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P Sudaka

Publications and source records attributed to P Sudaka.

At least 19 recordsLinked to original sources
PubMed1998

Renal neutral alpha-D-glucosidase has no role in transport of D-glucose derived from maltose hydrolysis.

To reinvestigate the "hydrolase-related transport" concept, neutral alpha-D-glucosidase, a membrane-bound disaccharidase of renal proximal tubule, was first purified from brush-border membranes and then asymmetrically reincorporated into egg phosphatidylcholine vesicles. Proteolytic treatments and immunotitration studies demonstrated that this enzyme was integrated in native and artificial membrane vesicles with a similar topology. The uptake of free glucose and glucose produced by maltose hydrolysis was studied using 1) proteoliposomes containing integrated neutral alpha-D-glucosidase, in combination with other membrane proteins, and 2) proteoliposomes containing only the other membrane proteins but incubated in a medium containing neutral alpha-D-glucosidase in its hydrophilic form. No modification was observed in the uptake of free D-glucose or D-glucose produced by maltose hydrolysis, regardless of enzyme localization. In contrast to previous findings on the hydrolase-related transport concept, these results rule out any participation of neutral alpha-D-glucosidase in the transport of free glucose or glucose produced by maltose hydrolysis. Hydrolytic activity and transmembrane transport appear to be two independent and sequential steps.

Animals↗
PubMed1994

Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex membranes in Western blot experiments. Taken together, our data support the idea that the enzyme is expressed under its tetrameric form in the membrane. The purified enzyme is able to exhibit a serine proteinase activity when it recovers its native tetrameric form. This high molecular weight tetrameric proteinase SP 220 K appears as a new member of the cell surface serine protease family.

Animals↗
PubMed1993

Oligomeric structure of the sodium-dependent phlorizin binding protein from kidney brush-border membranes.

Immunodetection of solubilized kidney brush-border proteins on Western blots using antibodies against the 70 kDa phlorizin binding component of sodium-glucose cotransporter allows to identify an additional protein band with apparent molecular mass of 120 kDa in the presence of reducing agent dithiothreitol. Antibodies specifically eluted from the 70 kDa protein still recognize the 120 kDa protein on Western blot. The lack of dissociation of the 120 kDa protein from native brush borders or Triton X-100 extract in the presence of dithiothreitol can be improved by an extended incubation at 25 degrees C; this protein is full dissociated when purified by electroelution from polyacrylamide gel and gives two subunits with apparent molecular masses of 70 and 60 kDa by Coomassie staining and Western blot analysis. The effect of dithiothreitol on the renal brush-border membrane phlorizin binding is studied; a decrease in the number of high-affinity phlorizin binding sites without modification of the affinity to the binding molecule is observed. These data suggest that the high-affinity phlorizin binding moiety of sodium-glucose cotransporter exists in the kidney as a dimeric structure.

Animals↗
PubMed1993

Simple column liquid chromatographic assay for serum neopterin.

A simple high-performance liquid chromatographic assay for serum neopterin with highly sensitive fluorimetric detection (limit 1 nM) is proposed. Comparison with results obtained by radioimmunoassay revealed a good correlation between the two techniques. The potential use of this method for the follow-up of liver transplant patients is discussed.

Adult↗
PubMed1993

Lane distortions in gel electrophoresis patterns.

Distortions effects were studied as a function of gel environment, apparatus design and buffer type. Outward lanes distortions pronounced in low conductivity buffers for both continuous buffer systems and stacking gels, were field strength dependent. In discontinuous buffer systems, the moving boundary of the Laemmli buffer system deformed depending on the environment. In the Michov buffer system, the high conductivity resolving gel surrounded by a low conductivity electrode buffer, displayed straight pathways until a field strength of 15 V/cm, permitting to obtain information about lanes distortions depending on the acrylamide matrix structure. A decreased ammonium persulfate concentration, e.g. to 0.03%, induced phase segregation in gels at low temperature during the run: a critical endpoint near 11 degrees C for a gel in the Michov buffer system under conditions of electrophoresis was supposed. Gel collapse resulted in inward deviations in the lanes of the pattern and also faint bands in the zones where the gel was retracted. Besides this effect of catalyst concentration, the inwardly distorted pattern depended on temperature, field strength, solvent and hydrolysis due to gel aging. Amendments for overcoming lane distortions are suggested.

Ammonium Sulfate↗
PubMed1992

Characterization and purification of a guanidinobenzoatase: a possible marker of human renal carcinoma.

Guanidinobenzoatases are cell surface-associated proteinases supposed to be involved in cancer metastasis, cell migration, and tissue remodeling. The main features of the guanidinobenzoatase associated with human renal carcinoma plasma membrane are weak membrane association, continuous cleavage of p-nitrophenyl-p'-guanidinobenzoate conversely to the site titration effect of this compound when used with trypsin, and a peculiar sensitivity to serine protease inhibitors, compatible with a poorly active form. Plasma membrane preparation followed by agmatine-trisacryl affinity chromatography allows the purification of guanidinobenzoatase to homogeneity with an apparent enrichment factor of 450. Purified guanidinobenzoatase appears as a single polypeptide chain of M(r) 80,000, likely stabilized by intrachain disulfides bonds. The properties of purified guanidinobenzoatase indicate that it is an original enzyme in spite of some similarities with plasminogen activators. Indeed, in addition to differences in substrate and inhibitor specificity, guanidinobenzoatase is not recognized by specific monoclonal antibodies directed against plasminogen activators or their single-chain precursors. Thus, human renal carcinoma guanidinobenzoatase appears to be an original enzyme whose activity is undetectable in the nontumoral tissue of origin. In this respect, use of purified guanidinobenzoatase would allow us to obtain specific tools to give new insights in cancer cell metastasis.

Adenocarcinoma↗
PubMed1991

Immunological recognition of sodium/D-glucose cotransporter from renal brush border membranes by polyclonal antibodies.

Antisera prepared in rabbit to a D-glucose-inhibitable phlorizin binding component of the pig kidney brush border membrane precipitated more than 90 percent of the D-glucose-inhibitable phlorizin binding activity from a Triton extract. These antibodies also stimulated D-glucose uptake by native brush border membranes at low D-glucose concentrations (1 mM) and inhibited it at higher D-glucose concentrations. Immunoblotting was used to locate polypeptide subunits of the glucose transporter in polyacrylamide gels of proteins extracted from the brush border membranes. The antibodies labelled the Mr 70,000 phlorizin-binding component in both reducing and non reducing conditions. Two additional polypeptides with relative molecular mass of 120,000 and 45,000 were also recognized under the same conditions; they might correspond, respectively, to another Na+/D-glucose cotransport unit and to a post mortem degradation product.

Animals↗
PubMed1991

A new device for measuring distribution of electric fields in isothermic gel electrophoresis.

Electrophoretic potentials inside a polyacrylamide slab gel were measured by a platinum electrode probe holder. These electrodes were inserted into the gel and the distribution in space of electric fields was explored by a multiplexing procedure. Horizontal and vertical arrays of electrodes enabled the evolution of average fields and local potential gradients to be monitored during electrophoretic migration. The results show the nonhomogeneity of mean field strength measurement along a slab gel, the dependence of the potential distribution according to the chemical composition of buffers, moving boundary detection and the resulting potential gradient variation resulting from conductance variation on either side of the boundaries. The role played by the gel and the electrode geometry in the map of potential distribution is underlined.

Electrodes↗
PubMed1990

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate.

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.

Animals↗
PubMed1990

Simultaneous preparation of basolateral and brush-border membrane vesicles from sea bass intestinal epithelium.

A method is described for simultaneous preparation of brush-border and basolateral sea bass enterocyte membranes using simple differential centrifugation and discontinuous sucrose gradient density centrifugation techniques. Basolateral membranes were purified with a Na+/K(+)-ATPase yield of about 11% of the original activity, with an enrichment factor of 12. The yield of maltase-glucoamylase, a specific marker of brush-border membranes, was also about 11% of the original activity, with 15-fold enrichment. The characteristics of these membrane preparations were determined. Electron microscopy analysis showed that these two membrane preparations were uniform in size and vesicular in nature. Orientation studies revealed that the luminal membrane vesicles were right-side out and 43% of the antiluminal membrane vesicles were sealed inside out. Investigation of D-glucose and L-leucine uptake showed that these two plasma membrane preparations retained their transport properties.

Animals↗
PubMed1990

Fast electrotransfer of human serum proteins in their native state from polyacrylamide thin gradient gels reinforced by textiles to polyvinylidene difluoride membranes. Rapid electrotransfer from thin gradient gels reinforced by textiles.

A fast electroblotting technique of native molecules electrophoretically separated in thin (0.25 to 0.5 mm) gradient gels, onto a high capacity membrane of polyvinylidene difluoride is described. Omitting methanol during transfer, the equilibration step is avoided and the same buffer is used in electrophoresis and transfer. As the gel reinforced by fabric never swells nor shrinks, and as all the bands are blotted, the transfer matrix exactly reflects the protein pattern of the original gel. Autoradiography is enhanced and electroelution is homogeneous in all parts of the gels. Significant improvement is noticed in binding proteins of molecular weight from about 20 kDa to more than 700 kDa, as suggested by complete electroelution of all native serum components.

Blood Proteins↗
PubMed1989

Purification and properties of neutral maltase from human granulocytes.

A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1----2), alpha (1----3) and alpha (1----4) glucosidic linkages. The highest activities were observed for alpha (1----4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.

Glycoside Hydrolase Inhibitors↗
PubMed1989

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

Animals↗
PubMed1989

[Physiologic variations in plasma osteocalcin in the child and the adolescent].

Osteocalcin, the most abundant of non-collagen proteins in bone, is produced by osteoblasts and incorporated into the bone matrix. A small amount is circulated in the bloodstream where it can be detected by radioimmunologic assay. Serum assays in 128 children and adolescents showed that serum osteocalcin levels accurately reflect the various stages of statural growth: peak levels were found at three different ages, i.e. infants (21.8 +/- 4.9 ng/ml), children over seven (19.4 +/- 5.2 ng/ml), and adolescents undergoing the early stages of puberty (22.5 +/- 4.2 ng/ml). Although significant differences were found between each age group and the preceding or following group, these results can be applied to individual patients only for longitudinal studies.

Adolescent↗
PubMed1988

Improved transfer of high molecular weight proteins using fabric-reinforced soft polyacrylamide gels.

Rapid transfer of electrophoretically separated, high molecular weight proteins from fabric-reinforced, soft, low-concentration polyacrylamide gels (3.5%T, 2.6%C) is described. Polyester fabrics proved useful for gel reinforcement during vertical slab gel electrophoresis. The fabric-reinforced gel retains its dimension during transfer, equilibration and drying, facilitating comparison of protein bands in the separation gel and transfer matrix.

Collodion↗
PubMed1988

LLC-PK1 cells express Na+-lactate cotransport in apical membranes after confluency.

L-[3H]lactate uptake was characterized in LLC-PK1 cell apical membrane vesicles obtained by intensive culture on microcarrier beads. The apical membrane preparation technique involved MgCl2 precipitation. Na+-dependent L-[3H]lactate uptake was present only after confluency; its appearance paralleled the subcellular localization of aminopeptidase in apical membranes. L-[3H]lactate uptake was Na+-dependent and electrogenic. Only the Na+-dependent component of L-[3H]lactate uptake was saturable with one family of independent carriers. The apparent affinity constant was 1.1 +/- 0.25 mM and the apparent maximal velocity was 29 +/- 3 nmol.mg-1.min-1. The Na+-lactate cotransport stoichiometry was 2 Na+ for 1 lactate. The specificity of the L-lactate transport system was compatible with that of the monocarboxylic acid pathway described previously in brush-border membranes of kidney cortex and discrete from the tricarboxylic acid carrier, the D-glucose transporter, and the general pathway for anions. The LLC-PK1 cell line appears to be a useful tool for study of the regulation of L-lactate uptake and biosynthesis of the renal monocarboxylic acid transporter.

Animals↗