Alexander Eichholz

Structural and functional organization of the brush border of intestinal epithelial cells: III. Enzymic activities and chemical composition of various fractions of tris-disrupted brush borders

Abstract The composition of subunits of the intestinal brush border has been studied. Fractions obtained after Tris disruption and subsequent density-gradient centrifugation were analyzed for their enzymic and chemical content. These fractions include a major membrane component, a minor membrane component, microvillus cores and two that are unidentified. The major membrane fraction contains all of the alkaline phosphatase, maltase, invertase, lactase, isomaltase, trehalase and leucyl naphthylamide hydrolase activities of the brush border. It also contains ATPase. A different ATPase activity is found in one of the morphologically identified fractions. It appears to be associated with a fibrillar material which can be formed from this fraction by appropriate treatment. Lactase activity is occasionally found to be differently distributed from the other disaccharidases; i.e. , an appreciable amount appears in the minor membrane component. No enzymatic activities were found in the fraction composed of microvillus cores. All fractions were analyzed for protein, carbohydrate, cholesterol, phospholipid and DNA. Cholesterol to phospholipid ratio of 1.1 was found in the major membrane fraction.

Studies on the organization of the brush border in intestinal epithelial cells: V. Subfractionation of enzymatic activities of the microvillus membrane

Abstract Isolated brush border membranes of the intestinal epithelial cell have been partially digested by treatment with low concentrations of papain. A multienzyme particle containing maltase, sucrase and isomaltase activities is released. Further treatment with papain releases leucyl naphthylamidase activity in particulate form. Trehalase activity remains with the residue of the membrane. The disaccharidase-containing particles can be separated from those containing leucyl naphthylamidase on Sephadex G-200 columns. Alkaline phosphate activity is rapidly destroyed by papain treatment. Leucyl glycine-splitting activity can be separated from leucyl naphthylamide-splitting activity by papain treatment. Thus there is now evidence for two brush-border enzymes involved in the digestion of peptides. The association of membrane enzymes with particles containing multiple activity is in accord with the view of a mosaic substructure of the membrane.

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised proteins separated 10-15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments. Electrophoresis of brush border proteins metabolically labelled with [14-C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity. The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.

Studies on the organization of the brush border in intestinal epithelial cells. VI. Glucose binding to isolated intestinal brush borders and their subfractions

Abstract A sensitive method for the measurement of specific glucose binding to isolated hamster intestinal brush borders has been developed. The method is based on the difference in binding of l -[ 14C]glucose and d -(su3H ]glucose as measured by retention of radioactivity on Millipore filters. Properties of the binding have been studied. Binding appears to be localized to brush borders. It exhibits a specificity towards a variety of test sugars which is different from the specificity of intestinal active transport. The K m of binding is 2 μM. Binding to intact brush borders shows no requirement for Na + . Binding is inhibited by Ca 2+ , Co 2+ , Fe 2+ , and Zn 2+ and activated by Mg 2+ and Mn 2+ . Binding to brush borders is highest in the distal portion of the small intestine. The distribution of binding among subfractions of the brush border has been studied. Possible interpretations of this binding are discussed.

Synthesis of photoaffinity labeling derivatives of d-glucose and d-galactose

Abstract Two new photoaffinity reagents having photoreactive groups attached to C-6 of d -galactose have been prepared. 6- N -(4-Azido-2-hydroxybenzoyl)- d -glucopyranosylamine and 6- N -(4-azido-2-hydroxybenzoyl)- d -galactopyranosylamine were synthesized by acylation of the protected amino sugars with 4-azidosalicoyl chloride or by treating the amine with 4-azidosalicylic acid. Subsequent iodination of the aromatic ring yields the diiodo derivative. Deprotection yields the sugar derivatized at C-6 by the diiodinated photoaffinity reagent. Photoaffinity reagents having high specific activity may be prepared by this procedure.

Transport of 5-thio-d-glucose in hamster small intestine

Abstract Segments of hamster small intestine have been used to study the interaction of 5-thio- d -glucose with the d -glucose transport system. By all current criteria, 5-thio- d -glucose is a substrate for the system. We have examined accumulation against a concentration gradient, substrate inhibition, phlorizin sensitivity, Na+ dependence, energy dependence and counterflow. The estimated K m value for 5-thio- d -glucose is of the order of 2.8 mM. Two interesting differences between d -glucose and 5-thio- d -glucose have been found. 5-Thio- d -glucose is less effective than d -glucose in inhibiting the specific binding of d -[3H]glucose to brush borders. 5-Thio- d -glucose is also a poor substrate for hexokinase. The effect of the sulphur substitution on possible ring conformation of the sugar is discussed with respect to the specificity of the transport system.

Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter

The photosensitive reagent 6-N-(4-azido-2-hydroxy-3,5-diiodobenzoyl)-D-glucosamine has been assessed as a potential photoaffinity label for the hexose transporter. Under zero-trans conditions, transport experiments performed in the dark reveal that the reagent inhibits the uptake of D-glucose in resealed human erythrocyte ghosts. Increasing the concentration of glucose in the transport medium has a protective effect, reducing the inhibition. Kinetic analysis indicates that the probe acts as a competitive inhibitor with high affinity for the erythrocyte hexose transporter (Ki between 0.07 and 0.2 microM). Exposure to a 280 nm filtered high intensity mercury-vapor lamp results in a rapid and efficient photolysis. At low concentrations of the probe, specific labeling of membrane preparations was observed. Autoradiograms of 10% SDS gels revealed the specific labeling of bands 4.51 and 6. This labeling was concentration-dependent and protected by D-glucose (not the L-isomer) and phloretin in the medium. When subjected to multiple exposures of low concentration of the photoaffinity reagent, apparent saturation was achieved.

Studies on the mechanism of active intestinal transport of glucose

Abstract d -[2-3H]Glucose is actively accumulated by hamster intestinal rings and everted sacs. The isolated glucose from the tissue and the serosal fluid showed no significant change in specific activity. These data rule out an active transport mechanism involving a covalent bond with the oxygen of the hydroxyl on C-2 of glucose. At the same time an exchange reaction of the 3H with water catalyzed by brush borders has been observed. This reaction is probably independent of glucose transport.