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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies were performed to characterize the binding1 of bile acids to intestinal brush border membranes. Total 14C-taurodeoxycholate binding was: 1) similar for brush borders prepared from jejunum and ileum, 2) linear with respect to monomer concentration, 3) uninhibited by a structural analog, and 4) not depressed by boiling or trypsin. A linear relationship existed between binding and the number of hydrogen bonds formed by a bile acid and the slope of the line corresponded to delta deltaF of 300 cal/mol. The binding of bile acids to the 105,000 x g supernatant fraction of sonicated brush borders was similar to the binding of phospholipid liposomes using gel chromatography. These data suggest that: 1) the kinetics and characteristics of binding of bile acid to ileal brush borders do not reflect the kinetics and characteristics of active ileal transport previously obtained in whole tissue preparations, but instead reflect the kinetics and characteristics of passive jejunal transport; 2) a determinant of binding is hydrogen bonding with water; 3) isolated intact brush borders are relatively polar membranes; and 4) binding to solubilized brush borders may represent partitioning between the aqueous phase and membrane lipid.
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PMID:Characterization of bile acid binding to rat intestinal brush border membranes. 1 8

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.
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PMID:Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium. 4 35

A genetically conditioned mouse model of exocrine pancreatic insufficiency (epi) has been used to study the effect of the absence of lumenal proteases on small intestinal mucosal proteins. The small bowel was divided into eight equal segments. Enzyme activity was increased only in the first three segments in the case of maltase, sucrase, and lactase (all mol wt above 200,000). Alkaline phosphatase (mol wt 145,000), trehalase (mol wt 95,000), and peptidase (mol wt 175,000) activities were unaffected in proximal segments from epi mice. Proximal brush border proteins were identified and measured quantitatively by sodium dodecyl sulfate acrylamide gel electrophoresis. Those enzymes with increased activity were associated with increased amounts of protein in epi mice. Double labeled studies of protein turnover revealed a longer half-life for large brush border proteins (mol wt above 175,000) in epi mice than in normal mice. Enterokinase activity (a marker for duodenal mucosa) was nearly absent from the duodenum of epi mice. Receptors for the intrinsic factor-vitamin B12 complex (markers for ileal mucosal) were present in the ileum equally in normal and in epi mice. Enterokinase activity can be induced in epi mice by feeding its substrate trypsinogen, but not by trypsin or chymotrypsinogen. Epi mice thus retain the ability to synthesize enterokinase. Pancreatic proteases play an important role in the turnover of certain large mucosal proteins and in the induction of enterokinase.
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PMID:Effect of exchange exocrine pancreatic insufficiency on small intestine in the mouse. 20 83

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

Bacterial extracts were prepared from cultures originating in chronic self-filling intestinal blind loops in rats. Their ability to remove active maltase molecules from isolated brush border membranes was studied in vitro. Twelve strains in 51 tested, belonging to one of three species, Bacteroides fragilis, Clostridium perfringens, and Streptococcus fecalis, possessed maltase-releasing activity. The ability to remove maltase correlated well with the ability to hydrolyze p-nitrophenyl-tert-butyloxycarbonyl-l-alaninate (NBA), an ester substrate rapidly hydrolyzed by elastase, but not with substrated favored by tryhsin and chymotrypsin. Maltase-releasing activity from C. perfringens was strongly inhibited by soybean trypsin inhibitor and to a lesser extent by lima bean trypsin inhibitor. Of four chloromethylketone active-site directed inhibitors tested with specificities for elastase, trypsin, and chymotrypsin, inhibition was maximal with elastase-specific inhibitors. In two species, activity was shown to be heat sensitive, and to be inhibited by concentration of the extract. In one species maltase-releasing activity was shown to be due to an enzyme of molecular weight at least 66,000 with the capacity to remove lactase, sucrase, and alkaline phosphatase, as well as maltase. The results indicate that anaerobic or facultatively anaerobic species, previously identified with the pathology of of the blind loop syndrome, contain proteases which are capable of removing components of the intestinal surface membrane. These proteases appear to have elastase-like substrate specificity and may be involved in the etiology of disaccharidase deficiency in bacterial overgrowth syndromes.
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PMID:Pathogenesis of mucosal injury in the blind loop syndrome. 35

The [3H] phlorizin-binding component of brush border vesicles was enriched in situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [3H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [3H] phlorizin binding entity. Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassie-stained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [3H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile. Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.
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PMID:Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions. 52 29

Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.
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PMID:Identification of a mucosal form of enteropeptidase in triton X-100 extracts of porcine duodenal mucosa. 55 56

Previous studies have established the existence of IgG receptors on the endodermal cells of the fetal rabbit yolk sac membrane (YSM). The present study partially characterizes these cell-associated receptors. The specific binding of rabbit IgG (IgGR) to freshly prepared cell homogenates, nuclei-free brush border preparations, and plasma membrane-rich fractions confirms that receptor function is associated with the endodermal cell, and indicates that this function is localized on its apical brush border, specifically on its plasma membrane. The protein nature of the receptor is demonstrated by the loss of specific binding capacity after treatment of formalin-fixed YSM with papain and trypsin. Evidence has also been adduced which indicates that membrane carbohydrate is not involved in receptor function. Thus, treatment of YSM with neuraminidase, beta-galactosidase, mixed glycosidases, as well as oxidation of YSM with periodate all are without effect on its capacity to bind IgGR. The interaction of IgGR with the receptor elements of formalin-fixed YSM is partially ionic in character. NaCl reversibly inhibits binding of IbGR by 60%. Divalent ions such as Ca++ are not involved in this receptor-ligand interaction since EDTA-treated YSM binds IgGR to the same extent as do controls. Receptor material on fixed YSM resists extraction by non-ionic detergents, deoxycholate, and chaotropic agents.
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PMID:Characterization of IgC receptors of the fetal rabbit yolk sac membrane: localization to subcellular fraction and effects of chemical agents and enzymes on binding. 55 7

Rat kidneys were disaggregated with 0.25% trypsin. Cell were separated by velocity sedimentation in a previously described isokinetic gradient, by isopycnic sedimentation, and by velocity sedimentation followed by isopycnic sedimetation. In some fractions from the isokinetic gradient, 71.8+/-2.4+ of the nucleated cells contained histochemically demonstrable alkaline phosphatase (HDAP); in semithin sections, 62.7+/-2.3% of these cells had brush borders. The correspondence between fractions enriched for cells with HDAP and fractions enriched for brush border suggested that HDAP might be a suitable marker for rat proximal tubule cells. These cell constituted 46.5+/-2.6% of the nucleated cells in the starting sample suspension of kidney cells, and 81.9+/-2.7% of nucleated cells in the purified fractions from the gradients. More than 98% of nucleated cells in these fractions excluded typan blue. Following isopycnin centrifugation, the purest fractions contained 87.3+/-1.5% nucleated cells with HDAP, 9.6+/-2.5% nucleated cells iwithout HDAP, and 3.1+/-2.5% red blood cells. These proximal tubule cells had densities of 1.036 to 1.052 g/ml. With rate-zonal separation followed by isopycnic separation, the purest gradient fraction contained 93.0+/-1.9% nucleated cell with HDAP, 6.0+/-2.3% nucleated cells with HDAP, and 1.0+/-0.9% red blood cells. These proximal tubule cells sedimented a density of 1.041 g/ml. More than 98% of these cells excluded trypan blue.
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PMID:Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of Ficoll in tissue culture medium. 84 13

Intestinal goblet cell mucus (GCM) was added to incubations of casein and trypsin (or chymotrypsin) to discover whether mucus could inhibit proteolysis. Contrary to expectation, GCM stimulated casein hydrolysis, reaching a maximum effect at a GCM to casein ratio (w/w) of 0.083. GCM did not contain proteolytic enzymes or proenzymes as contaminants, nor did GCM serve as a substrate for trypsin. Stimulation was not reduced by removing 85% of the sialic acid from GCM. Harsh physical treatment (boiling and freezing) of casein decreased (50%) the GCM effect, as did partial predigestion of casein by trypsin, and elevation of trypsin concentration beyond 3 mug per ml. Thus the undegraded structure of casein appeared to be important for the stimulation of proteolysis by GCM. GCM also enhanced the hydrolysis by trypsin of intestinal brush border membrane protein, but had no effect on the hydrolysis of hemoglobin, albumin, or benzoyl arginine ethyl ester. These results suggest that GCM reacts with specific substrates, in a fashion which promotes their digestion by trypsin or chymotrypsin.
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PMID:Stimulation of proteolytic digestion by intestinal goblet cell mucus. 111 51


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