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)

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.
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PMID:Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia. 264 45

We utilized antisera specific for murine IL 1 alpha and IL 1 beta proteins to characterize cell-associated IL 1 according to two distinct criteria, immunochemical detection of radiolabeled IL 1 polypeptides and inhibition of IL 1 activity in cell lysates. Evidence is presented that IL 1 alpha and IL 1 beta are each synthesized by LPS-stimulated but not by unstimulated murine macrophages and accumulate within the cell as intracellular precursors in sizes of 33 kDa and 37 kDa, respectively. As judged by the respective rates of synthesis, IL 1 alpha was about 4-fold more abundant than IL 1 beta. Most (greater than or equal to 95%) of the cell-associated IL 1 activity was inhibited in the presence of the antiserum to IL 1 alpha, suggesting that the precursor of IL 1 beta is mostly biologically inactive. Incubation of cell lysates with papain but not with trypsin or plasmin markedly stimulates an increase in the level of cell-associated IL 1 beta activity and leads to the cleavage of 15-16 kDa carboxyl-terminal fragments from the IL 1 beta precursor. Together, these data indicate that proteolysis of the IL 1 beta precursor is required to generate bioactive IL 1 beta molecules and provide a basis for further investigations of the specific role of proteinases in processing the IL 1 beta precursor to a bioactive form.
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PMID:Proteolysis of the native murine IL 1 beta precursor is required to generate IL 1 beta bioactivity. 265 10

Little is known concerning the mechanism involved in cell contact between the donor and recipient during conjugation in Neisseria gonorrhoeae. The formation of stable mating pairs during conjugation in Escherichia coli appears to require a specific protein as well as LPS in the outer membrane of the recipient cell. To attempt to identify the cell surface components necessary for conjugation in the neisseriae, we began a comparison of the outer membrane of Neisseria cinerea strains that can (Con+) and cannot (Con-) serve as recipients in conjugation with N. gonorrhoeae. There were no differences in outer-membrane protein profiles on SDS-polyacrylamide gel electrophoresis between Con+ and Con- strains that could be correlated with the ability to conjugate. However, whole outer membrane isolated from Con+ strains specifically inhibited conjugation while those from Con- strains did not. Proteolytic cleavage of outer-membrane proteins by trypsin, pronase or alpha-chymotrypsin abolished the inhibitory effect of Con+ outer membranes, suggesting that these outer membranes contained a protease-sensitive protein(s) involved in conjugation. Although periodate oxidation of Con+ outer-membrane carbohydrates did not abolish the inhibitory action of these membranes, purified LPS from both Con+ and Con- strains inhibited conjugation when added at low concentrations. These results suggest that conjugation requires the presence of a specific conjugal receptor that consists of both LPS and one or more outer-membrane proteins. Both Con+ and Con- strains contain the necessary LPS, but only Con+ strains contain the required protein(s).
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PMID:Role of outer-membrane proteins and lipopolysaccharide in conjugation between Neisseria gonorrhoeae and Neisseria cinerea. 315 96

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.
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PMID:Phosphorylation of intracellular precursors of human IL-1. 325 35

A high percentage (80-90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDM phi) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDM phi by means of specific staining. Mature PNA- thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDM phi. Since thymocyte survival was not at all affected by M phi culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by M phi. Similarly, treatment of M phi with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated M phi and LPS-activated M phi rather than nontreated M phi.
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PMID:Interaction between thymocytes and thymus-derived macrophages. II. Engulfment of thymocytes by macrophages. 326 11

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.
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PMID:Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release. 349 84

We and others have previously reported that human blood mononuclear cells release in culture certain substances that enhance the capacity of purified human blood eosinophils to kill the antibody-coated larvae of Schistosoma mansoni. The present study shows that this eosinophil cytotoxicity-enhancing activity (ECEA) is released by monocytes and T lymphocytes. Monocytes produce ECEA in resting and in LPS-stimulated cultures; T lymphocytes release such activity when stimulated by mitogens such as concanavalin A. Furthermore, the human monocytic line U-937 also releases ECEA-like activity when stimulated by LPS. The enhancing activity produced by monocytes has been partially characterized: it is sensitive to proteolysis by trypsin, relatively heat stable, and associated with molecules that have an apparent molecular weight of 14,000 to 65,000 daltons and isoelectric points of 3.8-3.9, 4.2, 4.5, 4.8-4.9. This shows that while ECEA produced by monocytes is heterogeneous in size and charge, it is probably different from interleukin 1.
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PMID:Activation of human eosinophils by monokines and lymphokines: source and biochemical characteristics of the eosinophil cytotoxicity-enhancing activity produced by blood mononuclear cells. 387 17

Purified precursor Hageman factor has been demonstrated to bind to soluble bacterial lipopolysaccharide (LPS, endotoxin) isolated from Escherichia coli 0111:B4, and this complex has been shown to have the capacity to convert prekallikrein to its active form. In addition, LPS-activated Hageman factor substantially reduces clotting times in XII-deficient plasma. The capacity to activate Hageman factor has been demonstrated to reside in the lipid A region of the LPS molecule. Activation of Hageman factor by LPS contrasts with fluid-phase activation (e.g., by kallikrein or trypsin) in that no cleavage to lower molecular weight fragments occurs. High concentrations of LPS inhibit the activity of Hageman factor, probably by a direct LPS-Hageman factor interaction.
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PMID:Direct evidence for Hageman factor (factor XII) activation by bacterial lipopolysaccharides (endotoxins). 437 13

It has been reported that polyclonal B cell stimulation results in formation of autoantibodies and immune complexes. We have previously reported that a polyclonal B cell activator (PBA) associated with alpha 2-macroglobulin (alpha 2M) is present in the serum of patients with rheumatoid arthritis and related diseases. Here we studied the possibility that patient alpha 2M (Pt-alpha 2M) carries a trypsin-like protease responsible for the PBA activity. This activity was determined by the Ig-turnover assay developed in our laboratories. The small molecular weight protease inhibitors, aprotinin (Trasylol, Bayer) and phenylmethylsulfonylfluoride (PMSF), and the large molecular weight soybean trypsin inhibitor (SBTI) were used. These inhibitors did not affect the PBA activity of dextran sulfate of LPS. However, as expected, trypsin had a PBA-activity which was blocked by all of the above mentioned inhibitors. A trypsin-normal alpha 2M complex (Tr-N alpha 2M) and PBA activity which was inhibited by PMSF or aprotinin but not by SBTI. The PBA associated with Pt-alpha 2M was also inhibited by PMSF or aprotinin but not by SBTI. Moreover, the Tr-N alpha 2M complex and the Pt-alpha 2M, but not that from normal donors, had esterase activity for p-toluenesulfonyl-L-argininemethyl ester. These data suggest a similarity between the Pt-alpha 2M and Tr-N alpha 2M complex. Thus, we concluded that the esterolytic activity is sufficient for PBA activity, that Pt-alpha 2M has esterolytic activity and that this PBA activity can be blocked by small molecular weight protease inhibitors.
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PMID:The effect of protease inhibitors on the polyclonal B cell activator from the serum of patients with rheumatoid arthritis. 617 49

The receptor specificity of H-2-restricted T lymphoblasts activated against trinitrobenzene sulfonate (TNBS)-coupled spleen cells was examined using an antigen binding assay. A population of Lyt-1+,2-T lymphoblasts acquired syngeneic Ia determinants during 4 days of primary culture with hapten-coupled stimulator cells. Syngeneic Ia was not reexpressed after trypsin treatment of the T cells, but was found after incubation with soluble Ia shed from lipopolysaccharide-activated blasts. Self-Ia binding was specific in that Lyt-1+,2- but not Lyt-1-,2+ cells acquired the antigen, and in that self-Ia bound more effectively than allogeneic Ia material. To determine the relationship of self-Ia binding to the recognition of foreign antigen, the binding of trinitrophenyl (TNP)-coupled plasma membrane vesicles by TNP-specific T cells was studied. TNP-vesicle binding occurred via TNP and H-2(Ia) molecules on the vesicles in that binding was inhibited with antibodies against TNP or H-2(Ia) molecules but not non-major histocompatibility complex (e.g., Ly-6.2) molecules on the vesicles. Complete inhibition of TNP-vesicle binding by an Iak-restricted TNP-specific T-cell line occurred with soluble TNP-lysine, but not an unrelated hapten, N-iodoacetyl-N-(5-sulfonic-1-naphthyl)ethylenediamine (I-AED)-cysteine. Conversely, I-AED-cysteine, but not TNP-lysine, inhibited binding of I-AED-coupled B6 vesicles by B6 anti-I-AED T cells. Significant, but weak inhibition of TNP-vesicle binding by the anti-TNP line was observed with glycoprotein preparations containing partially purified self-Ia molecules. However, inhibition was specific for I-Ak molecules, in that inhibition was lost after removal of I-Ak molecules from the glycoprotein preparation, and very little inhibition occurred with soluble glycoproteins prepared from thymocytes which contained very little Ia material or from LPS blasts of an unrelated H-2 haplotype. These results suggest a recognition model in which TNP and Ia determinants are recognized by neighboring receptor combining sites.
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PMID:Receptor specificity of Ia-restricted T lymphoblasts activated against trinitrobenzene sulfonate-coupled spleen cells: recognition of distinct trinitrophenyl and Ia moieties. 619 19


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