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)

A membrane fraction enriched in parathyroid hormone (PTH)-sensitive adenylate cyclase and sodium and potassium ion-activated (Na+, K+)-ATPase was prepared from bovine kidney. Tritiated PTH binding to this membrane fraction was dependent on both hormone and membrane protein concentration. Both total and specific binding of the hormone decreased significantly after 5 to 10 min of incubation at 22 degrees. PTH binding was highly specific, being sensitive to inhibition only with active forms of unlabeled hormone (native and 1-34 PTH). Specific binding showed a pH optimum of 7.3 to 7.5. Inhibition of binding of tritiated hormone by unlabeled PTH was also highly effective at pH 6.0, but this apparently specific binding was also inhibited by adrenocorticotropic hormone, insulin, glucagon, and vasopressin. Dissociation of bound hormone was demonstrated, and an apparent dissociation constant of 4.6 X 10(-2) min-1 was obtained. Specific binding was eliminated by pretreatment of the membranes with trypsin. The concentration dependence for inhibition of binding with unlabeled PTH was identical to that for activation of adenylate cyclase in this membrane preparation, and binding was also inhibited by concentrations of calcium in the 0.5 to 2 mM range.
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PMID:Binding of tritiated bovine parathyroid hormone to plasma membranes from bovine kidney cortex. 1 29

1. Complexes of human trypsin and human granulocyte elastase with alpha1-anti-trypsin and alpha2-macroglobulin were isolated and injected intravenously into human volunteers. 2. The elimination of alpha2-macroglobulin complexes with trypsin and elastase followed single-exponential functions with half-lives of 9 and 12 min respectively. The complexes showed no tendency to dissociate. 3. Complexes of alpha1-anti-trypsin with trypsin persisted in the circulation much longer, with a half-life of 3-5 h; complexes of alpha1-anti-trypsin with elastase had an intermediate half-life of 1 h. 4. Dissociation was observed of alpha1-anti-trypsin complexes with transfer of trypsin and elastase to alpha2-macroglobulin. 5. Dialysable radioactivity appeared in the urine soon after the injection of alpha2-macroglobulin complexes, which suggested a breakdown of complexes by cells in the reticuloendothelial system. Radioactivity over the liver achieved maximum values within 30-40 min after the injection of alpha2-macroglobulin complexes but not until 50-70 min after the injection of alpha1-anti-trypsin comlexes. 6. These results support the concept of a key position for alpha2-macroglobulin in the protective mechanisms against endogenous proteases.
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PMID:The disappearance of enzyme-inhibitor complexes from the circulation of man. 5 54

Binding of a denaturated polypeptide chain derived from chick skin collagen, the alpha 1(I) chain, by isolated membranes of human platelets has been demonstrated. The process is reversible, and time- and protein concentration-dependent. The binding is specific, with an association constant of 1.88 X 10(-6) M. Prior treatment of the isolated membranes with trypsin, chymotrypsin, and pronase, resulted in significant inhibition of the 14C-labeled alpha 1 chain binding, but neuraminidase or collagenase treatment had no effect. Dissociation of the bound radioactivity and subsequent chromatographic analyses on carboxymethylcellulose and agarose A-1.5m revealed that the alpha 1 chain was unaltered. Scatchard plot analysis suggested that there are approximately 20,000 binding sites per platelet. The binding of the alpha 1 chain was inhibited by a glycopeptide derived from alpha 1, alpha 1-CB5 and by purified glucosylgalactosyl hydroxylysine, but was not affected by other cyanogen bromide peptides of alpha 1, namely alpha 1-CB3, -CB4, -CB7, and -CB8. Kinetic studies demonstrated that inhibition by the hydroxylysine glycoside is competitive. Dose-response curves of platelet aggregation induced by alpha 1 and the binding of alpha 1 by platelet membranes correlate closely. These results indicate that there are specific binding sites for collagen alpha 1 chain on platelet membranes, and that the carbohydrate moiety of the alpha 1 chain plays a role in the binding. The findings also support the hypothesis that the chick skin alpha 1 chain mediates platelet aggregation and the release reaction by acting on platelet membranes.
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PMID:Binding of chick skin collagen alpha 1 chain by isolated membranes from human platelets. 97 74

Several methods for the dissociation of human tonsils into cell suspensions were compared. Dissociation of tonsils using 0-25 per cent trypsin gave both the largest number of total cells and the largest number of plasma cells per gram of tonsil. Lymphocytes and plasma cells were separated in a previously described isokinetic gradient of Ficoll in tissue culture medium. In the purest gradient fractions, lymphocytes were 97-2 plus or minus 1-9 per cent of nucleated cells. The purest gradient fractions contained 43-1 plus or minus 5-9 per cent plasma cells. More than 95 per cent of purified lymphocytes and plasma cells excluded Trypan Blue.
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PMID:A method for the separation of lymphocytes and plasma cells from the human palatine tonsil using sedimentation in an isokinetic gradient of Ficoll in tissue culture medium. 109 May 18

Dissociation of mixed trypsin (bovine plus porcine trypsin) complexes with chicken ovoinhibitor was used to investigate the nonequivalence of the two binding sites for trypsin on the inhibitor. Previous work has shown that 1 mol of trypsin dissociates much more rapidly than the 2nd from unmixed trypsin complexes, those containing 2 mol of one kind of trypsin, bovine or porcine, per mol of inhibitor. However, only approximately 0.5 to 0.6 mol of trypsin dissociated in the rapid step from the mixed trypsin complexes, those containing 1 mol each of bovine and porcine trypsin. Rates of the slow dissociation steps for the two types of complexes did not differ appreciably from each other. A general dissociation scheme is proposed, in which each of the 2:1 complexes can lose a trypsin molecule from either in two parrallel first order reactions, producing two different 1:1 complexes, which subsequently dissociate to yield free ovoinhibitor and a second trypsin molecule. In this scheme, both the earlier results with unmixed trypsin complexes and the preponderance (approximately 3:1) of slow dissociation from the mixed trypsin complexes can be rationalized if bovine trypsin is retained preferentially at one of the two trypsin binding sites on chicken ovoinhibitor, and porcine trypsin at the other. That is, one site allows rapid dissociation of porcine trypsin and slow dissociation of bovine trypsin, whereas the other allows rapid dissociation of bovine trypsin and slow dissociation of porcine trypsin.
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PMID:Preferred binding of bovine and porcine trypsins at two different sites on chicken ovoinhibitor. Reduced dissociation of mixed trypsin complexes. 117 53

Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.
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PMID:Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin. 124 51

Previous current-clamp work has shown that dihydropyrazole insecticides block sodium channels in tonic sensory receptors and in axons depolarized by high K+ external solutions and that hyperpolarization removes the block [Pestic. Sci. 28:389-411 (1990)]. Voltage-clamp studies on internally perfused crayfish giant axons were done to confirm and extend these observations. At -100 mV dihydropyrazoles had little effect on the sodium current, but at more depolarized potentials they blocked it from either face of the membrane. The onset of block following a holding potential change or during wash-in of a dihydropyrazole was very slow, with a time constant of several minutes, and, although block could be removed with a similar time course by hyperpolarization, the effects of the insecticides could not be reversed by prolonged washing. Dihydropyrazoles did not affect delayed rectifier potassium currents in the axon. The voltage-dependent block could be described as a uniform shift of the steady state (slow) sodium inactivation (S infinity) curve in the direction of hyperpolarization, indicative of selective binding to inactivated states of the channel. Using hyperpolarizing prepulses to remove slow inactivation, block of sodium channels by dihydropyrazoles could be measured directly at holding potentials as positive as -50 mV, and it could be demonstrated that block saturated near -70 mV, consistent with a dependence on slow inactivation. The data were fit to a model tha assumes the dihydropyrazole binds to the slow-inactivated state of the channel on a one to one basis. Dissociation constants obtained from this analysis were similar to those obtained from analysis of inhibition of the binding of [benzoyl-2,5-3H]-batrachotoxinin A 20-alpha-benzoate by the same dihydropyrazoles. In axons whose fast or slow inactivation gates had been removed by N-bromoacetamide or trypsin, respectively, dihydropyrazoles still blocked sodium current, indicating that dihydropyrazoles can block the channel as well as enhance the normal slow inactivation process.
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PMID:Slow voltage-dependent block of sodium channels in crayfish nerve by dihydropyrazole insecticides. 131 Jan 38

The kinetic properties and inhibitor sensitivity of human sperm phospholipase A2 (PLA2; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn-2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 mM lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA2 was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCl). Two molecular forms of PLA2 were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 microM and 0.64 mlU/mg protein and the other with respective constants of 630 microM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low-Km activity was less resistant to 60 degrees C preincubation at pH 7.5 (28% inactivation of low-Km activity after 45 min, as compared to no effect on high-Km activity). Quinacrine was a noncompetitive PLA2 inhibitor with Kis for low- and high-Km activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high-Km activity noncompetitively (Ki = 87 microM) and the low-Km activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low- and high-Km activities by p-bromophenacyl bromide were 0.28 mM and 0.032 min-1, and 0.73 mM and 0.066 min-1, respectively. PLA2 was inhibited by p-nitrophenyl-p'-guanidinobenzoate, at higher concentrations (10(-4)-10(-3) M) than required to inhibit trypsinlike proteinases; p-aminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA2 at concentrations from 2-5 mM but inhibited PLA2 (40-50%) at a concentration of 10 mM. MnCl2 (5mM) inhibited low- and high-Km PLA2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 microM), p-bromophenacyl bromide (20 microM), and MnCl2 (5 mM) were tested as inhibitors of the ionophore A23187-induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl2 (93%). The effect of MnCl2 was restricted to an interaction with A23187, rather than with PLA2; p-Bromophenacyl bromide inhibited (P less than 0.05) PLA2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA2 inhibition was poorly correlated with the acrosome reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization and inhibitor sensitivity of human sperm phospholipase A2: evidence against pivotal involvement of phospholipase A2 in the acrosome reaction. 226 93

High-affinity receptors for alpha 2-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4 degrees C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4 X 10(-4) min-1. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2 X 10(-2) min-1. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca2+ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4 degrees C was rapidly internalized (kint about 3 X 10(-1) min-1) after being warmed to 37 degrees C. Radioactivity released from the cells at 37 degrees C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k-1 about 1 X 10(-1) min-1) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of alpha 2-macroglobulin-proteinase complex into hepatocytes.
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PMID:Characterization of receptors for alpha 2-macroglobulin-trypsin complex in rat hepatocytes. 241 32

125I-labelled alpha 2-macroglobulin-trypsin (alpha 2MT) complex bound specifically to freshly isolated human blood monocytes at 4 degrees C but not to B or T lymphocytes, polymorphonuclear leucocytes, erythrocytes or thrombocytes. Binding of 12 pmol/l labelled alpha 2MT to freshly isolated monocytes was low. However, when monocytes were cultured in vitro, binding increased, reaching 10- to 20-fold higher specific binding by 2-4 weeks. Non-specific binding was absent. Considerable variations were observed in binding to monocytes from different cultures (individuals). The half-time of 125I-labelled alpha 2MT complex association to 14-days-old monocyte cultures was about 6 h at 4 degrees C. Dissociation of labelled complex after the addition of a saturing concentration of unlabelled complex was biphasic. About half of the labelled complex dissociated with a half-time of about 1 h, whereas the other half dissociated extremely slowly. At near steady state, half of the receptors were occupied at a complex concentration of about 200 pmol/l and Scatchard analysis showed that the data were adequately described by assuming one class of receptors. It is concluded that human monocytes express a marked increase in binding of alpha 2M complex when developing to macrophage-like cells in tissue cultures.
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PMID:Binding of alpha-2-macroglobulin trypsin complex to human monocytes in culture. 243 44


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