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Biomedical subjects

B Seetharam

Publications and source records attributed to B Seetharam.

At least 73 records · Page 4Linked to original sources
PubMed1986

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.

Animals↗
PubMed1986

Perthes' disease in South India.

One hundred sixty-five cases of Perthes' disease were observed during a 15-year period. The mean age at onset of symptoms was 9.89 years for boys and 8.71 years for girls. The overall male:female ratio was 2.58:1, with profound regional differences. Bilateral involvement was seen in ten children, and the right hip was more frequently affected in unilateral cases (R:L, 92:63). Fixed deformities were noted in 14 children. Profound restriction of hip movement was present in seven cases, and in three there was no movement at all. Seventy percent of the cases fell into Catterall's Group 3 or 4 and 20% could not be classified into any of the groups. Sixty percent of the children presented during the initial stage of the disease. One group of these children had features resembling tuberculosis, with fixed deformities, gross restriction of movement, and osteoporosis around the acetabulum. Their symptoms resolved without antitubercular treatment and their late radiologic features were those of healed Perthes' disease. Treatment planning was problematic because of the advanced age at onset, large proportion of girls, frequency of extensive involvement of the epiphysis, and socioeconomic factors.

Child↗
PubMed1985

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups.

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.

Alkaline Phosphatase↗
PubMed1985

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.

Animals↗
PubMed1985

Intestinal uptake and release of cobalamin complexed with rat intrinsic factor.

The mechanism of uptake of intrinsic factor (IF) and cobalamin (Cbl) by enterocytes and their subsequent fate have been uncertain. To examine this problem double-labeled IF X Cbl was added to small intestinal organ cultures. When 125I-IF X [57Co]Cbl was added to rabbit ileal explants, binding and internalization increased linearly for 24 h. After an 18-h chase with nonlabeled IF X Cbl, no 125I-IF returned to the cell surface. An amount of 35-45% of the internalized Cbl was found free, not bound to IF or any other protein. About 60% of both internalized ligands was bound to membranes but by a non-Ca2+-dependent bond, suggesting binding to a protein other than the brush-border receptor. Cobalamin was released from IF at pH 5.0 to the same degree (30%) as free Cbl was found inside the cell (35-45%). Neither pancreatic proteases nor ileal homogenates effected release of Cbl from IF. When cathepsins were added, the Cbl released was no greater than could be attributed to pH 5.0 alone. Chloroquine added to tissue explants did not alter the percentage of free intracellular Cbl. From these results we suggest that IF X Cbl is internalized and detached from the receptor within the enterocyte. The mechanism of release is not known but seems to require an acid pH (5.0). The Cbl is released in the mucosa, perhaps when the IF X Cbl complex enters a nonlysosomal cellular compartment with an acidic environment. There is no substantial recycling of IF to the brush-border membrane.

Animals↗
PubMed1984

Immunocytochemical localization of the intrinsic factor-cobalamin receptor in dog-ileum: distribution of intracellular receptor during cell maturation.

Absorption of cobalamin is facilitated by the binding of the intrinsic factor-cobalamin complex (IF-cbl) to specific receptors in the ileum. The physical and biochemical characteristics of this ligand-receptor binding reaction have been extensively studied, but little is known about the cellular mechanisms or receptor synthesis, intracellular transport, and expression on the microvillus surface membrane. We attempted to delineate these mechanisms by using ultrastructural immunocytochemistry to localize the IF-cbl receptor in the crypt, mid-villus, and villus tip regions of mucosal biopsies obtained from the ileum of anesthetized dogs. Prior to initiating the ileal localization studies, the antisera to purified canine IF-cbl receptor that was employed in our studies was shown to have specificity for site (e.g., ileal enterocytes vs. other cells within the gastrointestinal tract) and immunohistochemical specificity. Receptor synthesis in endoplasmic reticulum begins in crypt enterocytes, but continues in cells throughout the villus. In the mid-villus region synthesized receptor translocates vectorially to the microvillus surface associated with membranous vesicles and then inserts into the microvillus pit. Receptor remains fixed to the microvillus pit and does not distribute uniformly over the brush border membrane. All villus tip enterocytes contained IF-cbl receptor in microvillus pits, vesicles, and endoplasmic reticulum, but in addition extensive perinuclear membrane staining was evident as well as re-internalized receptor associated with multivesicular bodies. Basolateral membranes contained no receptor at any level of the villus. These observations suggest that the IF-cbl receptor (a) translocates to the apical cell surface at the mid-villus region by transport in vesicles, (b) directly inserts into and then remains fixed in microvillus pits, (c) is elaborated on the luminal surface most extensively in villus tip cells, and (d) although reinternalized, does not move IF and/or cbl to the basolateral cell surface.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Effect of bile and bile acids on binding of intrinsic factor to cobalamin and intrinsic factor-cobalamin complex to ileal receptor.

Human bile or commercially available bile acids bind [57Co]cobalamin (Cbl) due to the presence of "R"-type binders, which are normally present in the former and present as a contaminant in the latter. The competition of these R proteins for the binding of Cbl by intrinsic factor (IF) could explain the in vivo inhibition attributed previously to the bile acids themselves. R protein seems to be involved in the inhibition of Cbl binding because protease digestion of either bile or bile acid abolishes the Cbl binding ability. Moreover, antibody to R protein abolishes the inhibition. Bile or bile acids do not have a direct effect on either purified IF or the IF-Cbl receptor molecule, even though bile acids increase the attachment of IF-[57Co]Cbl to ileal brush-border membranes. These data demonstrate two steps where components of bile could affect Cbl absorption: the binding of Cbl to IF and of IF-Cbl to its ileal receptor. It is not clear whether these in vitro phenomena are important for the normal absorption of Cbl in vivo.

Animals↗
PubMed1982

Absorption and transport of cobalamin (vitamin B12).

This review apprises the reader of recent advances in cobalamin absorption and transport since 1970. This field has been of interest to gastroenterologists, hematologists, and biochemists as well as nutritionists. Clinical conditions leading to cobalamin deficiency present especially to the first two of these specialties. Many recent advances involve biochemical definition of the specific transport proteins themselves. Full understanding of the multiple clinical conditions associated with cobalamin deficiency requires some knowledge of these proteins. We do not discuss every paper in the field, especially the early literature (see 47, 61, 150). Despite rapid strides in understanding the mechanism of absorption and transport of cobalamin both in prokaryotic and eukaryotic cells, the complete sequence of events during cellular absorption at the plasma membrane level and events in the cells that mediate intracellular movement of cobalamin have not been elucidated completely. Here we emphasize the current state of knowledge about cobalamin absorption and transport in man and other mammalian systems, particularly the physiological role and biochemical nature of proteins involved (intrinsic factor, intrinsic factor-cobalamin receptor, transcobalamins).

Animals↗
PubMed1981

Interaction of receptor for intrinsic factor-cobalamin complex with synthetic and brush-border lipids.

The intestinal receptor for intrinsic factor-cobalamin complex integrates preferentially with cationic liposomes in such a way that its ligand binding sites are exposed to proteases and to antibody to the receptor. After integration the receptor could be solubilized from the liposomes by detergents and chaotropic salts. Kinetically, the brush-border and the liposome-bound receptor behaved similarly, and Triton X-100 had no effect either on Ka or Vmax, suggesting the absence of any effects of a lipid domain on ligand binding activity. However, acidic phospholipids caused inhibition when added to the receptor-ligand assay system in the absence of liposomes.

Animals↗
PubMed1980

Turnover of intestinal brush-border proteins during postnatal development in rat.

Protein turnover in brush-border membranes of rats during postnatal development has been studied by the double isotope technique. Unlike adult animals where only large proteins (mol wt > 140,000) show relatively rapid turnover, most brush-border proteins in 12-day-old rats show high 3H-to-14C ratios of leucine incorporation, consistent with rapid turnover. Lysosomal proteases, including cathepsin B, are partly responsible for this rapid turnover. This conclusion is based on the following findings: 1) In vivo treatment of 12-day-old animals with leupeptin, an inhibitor of cathepsin B, alters relative turnover rates, enzyme activity, and content of many brush-border proteins. Activities of maltase and trehalase rise while lactase falls. 2) Cathepsin B activity falls rapidly in intestine after the animals are 16 days of age, at a time when luminal pancreatic proteases are rising. Moreover, cathepsin B activity shows less latency in distal intestine at 12 and 16 days than at later ages or in proximal intestine. It is suggested that during postnatal development lysosomal enzymes, e.g., cathepsin B, play an important role in the turnover of intestinal brush-border proteins.

Aging↗
PubMed1980

Effect of pancreatic proteases on intestinal lactase activity.

The sensitivity of human intestinal lactase to pancreatic proteases was tested both in vitro and in vivo. Lactase specific activity in brush border membranes was decreased by 26%-27% during incubation with trypsin at pH 7.0 in patients with normal intestinal lactase levels, whereas in patients with lactase deficiency the inactivation was 75%. However, when lactase levels from deficient patients' mucosa were increased relative to trypsin during incubation so that they were comparable to the levels of activity in normal mucosa, inactivation of lactase in deficient patients was only 45%. Therefore, in these patients the greater in vitro lactase inactivation by trypsin could be explained at least in part by an increased trypsin/lactase ratio. Sucrase levels were decreased in vitro by trypsin (about 40%), but maltase activity was unaffected. The effect of pancreatic proteases was tested in vivo in patients with pancreatic insufficiency. After the addition of pancreatic enzymes (Viokase), lactase specific activity fell by 16% in patients with normal lactase, and by 38.5% in patients with lactase deficiency. In both groups of patients, lactase levels fell to a greater extent than did sucrase or maltase. These data demonstrate that pancreatic proteases can alter intestinal lactase activity in humans. Moreover, in lactase-deficient patients, lactase activity decreases to a greater extent than in patients with normal lactase, resulting in further deficiency of this enzyme.

Adult↗
PubMed1978

Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

Cobalamin (Cbl; vitamin B(12)) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R-type Cbl-binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50- and 3-fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t((1/2))'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein-Cbl/IF-Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150-fold decrease in its affinity for Cbl. Incubation of R protein-Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein-[(57)Co]Cbl and (125)I-R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. THESE STUDIES SUGGEST THE FOLLOWING: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.

Carrier Proteins↗