma membranes.

The functional sizes of the C-terminal isoprenylcysteine protein carboxyl methyltransferase (PCMT) from kidney cortex basolateral plasma membranes and yeast membranes have been estimated by the radiation inactivation and fragmentation method. Attempts to solubilize the methyltransferase with detergents were unsuccessful as they resulted in the irreversible denaturation of its enzymatic activity. The radiation inactivation sizes of the methyltransferases were 98 and 24 kDa for kidney and yeast, respectively. Kinetic experiments showed that irradiation affects the Vmax of the reaction but not the apparent Km for either S-adenosyl-L-methionine and N-acetyl farnesylcysteine. The functional size reported here for the kidney membrane is about 4-times larger than the size predicted for the Saccharomyces cerevisiae C-terminal PCMT deduced from the nucleotide sequence of its gene (28 kDa). These results suggest that mammalian methyltransferase has a functional size different from that of the yeast; tetramerization of monomers is one possible hypothesis for this difference.

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.

Regulation of phosphate transport by second messengers in capillaries of the blood-brain barrier.

Regulation of phosphate uptake by the blood-brain barrier was studied in isolated bovine capillaries. Dibutyryl cAMP, in the presence of 3-isobutylmethylxanthine, resulted in a dose-dependent inhibition of phosphate uptake. Phosphate influx, with or without 3-isobutylmethylxanthine, was not different. Inhibition of phosphate uptake was also observed when capillaries were preincubated with isoproterenol, parathyroid hormone, insulin and acidic or basic fibroblast growth factors. Treatment of capillaries with vasoactive intestinal peptide, prostaglandin E1, angiotensin II, epidermal growth factor and phorbol esters did not affect phosphate transport. Endothelin I increased phosphate uptake by 15%. Preincubation with cholera toxin also resulted in a dose-dependent decrease in phosphate uptake. In addition, pertussis toxin inhibited phosphate transport by 29%, but only in the presence of 3-isobutylmethylxanthine. These results demonstrate that generation of second messengers, following receptor stimulation, can induce physiological effects on capillary phosphate influx and suggest that G proteins may modulate this transport.

Palmitoylation of the glucose transporter in blood-brain barrier capillaries.

Palmitoylation of GLUT1 was investigated in brain capillaries. The glucose transporter was shown to be palmitoylated using [3H]palmitate labeling and immunoprecipitation. The labeling was sensitive to methanolic KOH or hydroxylamine hydrolysis, indicating the presence of an ester or thioester bond. The released fatty acid was analyzed by reverse-phase HPLC and was identified as [3H]palmitate. Specificity of the immunoprecipitation was assessed by competitive inhibition of anti-GLUT1 binding with a synthetic C-terminal peptide against which the antibody was raised. In vivo studies were performed using capillaries isolated from control rats, streptozotocin-induced diabetic rats and diet-induced hyperglycemic rats. Glycemia was increased 2- and 5-fold in the hyperglycemic and diabetic groups, respectively. GLUT1 expression was evaluated in the three groups by Western blot analysis. A 36% decrease in GLUT1 expression was observed in the diabetic group, while there was no significant variation in GLUT1 expression in the hyperglycemic group. Palmitoylation of GLUT1 was increased in both diet-induced hyperglycemic and diabetic groups. These results suggest that palmitoylation may be involved in the regulation of glucose transport activity in hyperglycemia.

Rapid activation of matrix metalloproteinase-2 by glioma cells occurs through a posttranslational MT1-MMP-dependent mechanism.

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Tyrosine protein kinase activity in renal brush-border membranes.

Tyrosine protein kinase (TPK) activity was detected in rat renal brush-border membranes (BBM) using poly(Glu80Na,Tyr20) as a substrate. Maximal TPK activity required prior detergent dispersion of the membranes with 0.05% Triton X-100 and the presence of vanadate, a potent inhibitor of phosphotyrosine protein phosphatases, in the phosphorylation medium. Optimal conditions for measurement of TPK activity were 10 mM of MgCl2 and MnCl2, at 30 degrees C and pH 7.0. TPK activity was inhibited by genistein, with a IC50 value of 15 microM, while no inhibition was observed in the presence of 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H7), an inhibitor of serine-threonine kinases. TPK activity was enriched 4-fold in the BBM fraction relative to cortex homogenate. It was co-enriched with BBM enzyme markers, but not with those of the basolateral membrane (BLM). The endogenous substrates of TPK in brush-border and basolateral membranes were determined by Western blot analysis using an antiphosphotyrosine monoclonal antibody (PY20). Various phosphotyrosine-containing proteins were found in the BBM (31, 34, 46, 50, 53, 72, 90, 118 and 170 kDa) and in the BLM (37, 48, 50, 53, 72, 90, 130 and 170 kDa). Addition of exogenous insulin receptor to BBM and BLM increased the phosphorylation of most of the substrates. Solubilization of the TPK activity from BBM with 0.5% CHAPS and subsequent gel filtration on Superdex 75 yielded two peaks of tyrosine protein kinase activity with apparent molecular masses of 49 and 66 kDa. These results provide evidence for a non-receptor TPK activity associated with the renal tubular luminal membrane.

Radiation-inactivation analysis of the oligomeric structure of the renal sodium/D-glucose symporter.

The radiation-inactivation size (RIS) of the rat renal brush-border membrane sodium/D-glucose cotransporter was estimated from the loss of transport activity in irradiated membrane vesicles. The RIS depended on the electrochemical conditions present when measuring transport activity. A RIS of 294 +/- 40 kDa was obtained when transport was measured in the presence of a sodium electrochemical gradient. Under sodium equilibrium conditions, the RIS was 84 +/- 25 kDa in the presence of a glucose gradient, and 92 +/- 20 kDa in its absence. In the absence of a sodium gradient, but in the presence of an electrical potential gradient, the RIS increased to 225 +/- 49 kDa. The 294 kDa result supports earlier suggestions that the Na+ gradient-dependent glucose transport activity is mediated by a tetramer. Individual monomers appear, however, to carry out glucose transport under equilibrium exchange conditions or when a glucose gradient serves as the only driving force. The electrical potential gradient-driven glucose transport RIS appears to involve three functional subunits.

The in situ size of the dopamine transporter is a tetramer as estimated by radiation inactivation.

Radiation inactivation analysis of the mazindol-sensitive binding of the dopamine transporter inhibitor, [3H]GBR-12935 to canine striatal membranes yielded a radiation inactivation target size of 278 +/- 16 kDa. This result, in conjunction with other findings in the literature demonstrating that the molecular mass of the dopamine transporter is approximately 70 kDa, suggests that the native form of the dopamine carrier in the neuronal membranes is a tetrameric assembly of identical 70 kDa subunits.

Matrix metalloproteinase inhibition by green tea catechins.

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.

Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells.

Polyclonal antibodies were raised in rabbits against a 14-amino acid portion of the gibbon ape leukemia virus human membrane receptor Glvr-1. This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Western blot. Using a standard Laemmli buffer system, under reducing conditions, a single band of approximately 85 kDa (designated p85) was immunodetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as well as a protein of 70-72 kDa were immunodetected. This protein was also present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same molecular masses were also detected in OK cell membranes following proteolysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using endoglycosidase F/N-glycosidase F did not alter the electrophoretic mobility of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85. In conclusion, these data demonstrate for the first time a Western blot detection of a type III phosphate transporter using polyclonal antibodies. They also suggest that, conversely to type I and type II phosphate transporters which are localized in the kidney, this third type of transporter is ubiquitous and probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, serving as a housekeeping Na+/Pi cotransport system. This is also the first report showing that p85 is not regulated in the same manner as type II phosphate transporters.

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose.

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport (kapp was 0.052 s-1 for the uptake of phosphate and 0.019 s-1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate (kapp = 0.0012 s-1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.

Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport.

Polyclonal antibodies raised against the 14-amino acid C-terminal portion of the rabbit renal brush-border membrane Na+/Pi cotransporter, as deduced from the nucleotide sequence of the cloned NaPi-1 gene, were used for Western blot analysis of renal brush-border membrane proteins from rat, rabbit and beef. Proteins of 65 kDa from the rat, 64 kDa from the rabbit, and 38, 66, 77, 92, 110, 176 and 222 kDa from the beef were specifically labelled. The affinity of the antibodies was much greater, however, for the proteins of the rat and rabbit than for those of the beef. The rat 65-kDa antigen was readily detected in brush-border membranes isolated from kidney cortex, but was absent from the basolateral membrane and the cytosolic and microsomal fractions of this tissue, in agreement with the subcellular localization of the Na+/Pi cotransporter. This antigen was however several-fold more abundant in the juxtamedullary portion of the cortex than in the outer portion. Despite a strong stimulation in phosphate transport, a low-phosphate diet had little influence on the amount of antigen detected. An additional peptide-displaceable band corresponding to a protein of 250 kDa appeared when beta-mercaptoethanol was omitted during electrophoresis, in agreement with the possibility that disulfide bonds may be involved in the regulation of renal phosphate transport activity.

Molecular size of the renal sodium/phosphate symporter in native and reconstituted systems.

The size of the renal sodium/phosphate symporter was estimated with the radiation inactivation technique in isolated bovine brush border membrane vesicles and after reconstitution in proteoliposomes. The functional unit of the native phosphate carrier had a radiation inactivation size of 172 +/- 17 kDa. Identical values were obtained for the reconstituted carrier whether it was irradiated before or after the formation of the proteoliposomes (161 +/- 9 and 159 +/- 11 kDa, respectively). The sodium-independent uptake of phosphate was not affected significantly by radiation doses up to 10 Mrad. This activity is therefore not due to the reconstitution of a large phosphate-binding protein such as alkaline phosphatase. Furthermore, bromotetramisole, a specific inhibitor of phosphate binding to this enzyme, had no significant effect on the uptake of phosphate by the proteoliposomes.