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.

Immunochemical analysis of high molecular mass urinary proteins.

Urinary high molecular mass proteins (fraction P) solubilized in Triton X-100 and by papain have been compared with the solubilized human renal brush border membrane proteins. Crossed immunoelectrophoresis of Triton X-100 fraction P extract, by means of two polyspecific antisera directed against either renal membrane or fraction P, revealed eleven immunoprecipitates antigenically identical with detergent renal membrane antigens. Among them, five hydrolases were identified by zymogram staining: microvillus aminopeptidase, maltase, trehalase, gamma-glutamyltransferase and alkaline phosphatase. Eight papain-solubilized fraction P proteins and Triton X-100-solubilized membrane extract presented 'identity' patterns in tandem crossed immunoelectrophoresis, but differed in their amphiphilicity, as demonstrated by the change of precipitation pattern on charge-shift caused immunoelectrophoresis. Among the eleven detergent-solubilized fraction P antigens, nine were proved to be amphiphilic proteins and six presented bidirectional charge shifting properties similar to those of renal membrane antigens. Quantitatively, five detergent fraction P proteins were found in the same amounts as in renal membrane extract, two in lesser amounts and four in greater. Moreover, the same two plasma proteins were identified in fraction P as in the renal membrane. Thus important similarities exist between the urinary fraction P and the native renal membrane.

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles.

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled D-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and D-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of D-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and D-glucose to the cotransporter.

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography.

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of D-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards D-glucose compared to other sugars tested was shown as well as the main features of D-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of D-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the D-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 A; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65,000. We conclude that this protein fraction is involved in D-glucose transport by renal brush borders.

Purification by affinity chromatography and characterization of a neutral alpha-glucosidase from horse kidney.

A horse kidney neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was purified about 580-fold with a yield of 33% by an affinity chromatography technique using the p-aminophenyl-beta-D-maltoside, a substrate derivative, as ligand. The purified enzyme, homogeneous in polyacrylamide gel electrophoresis, was a glycoprotein with a molecular weight of 280 000 as calculated by gel filtration and its isoelectric focusing points was found to be pH 4.1. The purified enzyme was able to hydrolyze various substrates having (alpha-1,2), (alpha-1,3), (alpha-1,4), and (alpha-1,6) glucosidic linkages. The V/Km ratio shows that the (alpha-1,4) linkages are the best substrates. The pKm of the purified enzyme determined at different pH values indicated that two ionizable groups with pK values 5.2 and 6.9 could be essential in the active site. Enzyme modification with cardodiimide abolished the maltase activity. The turanose, a substrate analogue, protected the enzyme against this inactivation.

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.

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.

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.

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms.

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.

[Catalytic properties of a neutral alpha-glucosidase from human kidney]. / Propriétés catalytiques de l'alpha-glucosidase neutre du rein humain

The catalytic properties of a neutral alpha-glucosidase purified to homogeneity from human renal cortex are described. The pH optimum was 6 (maltose and starch). It has a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1 leads to 2), alpha (1 leads to 3), alpha (1 leads to 4) and alpha (1 leads to 6) linkages. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 0,5 mM. It also hydrolyses polysaccharides as starch and glycogen. The Km and Vmax values for the various substrates were determined. The enzymes exhibited intrinsic transglucosylase activity. It catalysed glucosyl-transfer reaction from maltose to itself (disproportionation). Mixed substrate inhibition studies, inhibition studies and heat inactivation are interpreted in terms of the existence of at least two interacting sites on the enzyme.

Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.

Glucose transport by horse kidney brush borders. I.--Transport properties of brush border membrane closed vesicles.

Brush border membranes isolated from horse kidney cortex as closed right-side out vesicles show selective permeability when analyzed on sucrose and dextran gradients. These vesicles can actively accumulate D-glucose. The preservation of the glucose transport system is demonstrated by the following features: (a) the uptake and release rates of D-glucose are higher in the presence of a sodium gradient, showing that D-glucose transport is a sodium-dependent process; (b) this transport, specific for the D-isomer, is inhibited by phlorizin; (c) the D-glucose transport system is saturable; (d) no inhibition of D-glucose transport is found with C-mannose; (e) the D-glucose uptake is sensitive to osmotic variations.

Crossed-immunoelectrophoretic study on human renal brush border membrane vesicles.

The human kidney brush border membrane proteins were studied by crossed-immunoelectrophoresis. An antiserum against membrane vesicles was raised in rabbits and used in establishing a reference immunoelectrophoregram with the antigens released by Triton X-100. Among the precipitates observed, the following hydrolases were identified by zymogram staining: Microvillus aminopeptidase (EC 3..4.11.2), gamma-glutamyltransferase (EC 2.3.2.2), maltase (EC3.2.1.20) and trehalase (EC 3.2.1.28). Depletion of the antiserum with sealed, right-side-out vesicles was performed. No precipitates could be seen when the Triton X-100 extract was electrophoresed in a gel containing the depleted antibody. It is therefore suggested that the precipitation of membrane components by the complete antibody is mainly due to externally-located determinants and that the precipitates of the reference pattern correspond to membrane components pointing, at least in part, towards the tubular lumen. Evidence was also noted for a differential removal of antibodies directed against the different antigens. Such an observation could not be explained by the antigen accessibility nor by its amount in the membrane. Parallel crossed-immunoelectrophoresis of Triton X-100 and papain extracts gave rise to an "identity" pattern for only some antigens, particularly for microvillus aminopeptidase and maltase. It is thus strongly suggested that the papain-released form of these enzymes bears nearly all the antigenicity of the whole molecule.

A single-step centrifugation method for separation of granulocytes and mononuclear cells from blood using discontinuous density gradient of Percoll.

A rapid and reproducible method is described for the simultaneous purification of mononuclear and polymorphonuclear cells, based on the use of a discontinuous gradient of Percoll. Centrifugation at 600 X gav for 20 min resulted in separation of the mononuclear and polymorphonuclear cells into 2 distinct bands at the interfaces. The upper layer contained the mononuclear cells and the lower band highly purified polymorphonuclear cells. Both populations were purified about 97% without detectable cell alteration.

[Urinary elimination of multiple forms of gamma-glutamyltranspeptidase and aminopeptidase (author's transl)]. / Etude des formes d'élimination urinaire de la gamma-glutamyltranspeptidase et de l'aminopeptidase.

Concentrated and dialyzed 24-h urines of healthy persons were separated by 105000 X g ultracentrifugation into a pellet (P105) and a supernatant (S105) fraction. Chromatography of the P105 fraction on Sepharose 4B and 2B revealed that gamma-glutamyltranspeptidase and aminopeptidase activities had a molecular mass of 20 X 10(5) to 40 X 10(6), whereas in the S105 fraction soluble gamma-glutamyltranspeptidase and aminopeptidase had 86000 and 160000, respectively. Triton X-100 solubilization was performed on the P105 fraction and on a human renal cortex 40000 X g pellet, used as a reference. All the activity was recovered in both cases in a single peak of detergent gamma-glutamyltranspeptidase and detergent aminopeptidase eluted by filtration on Ultrogel Ac A22. Apparent molecular mass of Triton X-100 solubilized urinary and renal enzymes were 250000 and 243000 for gamma-glutamyltranspeptidase, and 298000 for both aminopeptidases. Protease solubilized forms were obtained by trypsic digestion of detergent urinary and renal forms. Both gamma-glutamyltranspeptidases were found to have an apparent molecular mass of 86000 on Sephadex G 150, which is identical to the value found for the S105 urinary gamma-glutamyltranspeptidase. The aminopeptidases had 238000 and 232000, which is a higher value than the molecular mass of the S105 urinary aminopeptidase. This letter could be a degraded form of the renal aminopeptidase. These findings suggest that gamma-glutamyltranspeptidase and aminopeptidase in the P105 fraction are similar to native renal enzymes. Evaluation of the P105 fraction enzymatic activities may thus be useful in the diagnosis of tubular damage.

Distribution of neutral maltase activity in mononuclear cells from different human lymphoid organs.

Neutral maltase activity has been determined in purified monocytes, T and B lymphocytes from human peripheral blood and major lymphoid organs. In peripheral blood as in all lymphoid organs studied, neutral maltase activity was found to be significantly higher in monocytes than in B lymphocytes. Neutral maltase activity was never detected in T lymphocytes whatever the methods used for their purification. Furthermore, in each case studied, B lymphocyte fractions represented all the activities measured in unseparated lymphocytes. Neutral maltase thus appears to be the first enzymatic marker useful for the recognition and characterisation of B lymphocytes.