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.

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.

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.