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)

Protease nexin-I (PN-I) is representative of a newly described class of serine protease inhibitors secreted by human fibroblasts, the protease nexins. Protease nexins form covalent complexes with their target proteases, subsequently binding to cells via specific receptors. PN-I preferentially binds thrombin, urokinase, trypsin, and plasmin, and its binding to thrombin is accelerated by heparin. We have previously described the production of a polyclonal antibody against PN-I which is able to block the binding of PN-I X proteinase complexes to cells and will immunoprecipitate metabolically labeled PN-I. Anti-PN-I was used to investigate the biosynthesis and regulation of PN-I in human fibroblasts. Unlabeled PN-I could compete for the binding of metabolically labeled PN-I to anti-PN-I, as shown by the elimination of the 43-kDa band representing PN-I on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs. Excision of this 43-kDa band from gels, followed by amino-terminal sequencing, showed a homogeneous protein that is homologous with that described by Scott et al. (Scott, R. W., Bergman, B. L., Bajpai, A., Hersh, R. T., Rodriguez, H., Jones, B. N., Barreda, C., Watts, S., and Baker, J. B. (1985) J. Biol. Chem. 260, 7029-7034). An analysis of the biosynthesis of the PN-I revealed that a lower Mr precursor exists intracellularly. This apparent rough endoplasmic reticulum form appears as a doublet on sodium dodecyl sulfate gels, as does mature PN-I. The PN-I precursor was also sensitive to endoglycosidase H, suggesting that it contains N-linked carbohydrates of the high mannose form. Mature PN-I is not sensitive to endoglycosidase H, but does contain 3 kDa of N-linked carbohydrate. PN-I appears to be constitutively secreted by fibroblasts. PN-I levels in conditioned media reach a steady state within 48 h, although PN-I synthesis maintains a constant rate. This steady state is due to the continuous uptake of PN-I from medium, presumably through a specific receptor.
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PMID:Biosynthesis of protease nexin-I. 377 29

Glial-derived neurite-promoting factor was found to be a slow-binding inhibitor of trypsin, urokinase, and thrombin. The kinetic mechanism of the inhibition differs among the three proteases. With trypsin and urokinase, an initial protease-factor complex formed which isomerized to a tighter complex. For thrombin, however, no initial complex was kinetically observed. The dissociation constants of the equilibrium complexes of the factor with trypsin, urokinase, and thrombin were 17, 280, and 18 pM, respectively, and the apparent second-order rate constants for the interaction of the factor with these enzymes were, respectively, 4.7 X 10(6), 1.2 X 10(5), and 2.1 X 10(6) M-1S-1. Heparin increased the rate at which the factor reacted with thrombin by over 40-fold to 8.9 X 10(7) M-1S-1 and decreased the dissociation constant of the complex by over 80-fold to 0.3 pM. The values obtained for the apparent second-order rate constants when compared with the kinetics of neurite induction by the factor indicate that the neurite-promoting activity of the factor is not due to the inhibition of urokinase but could be due to the inhibition of an enzyme with a specificity similar to that of thrombin or trypsin. Comparison of the values of the apparent second-order rate constants obtained for the factor with those obtained for protease nexin suggests that these two molecules are very similar in their inhibitory properties.
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PMID:Glial-derived neurite-promoting factor is a slow-binding inhibitor of trypsin, thrombin, and urokinase. 381 34

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.
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PMID:Protease nexin. Properties and a modified purification procedure. 399 57

Bovine corneal endothelial cells in culture bind internalize and degrade [125I]-trypsin. Binding involves the active site of trypsin and increases as a function of [125I]-trypsin concentration. Saturation is observed at a concentration of 0.5-1.0 micrograms ml-1. The cell surface binding of [125I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [125I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [125I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [125I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [125I]-trypsin X protease-nexin with a molecular weight of 64000 daltons. The cells, when incubated at 37 degrees C, appear to internalize the cell surface-bound [125I]-trypsin at a rate of 0.15-0.25 ng (10(6) cells)-1 min-1. Both the non-covalently cell surface-bound and the protease-nexin (PN) mediated-bound [125I]-trypsin are internalized by the cells, but the [125I]-trypsin X PN complexes contribute about 75% of the total amount of [125I]-trypsin internalized by the cells. The internalized [125I]-trypsin is degraded by the cultures at a rate of about 0.05 ng (10(6) cells)-1 min-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [125I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [125I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.
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PMID:Binding and processing of trypsin by cultured bovine corneal endothelial cells. 400 80

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 microM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 microM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.
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PMID:Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. 421 20

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods. The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture. It was found that ATP induced active disintegration of the axonemes after slight digestion. Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes. The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly. The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets. The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave.
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PMID:Effects of trypsin digestion on flagellar structures and their relationship to motility. 474 19

Recently we presented evidence that normal human foreskin fibroblasts (HF cells) limit the activity of secreted urokinase by secreting it as a proenzyme and by secreting protease nexin , an inhibitor of urokinase and certain other serine proteases (Scott, R.W., Eaton, D. L. Duran , N., and Baker, J.B. (1983) J. Biol. Chem. 258, 4397-4403). Using immunoaffinity chromatography we have now purified the HF cell urokinase proenzyme. It is a single 52-kDa polypeptide chain that is inactive toward both plasminogen and low molecular weight substrates. After proteolytic activation, this material (specific activity of 3 X 10(4) Committee on Thrombolytic Agents units/mg) is composed of two disulfide-bridged 33- and 19-kDa chains, and is thus similar to the predominant form of urokinase found in urine. Plasmin at 2 X 10(-10) M causes 50% activation of the proenzyme (1 X 10(-9) M) in 30 min at 37 degrees C. Thrombin and trypsin are one-twentieth as effective as plasmin. Activated HF cell 125I-urokinase forms sodium dodecyl sulfate stable complexes with purified protease nexin or protease nexin present in medium conditioned by HF cells. Purified protease nexin inhibits purified HF cell urokinase action on both plasminogen and low molecular weight substrates. The association rate constant for the reaction between protease nexin and HF cell urokinase is approximately 1.7 X 10(5) M-1 S-1. In contrast, the association rate constants for reactions between protease nexin and the one- and two-chain forms of tissue-type plasminogen activator are approximately 2 X 10(3) and approximately 3 X 10(4) M-1 S-1, respectively. The importance of protease nexin as a regulator of HF cell urokinase is supported by the finding that anti-protease nexin antibody potentiates the fibrinolytic activity of HF cell-conditioned medium incubated with plasminogen.
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PMID:Purification of human fibroblast urokinase proenzyme and analysis of its regulation by proteases and protease nexin. 637 53

Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.
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PMID:Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. 657 53

The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.
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PMID:Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin. 754 66

To elucidate the mechanism of oscillatory bending in cilia and flagella, we studied the effect of protease digestion on the response of axonemes to localized application of ATP. When the axonemes were treated with elastase and then reactivated locally by ATP iontophoresis, a pair of local bends were formed due to localized unidirectional sliding in the vicinity of the ATP pipette. Upon repeated application of ATP, the direction of bending with respect to the sperm head axis changed cyclically from side to side over several cycles. The bends were planar and similar to those observed in axonemes that had not been treated with elastase. In trypsin-treated axonemes, in contrast, repetitive local reactivation did not induce such cyclical bending; instead, it induced a bend that grew only in one direction upon repeated application of ATP. Moreover, the bends were not planar. Electron microscopy of these protease-digested axonemes showed that both the interdoublet (nexin) links and the radial spokes were disrupted, but the effects of these proteases were different; trypsin disrupted 60-70% of these structures whereas elastase disrupted 20-30% of them. In both cases, spokes no. 3 and no. 8 (and no. 7) were more resistant to digestion than the others, although they tended to be more resistant to elastase than to trypsin. The importance of radial spokes and interdoublet links in the generation of cyclical bending and the determination of the bending plane is discussed.
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PMID:Cyclical bending movements induced locally by successive iontophoretic application of ATP to an elastase-treated flagellar axoneme. 761 58


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