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)

Acidified extracts of rat antral stomach chromatographed on octadecylsilane cartridges contained material that inhibited the binding of [3H]Ro 5-4864 (4'-chlorodiazepam) and [3H]nitrenidipine to "peripheral-type" benzodiazepine receptors and dihydropyridine Ca2+-channel antagonist binding sites respectively. This material reduced the apparent affinities of both radioligands without significantly affecting the maximum number of binding sites. In contrast, the binding of [3H]diazepam, [3H]Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4] benzodiazepine-3-carboxylate), and [3H]3-carbomethoxy-beta-carboline to "brain-type" benzodiazepine receptors and [3H]dihydroalprenolol binding to beta-adrenergic receptors were unaffected by this material. Subsequent column chromatography on hydroxylapatite purified this material by greater than 2000-fold. This semi-purified substance was resolved by reverse phase HPLC as one u.v. adsorbing peak that inhibited both [3H]Ro 5-4864 and [3H]nitrendipine binding. The activity of this 16,000 dalton substance was destroyed completely by both heat treatment and pronase and partially reduced by trypsin. Furthermore, the inhibitory activity of this substance was enhanced by Ca2+ in a concentration-dependent fashion (0.1 to 10 mM). Comparison of TLC scans of 2-9,10[3H]dipalmitoyl-phosphatidylcholine incubated with either the HPLC purified material or authentic phospholipase A2(PLA2) (Naja naja) revealed that this substance has enzymatic properties indistinguishable from PLA2. These findings suggest that this endogenous protein may be a PLA2 isoenzyme which may modify both "peripheral-type" benzodiazepine receptors and dihydropyridine Ca2+-channel antagonist binding sites.
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PMID:Purification and characterization of an endogenous protein modulator of radioligand binding to "peripheral-type" benzodiazepine receptors and dihydropyridine Ca2+-channel antagonist binding sites. 282 15

Intrapancreatic activation of proteases is believed to play a major role in the pathogenesis of acute necrotizing pancreatitis. Several authors have questioned, however, the central role of trypsin in autodigestion of the pancreas. To clarify the direct effects of pancreatic enzymes and other related factors on acinar cells, we used the model of isolated pancreatic acini. Acini were prepared from male Wistar rats by collagenase digestion. Protein synthesis was measured by incubation of acini with [35S]methionine. Acini were resuspended thereafter in fresh buffer and further incubated for 30-90 min under various conditions [e.g., with pancreatic homogenates, ascites (from rats with pancreatitis induced by sodium taurocholate), pure pancreatic enzymes, and other factors]. The percentage of release of newly synthesized proteins into the culture medium was regarded as a biochemical parameter of cellular integrity. A morphologic score of cellular integrity was obtained via light microscopic evaluation of acini at the end of the various incubations by measuring the degree of cell lysis, loss of cell granules, ballooning, formation of vacuoles, and karyopyknosis. When normal [35S]methionine-labeled pancreatic acini were incubated with various factors, the percentage of release of labeled proteins into the medium was as follows: incubation with HEPES/Ringer's buffer, 1.8%; hemorrhagic pancreatic ascites, 3.8%; pancreatic homogenates, 2.0%; lipase, 1.8%; phospholipase A2, 3.0%; phospholipase A2 + lecithin, 3.2%; trypsin, 2.5%; 5% olive oil, 1.8%; ascites + olive oil, 78.3%; ascites + homogenized epididymal fat, 79.9%; lipase + olive oil, 32.0%; pancreatic homogenates + olive oil, 28.0%; diolein, 2.65%; and oleic acid, 62.9%. The cellular release of radiolabeled proteins showed an inverse correlation with cellular integrity as shown by light microscopy. We postulate that interstitial release of degradation products from triglycerides by lipase causes cellular disruption. Whereas phospholipase A2 and proteases do not seem to be very harmful in the early phases of cellular damage, lipase may play a major role in acute necrotizing pancreatitis.
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PMID:Role of pancreatic enzymes and their substrates in autodigestion of the pancreas. In vitro studies with isolated rat pancreatic acini. 291 45

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.
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PMID:Uncoupling of cholinergic synaptic vesicles by the presynaptic toxin beta-bungarotoxin. 294 72

The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A2 activity (PA2: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA2 activity optimally with trypsin concentrations of 1.0-1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA2 activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA2 activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA2 activation upon the addition of proteases, thus indicating that the increase in PA2 activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A2 that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A2 could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.
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PMID:Activation of phospholipase A2 of human spermatozoa by proteases. 297 29

Biologically active 125I-cytotoxin from Pseudomonas aeruginosa binds to plasma membranes from Ehrlich ascites tumor cells in a saturable manner. The Scatchard plot indicated a single binding site with a capacity of 260 pmoles/mg of membrane protein and a KD of 2 X 10(-8) M. Specific binding was dependent on temperature, pH and ionic strength. Thus constant levels of bound 125I-cytotoxin were attained either within 30 min at 30 degrees C or within 3 h at 4 degrees C. Binding was 30-fold higher at 4 degrees C vs 30 degrees C and 2-6-fold higher at pH 5.3 vs pH 8.3. Binding was not effected by 50 mM sugar or sialic acid. 300 mM sucrose, however, instead of phosphate buffer, reduced binding by 50%. Pretreatment of plasma membranes with trypsin or papain led to a significant decrease in 125I-cytotoxin binding. A pretreatment with phospholipase C or D had no effect, whereas phospholipase A2 induced a decrease by 34%. The collected data suggest that the binding site for 125I-cytotoxin within the plasma membrane from Ehrlich ascites tumor cells is a membrane protein. Correlation of 125I-cytotoxin binding and membrane action of the unlabelled cytotoxin can be observed through (a) increased lowering of the cellular K+ and Na+ gradient by decrease of medium pH, (b) decreased toxicity after substitution of ions by sugar, and (c) increased breakdown of cellular cationic gradient after temperature shift from 4 degrees C to 37 degrees C.
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PMID:Interaction of Pseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells. 300 83

Trypsin and chymotrypsin inactivated specific [3H]yohimbine binding sites in the partially purified human platelet membranes in a concentration- and time-dependent fashion. The maximal inactivation (70-80% of control) was incomplete regardless of the concentrations of the proteases used or the incubation time. Scatchard analysis of the binding data showed that the total number of binding sites was reduced, but the affinity of the receptor to the ligand remained unaffected. Pretreatment of the membranes with unlabeled yohimbine or epinephrine produced a 20-30% increase in the specific [3H]yohimbine binding; however, this treatment offered only a slight protection (10-15%) against trypsin-induced inactivation of [3H]yohimbine binding. Pretreatment with phospholipase A2 produced a complete inhibition, while pretreatment with phospholipase C resulted in only a partial (70-80% of control) reduction in [3H]yohimbine binding. The inhibitory effects were not reversed when the specific binding of [3H]yohimbine was carried out with membranes treated with phospholipases and subsequently washed with defatted bovine serum albumin, suggesting that products released from phospholipolysis were not involved in the inhibition of [3H]yohimbine binding. These results suggest that the integrity of the receptor proteins and phospholipids is necessary for the specific binding of the ligand to the alpha 2-adrenoreceptor proteins of the human platelet membranes.
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PMID:Effect of proteases and phospholipases on [3H]yohimbine binding to human platelet membranes. 301 56

A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.
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PMID:Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems. 302 20

Research has been carried out in order to clarify the chemical nature of cell receptors interacting with a fast growing strain of hepatitis A virus (HAV) producing a cytopathic effect on Frp/3 cells. Cell surface susceptibility to HAV attachment has been studied after treatment with enzymes acting on different chemical groupings. Results obtained showed a lowering of cell susceptibility to HAV infection following the action of phospholipase A2, phospholipase C, trypsin and beta-galactosidase. These data suggested that phospholipids, proteins and galactose participate to the cellular receptorial area for HAV.
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PMID:Study of the chemical nature of Frp/3 cell recognition units for hepatitis A virus. 302 55

A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.
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PMID:The complete amino-acid sequence of a basic phospholipase A2 in the venom of Bungarus fasciatus. 307 71

The binding of a series of glycosylated beta-galactosidases to a fraction rich in synaptic membrane of bovine brain was examined. beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal) was found the most effective in binding to synaptic membrane, followed by that modified with beta-D-glucopyranoside, whereas the enzyme modified with p-aminophenyl derivatives of alpha-D-galactopyranoside, alpha-D-glucopyranoside, and alpha- and beta-L-fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4 degrees C and the optimal pH was approximately 4.0. The binding of beta-D-Gal beta-gal was inhibited by free p-aminophenyl beta-D-galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 +/- 35 nM and 27.5 +/- 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.
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PMID:Specific binding of glycosylated beta-galactosidase to bovine brain synaptic membrane. 307 20


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