Bellur Seetharam

Development of intestinal brush border membrane proteins in the rat.

1. The proteins of the intestinal microvillus membrane have been studied during post-natal development in the rat (days 12--37). 2. In suckling animals (up to age 20 days), the majority of alkaline phosphatase, glucoamylase and lactase activities in the distal half of the intestine were located in the supernatant fraction (100000 X g, 60 min). These enzymes were attached to the membrane from the proximal intestine at all ages. 3. Alkaline phosphatase, maltase and lactase activities in the supernatant fractions chromatographed in Sephadex G-200 in positions similar to the corresponding membrane enzyme. Corresponding activities for lysosomal counter-parts of maltase and lactase present in the supernatant fraction chromatographed differently. Moreover, pH optimum of the soluble enzymes was 9.2 for phosphatase and 5.5--6.0 for glycoamylase and lactase. The soluble lactase and alkaline phosphatase were inhibited minimally by p-chloromercuribenzoate, and sodium fluoride respectively. L-Phenylalanine (20 mM) did inhibit the soluble phosphatase by 90%. Thus, the soluble enzymes are not mainly of the lysosomal origin, but have characteristics of membrane-bound enzymes. 4. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed 18 protein bands which were present in adult membranes. Two other proteins were unique for membranes of distal intestine in suckling rats. The proteins corresponding to known enzyme activity changed as expected with age (e.g. sucrase, maltase increased, lactase decreased). Most of the other proteins were also altered in amount during development. Thus, the changes in the microvillus membrane during development in the rat are not limited to specific enzymes.

Binding of intrinsic factor to ileal brush border membrane in the rat.

The rat ileal brush border membrane binds both free [125I]-intrinsic factor (IF) and the IF-[57Co]cobalamin (cbl) complex. This binding is observed with IF isolated from rat stomach, but not from IF isolated from hog, canine and human stomachs. The binding of rat-IF[57Co]cbl can be blocked with free rat IF but not with hog IF. The IF-cbl complex binds at a higher affinity (Ka = 0.15 X 10(9) M-1) compared to that of free IF (Ka = 0.9 X 10(9) M-1). Rat IF-cbl also binds efficiently to human and canine ileal membranes. While antibody to the canine ileal receptor blocks the binding of rat, human or hog IF-[57Co]cbl to human and canine ileal membranes, it does not affect the binding of rat IF-[57Co]cbl to rat ileal membranes. These findings demonstrate that the rat ileal receptor is different from canine and human ileal receptors.

Phase separation of rat intestinal brush border membrane proteins using Triton X-114☆

Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4 degrees C. Upon phase separation at 32 degrees C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and Ca2+-Mg2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Abstract Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.

Immunohistochemical survey of cobalamin-binding proteins.

Dietary cobalamin is bound to intrinsic factor (IF) and nonintrinsic factor (R protein) in the gastrointestinal tract. Distribution of IF has previously been described only in the stomach (parietal, chief cells), duodenum (mucous cells), and pancreatic juice. Cells containing R protein have been reported to be more widespread, including mucin-secreting cells of the intestinal tract. To further clarify the cells producing these cobalamin binders in the various organs, we surveyed a number of tissues in four species, with monospecific antisera raised against rat IF and hog R protein, using indirect immunoperoxidase histochemistry. A positive staining pattern was confirmed after blocking with the appropriate protein purified to homogeneity. Strong IF staining was noted over chief cells in the rat stomach, with weaker but substantial staining in the parietal cells as well. Intrinsic factor was found only in parietal cells in all other species examined. R protein was found in parietal cells of all species tested, except in the hog, where mucous cells of the basal crypt stained. The area of the brush border and apical cytoplasm in small intestinal epithelial cells was also positive for both IF and R protein, probably reflecting uptake. Accessory digestive organs, such as salivary gland and pancreas were also positive for both IF and R protein in the secretory ducts. Accessory digestive organs contribute to the secretion of IF as well as R protein, but the physiologic importance of this IF secretion is not clear.

Hydrophobic interactions of brush border alkaline phosphatases: The role of phosphatidyl inositol

Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.

Overexpression of an unstable intrinsic factor-cobalamin receptor in Imerslund-Gräsbeck syndrome.

Two sisters with Imerslund-Gräsbeck syndrome who presented with clinical features of cobalamin deficiency are described. Intrinsic factor-cobalamin receptor (IFCR) activity and protein levels were determined in ileal biopsy specimens by using radioisotope assay and immunoblotting, respectively. IFCR activities in ileal homogenates expressed as femtomoles of ligand binding per milligram of protein were 38 +/- 4 in control tissue, 494 +/- 24 in patient 1, and 94 +/- 7 in patient 2. However, when assayed in the presence of IFCR antiserum, the ligand binding was inhibited by > 90% in both normal control and the patients with Imerslund-Gräsbeck syndrome. Immunoblotting of total membranes from the biopsy specimen of these 2 patients failed to detect an immunoreactive band of molecular mass of 185 kilodaltons. These findings are at variance with reports of decreased IFCR activity and indicate a new phenotype in which an active but an unstable receptor is overexpressed in Imerslund-Gräsbeck syndrome.

Effect of lectins on the cobalamin-protein binding reactions: implications for the tissue uptake of cobalamin

Plant lectins have been thought to impair nutrient absorption, both by specific and nonspecific interference in the absorptive process. The main objective of this investigation was to study the effect of lectins on the various binding reactions involving cobalamin (cbl)-protein complexes and their receptors, and to identify the rate-limiting step important in maintaining tissue levels of cobalamin. Among the lectins tested in vivo, only concanavalin A (ConA) was able to inhibit the transport of cobalamin to the tissues and caused a 70% to 75% inhibition of [(57)Co] cobalamin transported to the liver and kidney. The inhibition of transport to the tissues was independent of route of administration of cobalamin, whether intragastric or systemic, and was not due to decreased gastrointestinal uptake. When tested in vitro, concanavalin A inhibited the binding of transcobalamin II-cbl to its receptor, but not the binding of cobalamin to intrinsic factor or intrinsic factor-cobalamin complex to the ileal receptor. These results suggest that late events during transcellular transport of cobalamin through the enterocytes is the rate-limiting step determining tissue levels of cobalamin and that ConA inhibits these latter events.

Interaction of Intestinal Disaccharidases with Phospholipids: Effect of Cholesterol

Although the rat intestinal brush border disaccharidases are the most easily solubilized protein components, the nature of the lipid-protein interactions in the membrane is incompletely understood. Phospholipid vesicles were prepared using the lecithin fraction from brush border membranes and synthetic lecithins. Addition of cholesterol to brush border lecithins enhanced the binding of disaccharidases, but not of alkaline phosphatase. The addition of cholesterol to synthetic lecithin vesicles enhanced the binding of disaccharidases only when added above the transition temperature of the lecithin used. The maximal effect occurred at an equimolar ratio of lecithin to cholesterol. Binding of disaccharidases to phospholipid vesicles was independent of charge or the nature of the polar head group, and the enzyme was inserted so that the catalytic domain was excluded from the lipid matrix. These results demonstrate that membrane attachment of disaccharidases is hydrophobic, involving primarily fatty acyl chains and an interaction with cholesterol. The membrane interaction does not seem to affect enzyme activity.

How does cobalamin (vitamin B12) enter and traverse mammalian cells

AbstractsThis overview is intended to present recent information available on the work done in understanding the mode of entry of cobalamin into and out of mammalian cells. I have focused primarily on the uptake of cobalamin bound to 3 proteins: intrinsic factor, transcobalamin II and R-proteins. Even though the nature of these cobalamin binding proteins, and the receptors involved in the internalization of these proteins is known, a total picture on the intracellular trafficking of cobalamin and the binding proteins is far from complete.