Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for the preparation of casein labelled with the 4-methylumbelliferyl fluorophore is described, and the product was used as a fluorigenic macromolecular substrate for a sensitive assay of the activity of proteinases. Nanogram quantities of trypsin, chymotrypsin, elastase and cathepsin D can be detected, but the substrate is unaffected by cathepsin B.
 ...
PMID:Fluorigenic method for the assay of proteinase activity with the use of 4-methylumbelliferyl-casein. 634 7

A method for the preparation of a radioisotopically labeled active-site directed reagent for proteases (125I-Tyr-Ala-Lys-ArgCH2Cl) is described, and an example of its use as a sensitive method for identifying trypsin-like proteases is provided. This high specific activity reagent was then used in an attempt to identify proteases in rat islets of Langerhans involved in the conversion of proinsulin to insulin. Previous studies have indicated that the endoprotease involved in proinsulin conversion is a cysteine proteinase and that 125I-Tyr-Ala-Lys-ArgCH2Cl affinity labels an islet crude granule fraction protein having a molecular weight of 31,500. Here we demonstrate, using a probe of higher specific activity, that the major affinity-labeled proteins of the islet crude granule fraction, when displayed by sodium dodecyl sulfate gel electrophoresis, have molecular weights of approximately 39,000 (5%), 31,500 (53%), and 5,000-6,000 (37%), with several other minor proteins (less than 5%) also labeled. The two predominant labeled proteins were mainly soluble rather than membrane bound, and they exhibited patterns of competition with various inhibitors that were similar to the pattern shown by the conversion of proinsulin to insulin in vitro. A rabbit antibody to rat liver cathepsin B immunoprecipitated both affinity-labeled 31,500 and 5,000-6,000 molecular weight proteins, and on the basis of this and structural considerations the 31,500 molecular weight cysteine protease is identified as cathepsin B. The 5,000-6,000 molecular weight peptide is an NH2-terminal, active site cysteine-containing, proteolytic fragment of the 31,500 molecular weight protein. Because cathepsin B is not per se a candidate for the proinsulin convertase because of its excessively broad substrate specificity, these studies suggest that a similar enzyme or a modified form of this enzyme is active within the secretory progranules, whereas the more typical cathepsin B may be largely confined to lysosomal contaminants in our granule preparations.
 ...
PMID:Identification of a 31,500 molecular weight islet cell protease as cathepsin B. 634 78

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum.
 ...
PMID:Partial purification and some properties of a new papain inhibitor from psoriatic scales. 639 31

At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.
 ...
PMID:Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187. 644 5

Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.
 ...
PMID:Characterization of porcine plasma fibronectin and its fragmentation by porcine liver cathepsin B. 645 32

When their membrane proteins were labeled with 125I by lactoperoxidase, dividing hepatoma cells lost radioactivity to the medium in a biphasic manner (T1/2 = 16-26 h, greater than 40 h). Lysosomotropic weak bases, chloroquine, and NH4Cl inhibited the rapid phase by 59%. More than 50% of the radioactivity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60-71% inhibited by lysosomotropic agents chloroquine and NH4Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH4Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-L-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by 21-24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 degrees C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lysosomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to agents which disrupt the cytoskeleton.
 ...
PMID:Degradation of surface-labeled hepatoma membrane polypeptides: effect of inhibitors. 648 99

At present, there is no established diagnostic method by which the metastatic ability of an individual prostatic cancer can be accurately predicted. Metastasis is a multistep process, the first critical step of which is invasion. Tumor invasion has been suggested to involve a variety of hydrolytic enzyme activities; therefore, the tumor levels of these activities might be indicative of the overall metastatic ability of the cancer. In order to evaluate if the quantitative levels of hydrolytic enzymes can be used to predict the metastatic ability of individual prostatic cancers, five different Dunning R-3327 rat prostatic adenocarcinoma sublines, with widely varying metastatic abilities, were assayed for the respective levels of a variety of hydrolytic enzyme activities (collagenase, trypsin-like, cathepsin B, neutral protease, N-acetyl-beta-glucosaminidase, chymotrypsin-like, leucine aminopeptidase, elastase, and plasminogen activator). These studies demonstrated that most hydrolytic activities are not elevated when going from normal prostate to prostatic cancer. In addition, only the levels of elastase and chymotrypsin-like activity were found to be consistently higher in highly metastatic prostatic cancers than in either the normal prostate or low-metastatic prostatic cancers. It was found that, by combining the relative activities of elastase and chymotrypsin-like activity and then dividing by the relative activities of N-acetyl-beta-glucosaminidase, a biochemical metastatic index could be constructed which accurately reflected the respective metastatic ability of the Dunning sublines.
 ...
PMID:Biochemical methods for predicting metastatic ability of prostatic cancer utilizing the dunning R-3327 rat prostatic adenocarcinoma system as a model. 653 99

Treatment of low-metastatic Lewis lung carcinoma cells (P-29) with dimethylsulfoxide (DMSO) in vitro enhanced their lung-colonizing ability. The effects of other highly polar compounds on the lung-colonizing ability of P-29 cells were examined. The following compounds were found to enhance the lung-colonizing ability of the cells: acetamide, N-methylacetamide, N-methylformamide, N,N-dimethylformamide, piperidone and hexamethylphosphoric triamide. Treatment of P-29 cells with DMSO or other polar compounds resulted in increases in activities of degradative enzymes, such as cathepsin B and plasminogen activator. The increases in cathepsin B and plasminogen activator activities were apparent after a 24 h treatment with DMSO and were suppressed by simultaneous treatment with cycloheximide, which suggested that they were due to syntheses of new proteins. DMSO-treated P-29 cells degraded [3H]leucine-labelled subendothelial matrix much more than did untreated cells. P-29 cells treated with DMSO or other polar compounds became attached to culture dishes more rapidly and were more resistant to detachment by trypsin treatment than untreated cells. A significant correlation was found between the degrees of adhesiveness of P-29 cells treated with various polar compounds and their lung-colonizing abilities.
 ...
PMID:Enhancement of lung-colonizing ability of cloned low-metastatic Lewis lung carcinoma cells by treatment with highly polar compounds. 654 Feb 47

Cathepsin L was capable of destroying rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructose 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PPi and Pi decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of Pi. Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and Pi. The thermostability of aldolase was also unaffected by these ligands. ATP and Pi had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and Pi are due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.
 ...
PMID:Inactivation of fructose-1,6-bisphosphate aldolase by cathepsin L. Stimulation by ATP. 669 88

The unknown enzymatic mechanism of enhanced protein breakdown in steroid myopathy was studied in functionally and biochemically different muscles of rabbits treated with dexamethasone for three weeks. After glucocorticoid administration the fast-twitch glycolytic semimembraneous muscle of treated animals was atrophied, whereas the weight of the slow-twitch oxidative soleus muscle was not altered. The specific activity of the lysosomal endo- and exopeptidases (cathepsin D, E, B and L, lysosomal carboxypeptidase A and dipeptidylpeptidase I) was increased about 2-fold in the atrophied white muscle. The activity of the cytosol enzyme Ca++-activated neutral proteinase was also elevated, whereas that of the other cytosol endopeptidase, chymotrypsin-like enzyme, was unaltered. The level of alanine aminopeptidase was only slightly increased. On the other hand, there were no unequivocal changes in protease activity in the soleus muscle. These findings are in agreement with the known differences in glucocorticoid-sensitivity of the various muscles. Our results suggest that the lysosomal proteolytic system and the Ca++-activated neutral proteinase may play an important role in the glucocorticoid-induced intracellular protein catabolism in muscle. The inhibitor capacities of cathepsin B and trypsin detectable in muscle cytosol were not altered after steroid treatment. Consequently, the increase in cathepsin B activity was not due to the loss of its inhibitor.
 ...
PMID:Proteases and proteinase inhibitors in experimental glucocorticosteroid myopathy. 676 81


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>