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)

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.
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PMID:Peroxidative modification of phospholipids in myocardial membranes. 235 24

Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells. 241 30

The Thomsen-Friedenreich antigen (T-antigen) is a cryptic disaccharide structure on human erythrocytes and is supposed to be expressed in an unhidden form on carcinoma cells. We tested the ability of four anti-T reagents (i.e., peanut agglutinin, human and rabbit anti-T antisera, and monoclonal anti-T antibodies) to agglutinate neuraminidase treated human erythrocytes and compared their capacity to bind to the surface of human carcinoma cell lines or neuraminidase treated lymphocytes. We found that all of the reagents strongly agglutinated neuraminidase treated erythrocytes. In contrast, only peanut agglutinin and the monoclonal antibody bound to the surface of carcinoma cell lines and to neuraminidase treated lymphocytes. Peanut agglutinin was inhibited by D-galactose and is known to be specific for the T-disaccharide. The determinants on erythrocytes, lymphocytes, and carcinoma cells, recognized by peanut agglutinin, are resistant to trypsin treatment. The monoclonal antibody was specifically inhibited by phenyl-beta-D-galactoside. The binding sites on erythrocytes and lymphocytes for the monoclonal antibody can be removed by treatment with trypsin or Pronase. On the other hand, the binding sites on carcinoma cells are resistant to trypsin but can be removed with Pronase. In contrast to the peanut agglutinin binding sites on carcinoma cells the structures recognized by the monoclonal antibody cannot be further increased by neuraminidase treatment. Human and rabbit anti-T antisera did not bind to tumor cell surface or to neuraminidase treated lymphocytes. Hemagglutination of human anti-T could be inhibited by asialofetuin; the specificity of rabbit anti-T could not be established in this study. Hemagglutination with both antisera is resistant to trypsin but partially sensitive to Pronase treatment. These results indicate that each of the reagents has a distinct specificity and recognizes different antigenic determinants on different molecules. Only peanut agglutinin and monoclonal anti-T antibodies are able to detect common structures on the surface of neuraminidase treated erythrocytes and carcinoma cell lines.
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PMID:Specificity of reagents directed to the Thomsen-Friedenreich antigen and their capacity to bind to the surface of human carcinoma cell lines. 241 54

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.
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PMID:Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. 242 14

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage-restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180-kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage-restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent-solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase-treated Daudi cells yielded a 130-kDa lentil lectin-binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDa) membrane proximal domain containing both of the common LC determinants and at least one N-linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.
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PMID:Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule. 242 43

At equilibrium, voltage-sensitive sodium channels normally are closed at all potentials. They open transiently in response to changes in membrane voltage or chronically under the influence of certain neurotoxins. Covalent modifications that result in chronic opening may help identify molecular domains involved in conductance regulation. Here, the purified sodium channel from electric eel electroplax, reconstituted in artificial liposomes, has been used to screen for such modifications. When the liposomes were treated with the alkaloid neurotoxin batrachotoxin, sodium-selective ion fluxes were produced, with permeability ratios PNa greater than PTl greater than PK greater than PRb greater than PCs. When the liposomes were treated with either of two oxidizing reagents (N-bromoacetamide or N-bromosuccinimide), or with Pronase or trypsin, ion-selective fluxes also were stimulated. These were blocked by tetrodotoxin and the anesthetic QX-314 in a manner suggesting that only modification of the cytoplasmic protein surface resulted in stimulation. Limited exposure to trypsin resulted in strong flux activation, with the concomitant appearance of peptide fragments with masses of approximately equal to 130, 70, and 38 kDa and fragments with masses of 45 and 24 kDa appearing later. We propose that characterization of these fragments may allow identification of channel domains important for inactivation gating.
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PMID:Reconstituted voltage-sensitive sodium channel from Electrophorus electricus: chemical modifications that alter regulation of ion permeability. 244 55

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.
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PMID:A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete. 244 70

Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.
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PMID:The peanut lectin-binding glycoproteins of human epidermal keratinocytes. 245 53

The binding of colicin E1 and its COOH-terminal channel-forming peptides to artificial membrane vesicles has an optimum at acidic pH values. The Mr 18,000 thermolytic peptide inserted into membrane vesicles at pH 4.0 has a limited accessibility to exogenous protease. It is converted by trypsin cleavage after Lys-381 and Lys-382 to a lower Mr 14,000 peptide. However, when the pH of a vesicle suspension to which peptide has been bound at pH 4.0 is shifted to 6.0, the accessibility to protease increased greatly. This was shown (i) by the large decrease in the amount of Mr 14,000 or Mr 18,000 peptide after the pH 4----6 shift and treatment with trypsin or Pronase, consistent with (ii) a previously observed decrease in membrane-bound radiolabeled peptide after protease treatment. (iii) When a photoactivable nitrene-generating phospholipid probe was used to label the colicin peptide inserted into the bilayer, the extent of labeling decreased by a factor of 3 when the pH was shifted from 4.0 to 6.5. (iv) Colicin peptide added to vesicles at pH 4.0 can "hop" to other vesicles if the pH and ionic strength of donor vesicles are successively increased. It is proposed that deprotonation of acidic residues in contact with the hydrophobic bilayer or the membrane surface destabilizes the inserted channel and causes it to be extruded from the membrane. The pH-dependent extrusion of the inserted colicin channel provides an example of dynamic properties of an intrinsic membrane protein.
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PMID:Dynamic properties of membrane proteins: reversible insertion into membrane vesicles of a colicin E1 channel-forming peptide. 245 8

1. Limited proteolytic digestion of rat liver microsomes (microsomal fractions) with trypsin (5 micrograms/ml), proteinase K (1.0 microgram/ml) and Pronase (20 micrograms/ml final concns.) resulted in abolition of GTP-dependent vesicle fusion. 2. Vesicle fusion could be partially restored to microsomes which had undergone limited tryptic digestion, by the addition of untreated microsomal vesicles. 3. GTP-dependent Ca2+ efflux from rat liver microsomes was also observed to be inhibited by limited proteolysis with trypsin and proteinase K. 4. Limited proteolysis of rat liver microsomes had no effect on subsequent GTP-dependent phosphorylation of polypeptides of Mr 17,000 and 38,000, and thus it is unlikely that the phosphorylation of these proteins is involved in GTP-dependent Ca2+ efflux and GTP-dependent vesicle fusion. 5. GTP binding by Gn proteins [proteins which bind GTP after transfer to nitrocellulose, as defined by Bhullar & Haslam (1986) Biochem. J. 245, 617-620] was inhibited by pre-treatment of microsomes with trypsin, proteinase K and Pronase at concentrations similar to those which abolished GTP-dependent Ca2+ efflux and vesicle fusion. 6. We suggest that one or more of the Gn proteins may be involved in the molecular mechanisms of GTP-dependent vesicle fusion and Ca2+ efflux in rat liver microsomes and that limited proteolytic digestion may be a useful tool in further investigation of these processes.
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PMID:The effect of limited proteolysis on GTP-dependent Ca2+ efflux and GTP-dependent fusion in rat liver microsomal vesicles. 249 9


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