R.K. Crane

Purification of the human intestinal brush border membrane

A method is described to obtain brush border,i.e. microvillus membranes, from human intestinal epithelial cells. It is based on the sequential use of CaCl2, differential centrifugation and Tris. The final preparation contains no DNA, no succinate dehydrogenase, traces only of (Na+K+)-ATPase, NADPH-cytochrome c reductase and β-glucuronidase and is characterized by a high specific activity in brush border membrane markers as well as its appearance on electron micrographs. The method may be applied to fresh or frozen intestine, to surgical specimens or peroral jejunal biopsies.

A rapid method for the isolation of kidney brush border membranes.

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca2+ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.

Phlorizin hydrolase: A β-glucosidase of hamster intestinal brush border membrane

Abstract A β-glucosidase activity catalyzing phlorizin hydrolysis to phloretin and glucose is present in the microvillus membrane of hamster intestinal brush border. The pH optimum, K m and the effects of Tris, galactono- and gluconolactones, sulfhydryl reagents and heavy metals were studied. This β-glucosidase is distinct from the other brush border glycosidases documented in the literature. The presence of this enzyme serves to rationalize the divergent observations that cellular entry of low concentrations of mucosally added phlorizin has not been detected although high concentrations have been reported to inhibit intracellular metabolism.

Active transport of myo-inositol and its relation to the sugar transport system in hamster small intestine

Incubation in vitro of segments of hamster small intestine has been used to study the mode of absorption of myo-inositol. By all current criteria, myo-inositol is actively transported. We have observed accumulation against a concentration gradient, energy-dependence, Na+-dependence, phlorizin sensitivity, and saturation kinetics with a Km of 0.14 mM. Sugars which interact with and undergo the translocation step of glucose transport are non-competitive inhibitors of the transport of myo-inositol. myo-Inositol has no effect on the d-glucose transport system. Phlorizin interacts competitively with the myo-inositol binding site with an affinity 10–100-fold less than it has for the common sugar binding site. These results indicate that the pathway for myo-inositol to cross the brush border membrane is not entirely the same as the d-glucose pathway. However, they also indicate that there is an interaction between the two at the level of translocation.

Studies on the transport of glucose from disaccharides by hamster small intestine in vitro. I. Evidence for a disaccharidase-related transport system☆

In the presence of saturating concentrations of free d-glucose, total glucose uptake was enhanced beyond the theoretical V for free glucose uptake when disaccharides were incubated with intestinal rings. This phenomenon was not seen when glucose 1-phosphate was the substrate. Analogs of d-glucose transport system, galactose and β-methyl glucoside, had an inhibitory effect on glucose uptake from sucrose and their uptake was in turn inhibited by the disaccharide. This inhibition was non-competitive. The effect of phlorizin on glucose uptake from sucrose was 2-fold, competitive at low concentrations and non-competitive at high concentrations. Sucrose did not induce counterflow of the preloaded β-methyl glucoside. These observations indicate that with a disaccharide as the substrate, there is a component of glucose transport which is in addition to the monoscaccharide-transport system and that this could arise as a consequence of the association of disaccharidases with the brush border membrane.

Modification of an assay for trypsin and its application for the estimation of enteropeptidase

A sensitive method for the estimation of trypsin and enteropeptidase is described. The use of alpha-N-benzoyl-DL-arginine-p-nitroanilide as substrate in combination with the Bratton-Marshall reaction increased the sensitivity of an established method to a degree which permits the determination of enteropeptidase at considerably lower activity levels than heretofore such as those expected in brush border membrane preparations from peroral biopsies of human small intestine.

Studies on the transport of glucose from disaccharides by hamster small intestine in vitro. II. Characteristics of the disaccharidase-related transport system.

Abstract The presence of disaccharidase-associated transport different from the Na + -dependent monosaccharide transport system in the hamster small intestine is confirmed. The transport of glucose from sucrose is substantially independent of Na + . In the absence of Na + , glucose released from sucrose does not mix with a pool of added free glucose but is directly transferred. Maltose, isomaltose and trehalose acf similarly to sucrose. Both moieties released from sucrose, glucose and fructose, are transferred. The extent of uptake is not related to total sucrase activity. The possibility is considered that the brush border disaccharidases may subserve a translocating “carrier” function for a part of the products of their enzymic action.

An analysis of the d-glucose influx kinetics of in vitro hamster jejunum, based on considerations of the mass-transfer coefficient

1. A study designed specifically to investigate the effects of unstirred layers on the apparent glucose-influx kinetics of hamster jejunum was conducted. 2. The apparent V was 12.81, 10.71, 9.75, 10.17 and 9.33 mumol/cm-2 - h while the apparent Km was 7.42, 3.95, 1.87, 0.93 and 0.5 mM, respectively, when the rate of shaking the incubation flasks was 40, 80, 120, 160 and 200 cycles/min. 3. Extrapolation of the slope and reciprocal intercept of Lineweaver-Burke plots of the data to infinite shaking rate is mathematically justified to yield the slope and intercept of a Lineweaver-Burk plot which is uncomplicated by unstirred layers. These extrapolations were found to have a regression coefficient = 1 when plotted as (intercept)-1 or slope = b0 + b1b-(shake)-2 where b = 2.764 for the slope plot and 6.626 for the (intercept)-1 plot. From the values of b0 one obtains a Km of 0.41 and a V 0f 0.35 which should represent the true kinetic parameters for glucose influx into this tissue under the experimental conditions employed. 4. Values of the theoretical flux expected on a basis of unstirred-layer thickness which was calculated from the relation Cb (for J = V/2) = Km + 0.5 V/Kd agreed with the experimental values of J in some instances but the 95% confidence interval of the theoretical and experimental values did not overlap in many instances at low shaking rates and low concentrations of glucose. 5. A factor theta representing the error between the theoretical and experimental values was found to fit the relationship 1n(theoretical J) = - 3.8 + 5.77 (1/theta) with a regression coefficient of 0.98 and was proposed to be due to one or more of the following parameters: (1) a villus tip to base gradient of transport (influx) activity; (2) a dependence of brush-border influx area on substrate concentration in the bulk incubation media; and (3) an end-product inhibition of the overall transport rate. 6. It is apparent from the data that the flux of glucose across the unstirred layer is ordinarily the rate-limiting step in the trans-brush-border transport of this sugar by hamster jejunum when less than saturating concentrations of glucose are used. At high shaking rates the contribution of the unstirred layer is reduced.

Evidence for an intermediate step in carrier-mediated sugar translocation across the brush border membrane of hamster small intestine

The following observations have been made about the interactions of 6-deoxy-l-galactose with the Na+-dependent sugar transport system of hamster small intestine. 1. 1. 6-Deoxy-l-galactose does not appreciably enter the intracellular spaces of incubated intestinal segments and phlorizin is devoid of action on this minimal entry. 2. 2. 6-Deoxy-l-galactose is a competitive inhibitor of the Na+-dependent sugar transport system with a Ki of about 20 mM. 3. 3. 6-Deoxy-l-galactose does not elicit counterflow of a known substrate, l-glucose, under conditions where counterflow is elicited by a substrate, 1,5-anhydro-d-glucitol, of approximately equivalent affinity. 4. 4. The i of 6-deoxy-l-galactose increases with the reduction in Na+ concentration as does the Km of 3-O-methylglucose. Consequently, the ratio Km3-O-methylglucose/Ki 6-deoxy-l-galactose remains constant over a 5-fold range of Na+ concentration; that is, the competitive interaction of 6-deoxy-l-galactose is Na− dependent. 5. 5. 6-Deoxy-l-galactose acts like mannitol in transmural potential studies; that is, it induces a streaming potential only. It does not induce Na+ movement despite the fact that its carrier interaction is Na+-dependent. From these observations the inference is drawn that mobility of the carrier-substrate complex which results in translocation of a substrate involves a non-covalently bonding transformation of the complex; a transformation which cannot take place when 6-deoxy-l-galactose is bound to the carrier, 6-Deoxy-l-galactose forms an abortive complex.

Proteins and glycoproteins of the human intestinal brush border membrane.

Abstract Proteins associated with human jejunal and ileal epithelial brush borders and their subfractions were solubilized with dodecyl sulfate and separated by electrophoresis on acrylamide gels. Twenty-three bands, at least, have been found in the brush border membrane protein patterns, corresponding to a heterogeneous group of polypeptides of molecular weights ranging from 25 000 to over 400 000. The protein patterns obtained from jejunum and ileum are strikingly different. Thirty bands have been found in the protein subunit patterns with the most prominent band centered at 46 000–50 000 daltons. Two classes of proteins were present in the bursh border, the largest polypeptides belonging to the membrane, the smallest to the core fraction. About 13 glycoproteins with apparent molecular weight varying from 50 000 to over 400 000 have been detected in the brush border membrane. Most of them have been identified as membrane proteins.