Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A macromolecule which binds intrinsic factor saturated with vitamin B12 has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor 'receptor' was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[57Co]vitamin-B12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[57Co]B12 alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [57Co]B12. The receptor did not bind intrinsic factor-[57Co]B12 below pH 5 while binding was observed to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[57Co]B12 to the receptor and this inhibition could be reversed by the addition of excess Ca2+. Mg2+ and Mn2+ were less effective than Ca2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B12/min with a Km of 1.36 x 10(-10) M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B12 than for intrinsic factor free of vitamin B12. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.
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PMID:Solubilization, partial purification and radioassay for the intrinsic factor receptor from the ileal mucosa. 1 Sep 57

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.
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PMID: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. 2 56

Crude preparations of hog gastric intrinsic factor or their own previously collected gastric juices administered with labeled vitamin B12 did not enhance vitamin B12 absorption in patients with vitamin B12 malabsorption secondary to pancreatic insufficiency. However, when these sources of gastric intrinsic factor were incubated with three times crystallized preparations of insolubilized bovine trypsin or chymotrypsin, the proteolytic enzymes were removed by centrifugation, and the preparations of gastric intrinsic factor were readministered to these patients, the absorption of vitamin B12 was markedly enhanced. Studies of hog gastric intrinsic factor before and after exposure to proteolytic enzymes failed to show any difference on Sephadex chromatography or polyacrylamide gel electrophoresis or on its affinity for vitamin B12 or the ileal receptor in guinea pigs. These investigations demonstrate that: (1) gastric intrinsic factor as secreted by subjects with pancreatic insufficiency or obtained from hog pyloric mucosal extracts is ineffective in promoting vitamin B12 absorption in patients with pancreatic insufficiency, (2) incubation of crude preparations of gastric intrinsic factor with insolubilized pancreatic proteases modified these preparations of gastric intrinsic factor in an as yet undefined manner, allowing them to enhance vitamin B12 absorption, and (3) in vitro studies using gut sacs or brush border preparations do not reflect the abnormality in vitamin B12 absorption associated with pancreatic dysfunction.
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PMID:Evidence that pancreatic proteases enhance vitamin B12 absorption by acting on curde preparations of hog gastric intrinsic factor and human gastric juice. 31 82

Pancreatic extract (PE) contained small-molecular, thermo-stable as well as macro-molecular, thermo-labile factors capable of reducing the uptake of 57CoB12 bound to rat intrinsic factor by perfused rat intestinal segments (p less than 0.01 and p less than 0.01). Neither non-radioactive vitamin B12 nor non-pacreatic protein reduced the 57CoB12-uptake (p greater than 0.5 and p greater than 0.1) Crystalline trypsin and trypsinogen, but not chymotrypsin, also inhibited the uptake (p less than 0.05, p less than 0.02 and p greater than 0.05). The tryptic inhibition was abolished by soybean trypsin inhibitor (p greater than 0.05).
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PMID:Pancreatic extract and the intestinal uptake of vitamin B12. II. Inhibitory effect of trypsin and trypsinogen. 84 84

Two groups of biological methods are commonly used to evaluate the exocrine pancreatic function: tests which require tubes for the collection of duodenal juice and the tubeless tests which are indirect tests of pancreatic function. In this study we have attempted to improve a new test: the test of haptocorrin degradation (THD). This test measures the transfer of labelled cobalamin from haptocorrin to the intrinsic factor which is provoked by the degradation of the haptocorrin by proteases in the duodenal juice. We present the results of this test in 90 patients with chronic pancreatitis. THD was first assayed with basal duodenal juice collected by naso duodenal tubing during secretin cerulein stimulation. In this study the sensitivity and specificity of THD was 0.86 and 0.93, respectively. In the second part of this study we demonstrated that the means of collecting duodenal juice had no effect on the results of THD. Duodenal juice was collected during a secretin cerulein test or during a routine upper gastrointestinal endoscopy after pancreatic stimulation with secretin. The sensitivity and specificity of THD was 0.90 and 0.94, respectively, when duodenal juice was collected during endoscopy. THD was significantly correlated with the NBT-PABA test, steatorrhea, and with the activity of trypsin and chymotrypsin in the duodenal juice. In this study, NBT-PABA was less sensitive than THD for the diagnosis of chronic pancreatitis (sensitivity was 0.70 and 0.89, respectively). The specificity of THD was estimated at 0.94. THD seemed to be a valuable adjunct to test pancreatic function. As upper gastrointestinal endoscopy is usually performed in patients with proved or suspected chronic pancreatitis, THD seems to have a place of choice among the other tests of pancreatic exocrine function. Further evaluation of this test by a multicentric prospective trial is now needed.
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PMID:[Evaluation of exocrine pancreatic function by the haptocorrin degradation test of the duodenal fluid collected by endoscopy]. 273 91

An in-vitro test of degradation of haptocorrin, a cobalamin-binding glycoprotein, was used to diagnose exocrine pancreatic dysfunction. This radioisotopic test (TDH) required only 50 microliters duodenal juice collected during endoscopy after stimulation with 1 U/kg secretin intravenously. The initial reaction mixture, composed of salivary haptocorrin saturated with cobalt-57-labelled cyanocobalamin and unsaturated intrinsic factor, was incubated with 25 microliters duodenal juice. The percentage of degraded haptocorrin was estimated from the proportion of labelled cyanocobalamin that was transferred from haptocorrin to intrinsic factor. The TDH result was 41.6 +/- 31.7% (SD) in a group of chronic pancreatitis patients (n = 22) and 91.5 +/- 4.8% in the control group (n = 47). The sensitivity and specificity for exocrine pancreatic dysfunction were estimated as 0.91 and 0.96, respectively, for a lower limit of normal values of 81.7%. A hyperbolic relation was found between the TDH and the trypsin or chymotrypsin activity in duodenal juice (p less than 0.001). In this study, the N-benzoyl-tyrosyl-p-aminobenzoic acid test was less sensitive than the TDH, since its result was abnormal in only 64% of the patients. The TDH was easier to carry out and less time-consuming than the determination of pancreatic enzyme output in duodenal juice collected after hormonal stimulation.
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PMID:In-vitro test of haptocorrin degradation for biological diagnosis of exocrine pancreatic dysfunction using duodenal juice collected during endoscopy. 287 85

The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.
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PMID:Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor. 305 9

the absorption of vitamin B(12) in many animals requires its prior association with intrinsic factor (IF) and attachment to a specific receptor in the intestine. Employing Triton X-100, we have solubilized from guinea pig ileum a factor that binds intrinsic factor-vitamin B(12) complex (IF-B(12)). This binding factor was soluble to the extent that it was not sedimented by centrifugation at 100,000 g for 1 h and was small enough to enter the included volume of a Sepharose 4-B column. Furthermore, the ileal extract contained no microfine particles of membrane upon electron microscopic search. When a portion of the extract was incubated with a mixture of gastric juice and (57)Co-labeled vitamin B(12), a portion of the radioactivity was excluded from a Sephadex G-200 column. When gastric juice from a patient with a congenital abnormality of IF that prevented its binding to intestine was substituted for normal human gastric juice, radioactivity was not excluded from the gel, indicating failure of this abnormal IF-B(12) to bind to the intestinal extract. These data suggested the presence of a specific binder of IF-B(12) in the ileal mucosal extract. The reactions of normal IF-B(12) with the solubilized binding factor and with the membrane-bound "receptor" had several characteristics in common, including calcium dependence, temperature independence, and pH optimum near neutral. Extracts from the distal intestine showed more activity than did those from the proximal. The solubilized binding facter seemed specific for IF-B(12) in that it was not blocked by prior incubation with excesses of either free vitamin B(12) or IF. Binding activity of the extract was decreased by incubation at pH 2.0, by heating to 56 degrees C, and by incubation with chymotrypsin and dithiothretiol. Incubation with trypsin, neuraminidase, and sulphydryl blockers did not affect it. The Triton X-100 extract of guinea pig ileal mucosa contains a specific binding factor that probably is the receptor for IF-B(12). This appears to be a protein with function dependent on peptide and disulphide linkages.
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PMID:Solubilized receptor for intrinsic factor-Vitamin B12 complex from guinea pig intestinal mucosa. 485 16

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.
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PMID:The cobalamin-binding protein in zebrafish is an intermediate between the three cobalamin-binding proteins in human. 2253 67

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.
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PMID:A single rainbow trout cobalamin-binding protein stands in for three human binders. 2287 37


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