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)

The effect of sodium sulphite, cysteine, glutathione, mercaptoethanol and dithioerythritol (0.1-10 mmol l-1) on the activity of proteases of Microsporum gypseum was studied using azocasein, cross-linked bovine serum albumin and keratin as substrates. With the substrate without disulphide bonds (casein) no stimulation was found, and reducing agents inhibited proteolysis in most cases. With the remaining two substrates, all substances enhanced the activity of proteases probably through the cleavage of the substrate disulphide bonds. Sulphite was more effective than the four used thiols and enhanced the activity against serum albumin up to 3.2 times and against keratin up to 2.9 times. Using sulphitolysed sheep wool, keratinolytic activity increased after sulphitolysis of more than 20% of disulphide bonds. With the fully sulphitolysed wool the activity increased 43 times. The obtained results support the author's hypothesis on keratin degradation by sulphite excretion prior to attack by fungal proteases. Stimulation of proteolysis and keratinolysis by cleavage of disulphide bonds is not specific for dermatophytic proteases because trypsin and pronase behaved similarly in the experiments.
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PMID:Effect of reducing agents on proteolytic and keratinolytic activity of enzymes of Microsporum gypseum. 128 10

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.
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PMID:Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). 131 59

Protease activity is associated with the coadhesion of Actinomyces viscosus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative functions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesion assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was measured by the decrease in turbidity caused by the absorption of A. viscosus cells from aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesion. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproteolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivalis cells and fimbriae, by preimmune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzymatically "prime" A. viscosus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.
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PMID:Inhibition of Actinomyces viscosus--Porphyromonas gingivalis coadhesion by trypsin and other proteins. 132 96

Oxidative damage to bovine serum albumin (BSA) was induced by hydroxyl radical (HO.) generating systems of xanthine oxidase (XO) + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. Formation of bityrosine and loss of tryptophan were observed in the ascorbate + EDTA-Fe3+ system and carbonyl formation was induced by both systems. Mannitol and ethanol very strongly inhibited the carbonyl and/or bityrosine formation, indicating that the oxidative damage to BSA was due to HO(.). The sulfhydryl (SH) groups of BSA were very sensitive to the XO + EDTA-Fe3+ but not to the ascorbate + EDTA-Fe3+ system. Catalase but not hydroxyl radical scavengers or superoxide dismutase strongly inhibited the loss of SH groups, indicating that H2O2 is involved in their oxidation. Fragmentation of BSA was observed during exposure to the XO + EDTA-Fe3+ and ascorbate + EDTA-Fe3+ systems and the products presented a broad band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Little formation of amine groups was observed in these systems, indicating that little peptide bond cleavage occurred. BSA exposed to the ascorbate + EDTA-Fe3+ system was more readily degraded by trypsin than that exposed to the XO + EDTA-Fe3+ system. Elastase degraded BSA exposed to the ascorbate + EDTA-Fe3+ system but not to the XO + EDTA-Fe3+ system.
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PMID:Oxidative damage to bovine serum albumin induced by hydroxyl radical generating systems of xanthine oxidase + EDTA-Fe3+ and ascorbate + EDTA-Fe3+. 133 12

Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. 134 62

T-kinin (Ile-Ser-Bradykinin) has been isolated only from the plasma of the rat and it is unclear whether the peptide, or its biosynthetic precursor, T-kininogen, circulates in the human. An NH2-terminally directed antiserum to T-kinin was raised in rabbits using an immunogen prepared by coupling the free -SH group of T-kinin extended from its COOH-terminus by a cysteinyl residue to an -NH2 group on human serum albumin. A radioimmunoassay was developed using this antiserum and 125I-labelled [Tyr10]T-kinin as tracer that was sensitive (least-detectable concentration 3 fmol/tube) and relatively specific for T-kinin (cross-reactivity with bradykinin and kallidin less than 1%). Treatment of rat plasma with an excess of trypsin in the presence of a kininase inhibitor generated T-kinin immunoreactivity equivalent to 455 +/- 71 pmol/ml (mean +/- S.E.M.; n = 9) and this immunoreactivity was eluted from a reversed-phase HPLC column as a single peak with the same retention time as synthetic T-kinin. In contrast, treatment of plasma from healthy human subjects (n = 8) and from patients (n = 8) with inflammation due to acute or chronic gastrointestinal disease under the same conditions did not generate any detectable T-kinin immunoreactivity. It is concluded, therefore, that T-kininogen, the biosynthetic precursor of T-kinin in the rat, is either absent from the plasma of human subjects or is present in a concentration less than 30 fmol/ml. Similarly, T-kininogen is probably not an acute phase reactant in humans.
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PMID:Measurement of T-kinin in rat plasma using a specific radioimmunoassay. 143 85

Albumin binding to the endothelial surface apparently initiates its transcytosis via plasmalemmal vesicles and also increases capillary permselectivity. Several albumin-binding proteins, which, we call gp60, gp30, and gp18, have been identified; however, their functional relationship to each other is unclear. In this study, we show that gp30 and gp18 are both variably expressed by cultured rat fibroblasts, smooth muscle cells, and endothelial cells and are present in all tissues examined (heart, lung, skeletal muscle, diaphragm, duodenum, kidney, fat, brain, adrenal, pancreas, and liver). The binding of albumin-gold complexes (A-Au) to gp30 and gp18 was compared with that of native and modified albumins. Monomeric native bovine serum albumin (BSA) interacted much less avidly than A-Au and BSA that was chemically modified by formaldehyde (Fm-BSA) or maleic anhydride (Mal-BSA). Mal-BSA and A-Au have similar affinity constants for gp30 and gp18 (KD approximately 3-7 micrograms/ml (50-100 nM)), which is 1000-fold greater than BSA. These interactions were Ca(2+)-independent but sensitive to pH (< 6.0) and high salt concentrations (> or = 1.0 M). Comparative biochemical characterization provided evidence of conformational changes for Mal-BSA, Fm-BSA, and A-Au. Anti-native BSA serum recognizes BSA much more avidly than modified BSA. Mal-BSA, Fm-BSA, and A-Au are much more sensitive to trypsin digestion than BSA. Cellular processing was also examined. A-Au and Mal-BSA bound at the endothelial cell surface were degraded, whereas BSA was not. Our results indicate that: (i) gp30 and gp18, unlike gp60, are expressed in all tissues tested regardless of the type of endothelia lining the microvasculature and the local mechanism of transendothelial albumin transport; (ii) BSA conformationally modified by either surface adsorption or chemical means not only interacts more avidly with gp30 and gp18 than native albumin but also is preferentially degraded by the cells; (iii) A-Au and native albumin are not equivalent probes for detecting albumin interaction sites; and (iv) gp30 and gp18 exhibit binding behavior resembling scavenger receptors. The possible roles of gp30 and gp18 in albumin binding, transcytosis, endocytosis, and even protein catabolism are discussed.
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PMID:Preferential interaction of albumin-binding proteins, gp30 and gp18, with conformationally modified albumins. Presence in many cells and tissues with a possible role in catabolism. 144

Morphologically and functionally intact mitochondria were isolated from the porcine pancreas using a conventional, differential centrifugation method. The homogenate of the porcine pancreas made in a medium containing 0.25 M sucrose, 10 mM Tris-Cl, pH 7.4, 0.5% bovine serum albumin and 1 mM EDTA, in the presence or in the absence of dibucaine and trypsin inhibitor, was centrifuged for 10 min at 700xg. The supernatant was centrifuged for 10 min at 7000xg, and the pellet was washed three times. Trypsin activity of mitochondria isolated in the absence of dibucaine and trypsin inhibitor was as low as that of mitochondria isolated in the presence of dibucaine and trypsin inhibitor, and a major part of the activity remained inactivated. Phospholipase A2 activity of the former mitochondria was as low as that of the latter, and remained unchanged up to 8-10 hr at 4 degrees C. The presence of bovine serum albumin and EDTA in the respiration medium was absolutely required to obtain good respiratory controls of those mitochondria. These data suggest that well-coupled mitochondria can be obtained from the pancreas by a conventional isolation procedure without activating the major part of trypsin.
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PMID:Isolation and some biochemical properties of porcine pancreas mitochondria. 151 53

A number of enzymatic properties of fish pylochymopsin and bull chymopsin have been studied. Hydrolysis of synthetic ethers of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine by these chymopsins depending at the time and concentration of preparations has been studied. It was found that bull chymopsin is the most active one. It was shown that concentrations of 2 to 6 micrograms/ml of bull chymopsin and of 15 to 20 micrograms/ml of fish enzyme were optimal for synthetic substrate BTME hydrolysis. The significant trypsin activity was revealed in the both preparations on a number of synthetic amides. In contrast to the bull chymopsin the treatment of fish pylochymopsin by TPCK did not completely remove the chymotryptic activity of pylochymopsin. It was shown that tryptic activity in the both preparations was completely removed with TLCK. The time and concentration dependence of the autolysis in both chymopsins has been studied. It should be noted that this process is negligible for fish pylochymopsin in contrast to bull chymopsin. Stabilization of both proteases in aqueous solution at room temperature has been studied. Stabilization of the chymopsins in solution is achieved by the addition of various protein preparations including casein and serum albumin. The degree of stabilization by these proteins was achieved at 2% concentration.
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PMID:[Enzymatic activity of chymopsin of various origin]. 151 48

Treponema denticola surface proteins were studied for their biochemical and biological characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins. Antiserum raised against whole cells of T. denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins. Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell. SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa. Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole-cell antiserum and anti-53-kDa-protein antibody. The aggregates dissociated into their subunits after heating to 70 degrees C. Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5. The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested. A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T. denticola ATCC 35405. The recombinant protein had exactly the same molecular mass as the major 53-kDa T. denticola surface protein and reacted with antisera raised against this protein. The role of T. denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay. Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T. denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed. Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA. Our results suggest that the 53-kDa major surface protein of T. denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species.
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PMID:Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen. 156 96


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