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)

Polymorphism of platelet membrane glycoprotein Ib (GP Ib) has been described in both normal and functionally abnormal platelets. In this report we have investigated the polymorphism in one Australian Caucasian family with normal platelet function in which the following phenotypes were found: father, BC; mother, CD; son, BC; and daughter, BD; thus establishing a genetic basis for this phenomenon. The daughter's phenotype BD is highly distinctive due to the double-band pattern obtained by gel analysis. Platelets from the daughter were digested with either trypsin or elastase and the GP Ib cleavage products were examined by immunoprecipitation with anti-GP Ib-IX complex monoclonal antibodies against each of the major tryptic domains. On the basis of these studies, we have determined that the GPIb polymorphism in this family resides in the 85-kDa, macroglycopeptide region of the alpha-chain of GP Ib and not in either the 45-kDa, N-terminal region of GP Ib alpha or the membrane-associated region of the complex.
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PMID:Polymorphism of platelet glycoprotein Ib associated with variability of the 85-kDa macroglycopeptide region. 152 1

Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.
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PMID:Characterization of lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase. 165 13

The molecular basis for binding of alpha-macroglobulin-proteinase complexes to the human two-chain 500/85-kDa (alpha/beta) alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein was analyzed. Ligand blotting experiments showed that a 40-kDa protein, present in the affinity-purified alpha 2MR preparation, is bound to the alpha 2MR alpha-chain and released by heparin. Removal of the 40-kDa protein resulted in a 3-5-fold increase in binding of alpha 2M-trypsin. Nitrocellulose-immobilized pure two-chain alpha 2MR was incubated with human alpha 2M-trypsin, containing four identical subunits, and two monovalent ligands: rat alpha 1-inhibitor-3-chymotrypsin and the 18-kDa receptor binding fragment of the alpha 2M subunit. Binding of alpha 2M-trypsin to the alpha-chain of immobilized alpha 2MR was composed of a high (Kd = 40 pM at 4 degrees C) and a low (Kd = 2 nM) affinity component. alpha 1-Inhibitor-3-chymotrypsin bound to the same sites but with one component (Kd = 0.4 nM). Competition-inhibition experiments and dissociation experiments, using ligands with different valences, as well as experiments with alpha 2MR immobilized at different densities, led to the following model. The low (Kd = 2 nM) affinity of alpha 2M-proteinase is prevalent when only one of the four domains binds to alpha 2MR, i.e. when the receptor density is low or when neighboring receptors are occupied. The high (Kd = 40 pM) affinity is achieved by binding of at least two domains to adjacent receptors.
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PMID:Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors. 171 12

A monoclonal antibody (Mab), 45J, which reacts with intact fibrinogen, has been employed to demonstrate the interaction of the carboxy terminal regions of the A alpha-chain in non-denatured plasma fibrinogen. The 45J Mab recognizes an epitope in the mid section of the carboxy terminal end of the A alpha chain. The epitope is destroyed by plasmin and trypsin digestion. The 45J Mab and a horseradish peroxidase conjugate of the 45J Mab (45J-HRP) were used in an ELISA to demonstrate that the antibody could recognize two copies of the same epitope on purified fibrinogen or denatured plasma fibrinogen. Fibrinogen in non-denatured plasma could not be detected by this single antibody ELISA. This immunochemical study demonstrates that only one copy of the epitope on the C-terminal protuberance of the A alpha-chain is exposed in non-denatured plasma. However, once the plasma fibrinogen has been denatured, as in the purification process, both copies of the epitope are available for antibody binding. This finding suggests that in plasma there is an intramolecular interaction between the carboxy terminal ends of the fibrinogen A alpha-chains which can be destroyed by denaturation.
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PMID:Immunochemical evidence for intramolecular interaction of the carboxy terminal A alpha-appendages of plasma fibrinogen. 172 3

Externally oriented protein kinases are present on the plasma membrane of the human parasite, Leishmania. Since activation of complement plays an important role in the survival of these parasites, we examined the ability of protein kinases from Leishmania major to phosphorylate components of the human complement system. The leishmanial protein kinase-1 (LPK-1) isolated from promastigotes of L. major was able to phosphorylate purified human C3, C5 and C9. Only the alpha-chain of C3 and C5 was phosphorylated. The beta-chain appeared not to be a substrate for this enzyme. C3b which is formed by proteolytic cleavage of C3 was not phosphorylated by LPK-1. Trypsin treatment of phosphorylated C3 (P-C3) resulted in the disappearance of 32P from the alpha-chain. This was correlated with the conversion of the C3 alpha-chain to the alpha'-chain of C3b, and the appearance of a 9 kDa 32P fragment comigrating with the C3a fragment of C3. P-C3 was more resistant to cleavage by trypsin than nonphosphorylated C3. LPK-1 phosphorylated purified C3a and two synthetic peptides, C3a21R and YA-C3a10R, derived from its COOH-terminal end, which contain the C3a binding site to leukocytes and platelets. LPK-1 did not phosphorylate C3a8R. Phosphoamino acid analysis of the synthetic peptides indicated that serine 71 of C3a was phosphorylated by LPK-1. Treatment of C3 with either methylamine or freeze-thaw C3 (H2O) prevented phosphorylation by the LPK-1 suggesting that substrate conformation may be involved in recognition by the leishmanial enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Leishmanial protein kinases phosphorylate components of the complement system. 175 17

The cleavage of purified, functionally active rabbit C3 by cobra venom factor and trypsin was analysed by reducing and non-reducing sodium dodecyl sulphate electrophoresis and autoradiography. The specific aim of the study was to compare these reactions to those that occur with human C3. Analysis showed that the pattern of breakdown was very similar to that for the human protein: while the beta-chain remained intact, there was step-wise degradation of the alpha-chain to form C3a, C3b, iC3b and C3c, all of which could be identified by gel analysis. The metabolic behaviour of three of these cleavage products, C3a, C3b and iC3b, was then examined in vivo using dual isotope techniques. Rabbits were studied simultaneously with 131I-C3 and 125I-labelled C3 breakdown products. Analysis of plasma and urine radioactivity for the subsequent 72 h showed that all three breakdown proteins had rapid rates of catabolism in vivo compared to the native molecule. Specifically, 93 and 98% of C3b and iC3b, respectively, were eliminated from the plasma compartment within 10 h of injection. C3a was completely eliminated within 10 h. By comparison, native C3 showed a half-life of 29 +/- 3 h (mean +/- SD) and a fractional catabolic rate of 4.30 +/- 0.75%/h. The data support the use of this species in studies of complement behaviour in models of human immune disease and further clarify the basis for changes in plasma C3 concentration that accompany active immune complex- and antibody-mediated activity, in vivo.
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PMID:Activation of rabbit C3: studies of the generation of cleavage products in vitro and of their metabolism in vivo. 180 45

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.
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PMID:In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways. 230 32

Rat blood plasma contains three high molecular weight thiol ester-containing proteinase inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2-macroglobulin (alpha 2M). Rat serums have been analyzed using a two-dimensional gel electrophoretic technique which optimizes recovery of high molecular weight proteins. alpha 1M, and (alpha beta)4-tetramer in native solution, separated in the second sodium dodecyl sulfate-containing electrophoretic dimension as a disulfide-linked (alpha beta)2-dimer with an approximate Mr of 360 kDa. alpha 1I3 separated in the gels as a single 190-kDa polypeptide. It is also a monomer in native solution by ultracentrifugation criteria. Native rat alpha 2M is a tetramer, but it separates in the gels as a disulfide-linked dimer with an Mr of approximately 360 kDa. The kinetics of changes in concentration of these proteins during the induction of polyarthritis was also measured by quantitative immunoelectrophoresis. In rats with adjuvant-induced polyarthritis, the concentration of alpha 1I3 dramatically decreases and alpha 2M appears and continues to increase in a biphasic manner for 2 weeks. The alpha 1M concentration remains relatively constant. All three macroglobulins were purified utilizing modern rapid chromatographic techniques, and parallel comparisons of their native physicochemical properties were carried out. The N-terminal sequence of the alpha-chain of rat alpha 1M was also shown to share sequence homology with that of alpha 2M. In agreement, Esnard et al. (Esnard, F., Gutman, N., El Moujahed, A., and Gauthier, F. (1985) FEBS Lett. 182, 125-129) recently reported that alpha 1I3 also contains a thiol ester bond, as do alpha 1M and alpha 2M, since it reacts covalently with [14C]methylamine and is cleaved autolytically at 80 degrees C. We have examined negatively stained preparations of native, trypsin-treated, and methylamine-treated human alpha 2M, rat alpha 2M, and rat alpha 1M in the electron microscope. Trypsin appears to convert globular ring-shaped native molecules to rectangular box-like structures, in agreement with the conclusions of a recent report on human alpha 2M (Tapon-Bretaudiere, J., Bros, A., Couture-Tosi, E., and Delain, E. (1985) EMBO J. 4, 85-89).
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PMID:Three high molecular weight protease inhibitors of rat plasma. Isolation, characterization, and acute phase changes. 243 67

We investigated in detail the previously described capacity of pseudohyphae of Candida albicans to bind C3-coated particles. We show that the expression of the C3bi receptor of C. albicans was dependent upon the growth temperature of the fungi. C. albicans grown at 30 degrees C bound strongly to EAC1423bi, whereas those cells grown at 38.5 degrees C were completely devoid of this capacity. The molecule responsible for the attachment of EAC1423bi was heat labile and trypsin sensitive. Several, but not all, monoclonal antibodies to the alpha-chain of human complement receptor type 3 (CR3) stained C. albicans, and this reactivity was expressed in parallel with the capacity of C. albicans to bind EAC1423bi, i.e., both were dependent on the growth temperature of the fungi and were trypsin sensitive. In contrast to CR3, the binding of EAC1423bi to C. albicans did not require the presence of divalent cations. Rabbit immunoglobulin G antibodies directed against C. albicans inhibited the binding of EAC1423bi to C. albicans but not to human CR3. These inhibiting IgG antibodies recognized antigens expressed on the surface of pseudohyphae but not those of yeast cells. OKM-1, a monoclonal antibody to human CR3 inhibited the attachment of EAC1423bi to CR3 and also to C. albicans. OKM-1 precipitated a 130-kilodalton band from solubilized 125I-labeled C. albicans. We conclude that the complement receptors on C. albicans and human CR3 were antigenically related but not identical and that they differed in their functional characteristics.
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PMID:C3bi-binding protein on Candida albicans: temperature-dependent expression and relationship to human complement receptor type 3. 252 78

Nascent polysome-associated type I procollagen pro-alpha-chains isolated from chick embryo tendon fibroblasts were examined for their proteinase resistance. The distribution of chain sizes and their proteinase resistance were also determined following chain elongation in an in vitro readout system in the absence of chain initiation factors. Chains were labeled with [14C]proline in the cells and with [3H]proline in the readout system. Differences in the ratios of 14C to 3H in the double-labeled nascent chains before and after chymotryptic digestion, determined by slicing and counting polyacrylamide gels after electrophoresis, permitted analysis of the relative stabilities of in vivo and in vitro elongated portions of the chains. In confirmation of earlier work, the polysome-bound nascent procollagen contained chymotrypsin, chymotrypsin plus trypsin, and pepsin-resistant alpha-chain size components. The readout system data showed that the full length chains produced in the cell were more resistant to digestion than the fully elongated readout-completed chains. The protease resistance of the chains was taken to indicate the registration of the chains prior to the induction of helix formation during the isolation procedure. These data support the model in which chain selection and folding are facilitated by the organization of the attachment of the ribosomes to the endoplasmic reticulum surface.
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PMID:The coordinate synthesis and cotranslational assembly of type I procollagen. 264 82


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