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)

We recently reported the isolation and identification of a Giardia lamblia-specific antigen (GSA 65) that is shed in the stool of giardiasis patients. In the present study, this antigen was affinity purified from sonic extracts of axenically cultured G. lamblia trophozoites and characterized to better understand its biological function and its potential usefulness in the design of coprodiagnostic assays for giardiasis. GSA 65 was resistant to proteolytic digestion with trypsin, chymotrypsin, and protease but was sensitive to treatment with NaIO4 as assessed by Western blotting. This antigen was also stable during prolonged storage at 4 and -20 degrees C in 10% Formalin or distilled H2O as assessed by counterimmunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing gel banding patterns, in conjunction with protein and carbohydrate assays and lectin binding studies, confirmed that this antigen is a highly glycosylated glycoprotein. The resistance of GSA 65 to proteolytic degradation, together with previous immunofluorescence data that indicate the antigen is an integral part of the G. lamblia cyst wall, suggests that this molecule may play a role in maintaining the integrity of the cyst in vivo. The ability of GSA 65 to maintain its antigenic structure under a wide variety of conditions makes it an ideal antigen around which to design sensitive immunodiagnostic assays for giardiasis.
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PMID:Physical and chemical characterization of a Giardia lamblia-specific antigen useful in the coprodiagnosis of giardiasis. 353 98

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.
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PMID:Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles. 382 12

The chemical structure of carcinoembryonic antigen (CEA) and two closely related antigens, normal fecal antigen-2 (NFA-2) in normal adult feces and nonspecific cross-reacting antigen-2 (NCA-2) in the meconium, were further analyzed comparatively. The NH2-terminal amino acid sequence of NCA-2 was newly determined to position 18 and found to be identical to that so far determined for CEA- and NFA-2. After proteolytic digestion with chymotrypsin or protease V8, the digests of these antigens showed two groups of fragments upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One consisted of the sharply banded fragments which were identical in all antigens and stained only with Coomassie brilliant blue (CBB) (five bands in the range 2500-10,000 daltons for chymotrypsin and 11 bands in the range 8000-35,000 daltons for protease V8, respectively), and the other consisted of the dispersed fragments which had variable mol. wts in the range 10,000-100,000 and were stainable with both CBB and periodic acid-Schiff reagent. Elution profiles of CEA, NFA-2, and NCA-2 from lectin columns, especially from concanavalin A-Sepharose columns, suggested some differences in oligosaccharide chains between them. These results indicate that the fundamental chemical structure of these antigens seems to be very similar to one another and is divided into two parts; an homologous portion(s) which is common to all three antigens and contains no sialylated sugar components, and a heterogeneous portion(s) which is variable among these antigens and contains sialylated sugar components.
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PMID:Further comparative studies on chemical properties of carcinoembryonic antigen in tumor tissues and closely related antigens in adult feces and meconium. 388 29

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.
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PMID:Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas. 404 99

The soybean Bowman-Birk inhibitor (BBI), a polypeptide of MW 8,000, has a specificity directed against trypsin and chymotrypsin. BBI was localized at the ultrastructural level by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledon and embryonic axis, BBI was found in all protein bodies, the nucleus and, to a lesser extent, the cytoplasm. Contrary to the Kunitz trypsin inhibitor (Horisberger and Tacchini-Vonlanthen 1983), BBI was not present in the cell wall but was found in the intercellular space. Intensity of marking in cotyledons of four-day-old seedlings was similar with the exception of the intercellular space which was free of BBI. In two lines lacking the Kunitz inhibitor (P.I. 157440 and 196168), data indicated that marking intensity was similar to that of cv. Maple Arrow. In contrast, in varieties lacking the lectin (Norredo, T-102) marking was more intense than in cv. Maple Arrow.
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PMID:Ultrastructural localization of Bowman-Birk inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method. 630 85

A chymotrypsin-derived and 125I-labelled 125-kDa fragment of human plasma fibronectin which contained the cell binding site, was only weakly bound by peritoneal macrophages of guinea pigs and binding was not saturable. In presence of wheat germ lectin binding increased proportionally to the logarithm of the lectin concentration. Association of 125I-fragment with cells was partially prevented by non-labelled fragment indicating a saturable receptor-ligand interaction. An apparent affinity constant of about 2--4 x 10(-5) M was evaluated. A considerable fraction of the cell-bound 125I-fragment resisted removal by proteases suggesting that it was internalized. In order to investigate an influence of wheat germ lectin on the binding of 125I-fibronectin by the cells the macrophages were preincubated with the lectin followed by washing and evaluation of 125I-fibronectin binding. A simultaneous incubation of the cells with 125I-fibronectin and lectin was impractical due to partial interaction of the two proteins giving rise to some unspecific precipitates. Preincubation with wheat germ lectin considerably improved the capacity of the macrophages for binding of 125I-fibronectin. Again the binding of 125I-labelled protein could be restricted by unlabelled one. N-acetyl-glucosamine inhibited the binding of 125I-fibronectin by wheat germ lectin-treated cells if applied in the preincubation phase and more effectively, if applied in the final 125I-fibronectin binding assay. N-Acetylneuraminic acid also inhibited this step. In addition to wheat germ lectin concanavalin A was capable of generating fibronectin receptors on the cell surface. Soy bean lectin, however, was ineffective.
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PMID:Generation of fibronectin receptors on macrophages by wheat germ lectin. 631 9

High-affinity binding sites for the potent 1,4-dihydropyridine calcium channel blocker [3H]-nimodipine were solubilized from guinea-pig skeletal muscle microsomes with digitonin and CHAPS [3-(3-cholamidopropyl)-dimethyl-ammonio-l-propanesulfonate]. Detergent-solubilized binding sites could not be sedimented by centrifugation (50,000 X g, 4 h), passed freely through 0.2 micron nitrocellulose filters and were stable at 4 degrees C with half-lives of greater than 60 h. The solubilized 1,4-dihydropyridine binding sites were precipitable with polyethyleneglycol 6000 on Whatman GF/C filters. Saturation analysis of solubilized microsomes with [3H]-nimodipine revealed a single class of binding sites (Bmax = 0.5 to 1.7 pmol per mg of protein) with a KD of 2.2-3.6 nmol/l at 37 degrees C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k-1 = 1.5 min-1, at 37 degrees C). The solubilized drug receptors discriminated between the optical enantiomers of chiral 1,4-dihydropyridine calcium channel blockers, (-)- and (+)D-600 as well as between l-cis and d-cis-diltiazem. d-cis-Diltiazem stimulated the binding of [3H]-nimodipine (ED50:3.6 mumol/l), by increasing the Bmax and slowed the dissociation rate of the labelled 1,4-dihydropyridine calcium channel blocker. The solubilized binding sites were sensitive to pronase, alpha-chymotrypsin and phospholipases A and C indicating their protein nature as well as their lipid requirement. Chelation of endogeneous divalent cations by EDTA, EGTA or CDTA inhibits high-affinity [3H]-nimodipine binding, demonstrating that divalent cations are required for high affinity [3H]-nimodipine binding. Detergent-solubilized binding sites are adsorbed by several sepharose-immobilized lectins, including concanavalin A, wheat germ agglutinin and lentil-lectin but not by helix pomatia lectin. Preparative chromatography on concanavalin A sepharose was performed and the adsorbed [3H]-nimodipine binding sites were selectively eluted by alpha-methylmannoside; NaCl (1 mol/l) being completely ineffective as elutant. The purification factors by this method were 17-40-fold. The binding sites could be also purified (up to 10-fold) by sucrose density centrifugation. The S20, w value of the drug receptors is 12.9 s. It is concluded that the 1,4-dihydropyridine binding sites of the putative calcium channel are intimately associated with carbohydrate containing structures. Since the detergent-solubilized material shows allosteric regulation of 1,4-dihydropyridine binding, interaction with chemically different classes of calcium channel blockers, metalloprotein nature and a S20, w value which is indicative of structure large enough to span the membrane, we conclude that we have solubilized and partially purified the putative calcium channel.
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PMID:Solubilization and partial purification of putative calcium channels labelled with [3H]-nimodipine. 631 49

The importance of the red cell membrane sialoglycoproteins in the invasion of P. falciparum merozoites has been assessed. Human erythrocytes deficient in glycophorin A (En(a-)cells) or B (S-s-U-, S-s-U+ cells) showed significant resistance to invasion. Treatment of normal erythrocytes with trypsin and chymotrypsin also reduced invasion. These results indicate that determinants carried on glycophorins A, B and C play an essential role in the successful invasion into human red cells. Sugar components present on glycophorin, in particular N-acetyl glucosamine and N-acetyl galactosamine, as shown by specific sugar and antibody inhibition studies, appear to act as important determinants for attachment to the erythrocyte. This implicates a protein(s) on the merozoite surface membrane which has the properties of a lectin.
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PMID:Merozoites of P. falciparum require glycophorin for invasion into red cells. 637 Apr 71

The complete amino acid sequence of the major alpha subunit of the lectin from seeds of Dioclea grandiflora was determined. The sequence was deduced from analysis of peptides derived from the native alpha subunit by digestion with trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and pepsin; and from larger peptides produced by digestion of the citraconylated protein with trypsin. The alpha subunit consists of a single polypeptide chain of 237 amino acids which differs from the sequence of concanavalin in 53 positions. Significant levels of heterogeneity were observed in five positions in the sequence.
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PMID:The complete amino acid sequence of the major alpha subunit of the lectin from the seeds of Dioclea grandiflora (Mart). 638 25

The amino sugars glucosamine, galactosamine and mannosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction. The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectin-like activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.
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PMID:Effect of amino sugars on platelet aggregation and on fibrinogen binding. 649 68


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