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.

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.

[Transplantation of a segmental small intestine in dogs. Comparison between jejunal graft and ileal graft]. / Transplantation d'intestin grêle segmentaire chez le chien. Comparaison d'un greffon jéjunal et d'un greffon iléal.

The purpose of this study was to compare heterotopic jejunal and ileal allografts in the dog under cyclosporine A. Fourteen allografts (8 ileal and 6 jejunal) were successfully performed. There was no case of graft-versus-host disease in this series. Six allografts (42.5 percent), 3 jejunal (50 percent) and 3 ileal (37.5 percent), were rejected during the first 3 months (NS). Eight allografts, 5 ileal and 3 jejunal, were tolerated up to 3 months and then removed; 2 ileal and 2 jejunal were normal; 2 ileal and 1 jejunal showed signs of chronic rejection, and 1 ileal signs of advanced rejection. It is concluded that there is no major difference between jejunal and ileal allografts in the dog.

Jejunal versus ileal segmental allografts in the dog: comparison of immunologic and functional results.

Segmental small-bowel grafts have been advocated as a means of reducing the incidence of rejection and graft-versus-host disease in small-bowel transplant recipients. This study compared the results achieved with heterotopic segmental allografts of the jejunum and the ileum that used 120 cm Thiry-Vella loops in a dog model. Immunosuppressive therapy consisted of 25 mg cyclosporine/kg/day. Results were monitored by histologic examinations, function tests (maltose and xylose absorption), and brush-border enzyme assays. Thirty-three dogs were randomized for use as a donor (n = 11) or recipient of a jejunal allograft (n = 11) or an ileal allograft (n = 11). Eight allografts were technical failures and were excluded from analysis. Fourteen allografts were successful (eight ileal, six jejunal). No case of graft-versus-host disease was observed. Six allografts (42.5%, three jejunal [50%] and three ileal [37.5%]) were rejected during the first 3 months (not statistically significant). Eight allografts (five ileal, three jejunal) were tolerated for up to 3 months and were removed. Two ileal and two jejunal allografts appeared grossly normal at surgical removal, but two ileal and one jejunal allografts exhibited signs of chronic rejection, and one ileal allograft showed advanced rejection. The jejunal and ileal allografts had similar clinical courses, as were revealed by immunologic reactions and functional parameters. We conclude that there is no major difference between jejunal allografts and ileal allografts in the dog.

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.

[Segmental small intestine transplantation in dogs: comparison between a jejunal graft and an ileal graft]. / Transplantation d'intestin grêle segmentaire chez le chien: comparaison d'un greffon jejunal et d'un greffon iléal.

The aim of this study was to compare segmental grafts of jejunum and ileum in a dog model. 14 segmental grafts, 8 ileal (Il. A) and 6 jejunal (Jej. A.), were successfully allografted as 120 cm-Thiry-Vella segments. Immunosuppressive therapy consisted of cyclosporin 25 mg/kg/day per os. Monitoring was performed by histology and absorption (maltose and xylose) studies as well as analysis of brush border enzymes. No cases of Graft-versus-host disease were observed. Six allografts (42.5 per cent) including 3 Jej. A. (50 per cent) and 3 Il. A. (37.5 per cent) were rejected during the first three months. Eight allografts (5 Il. A. and 3 Jej. A.) were tolerated for up to 3 months and were removed: 2 Il. A. and 2 Jej. A. were normal, while 2 Il. A. and one Jej. A presented with signs of chronic rejection and one Il. A. with advanced rejection. Jej. A. and Il. A. showed a similar course, by means of immunologic reactions as well as functional characteristics. It is concluded that there is no major difference between Jej. A. and Il. A. in the dog. Because of the specialized absorptive functions of the ileum and its adaptative properties, ileal segmental grafts should be preferred to jejunal grafts for the treatment of short-gut syndrome.

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.