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 role for second messenger-regulated protein kinases in the early post-IL-3 receptor signal transduction pathway was investigated in the mast cell/megakaryocyte line R6-XE.4. The activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was assessed by the ability of the enzyme to phosphorylate histone H1 in the presence of calcium, diacylglycerol, and phosphatidylserine or after proteolytic activation of PKC with trypsin. In high serum-supplemented cells, but not in cells that were preincubated in serum-deficient media for 6 h, subsequent treatment for 15 min with synthetic IL-3 (10 micrograms/ml) caused up to a sixfold increase in the calcium- and lipid-stimulated histone H1 phosphorylating activity of particulate-associated PKC after fractionation on MonoQ. However, there was no corresponding reduction of cytosolic PKC activity. Therefore, IL-3 appeared to modify the activity of preexisting membrane-associated PKC rather than eliciting its recruitment from the cytoplasm in R6-XE.4 cells. This was in contrast to the situation with FDC-P1 cells, where IL-3 induced PKC translocation. IL-3 also stimulated a cytosolic protein kinase that phosphorylated a synthetic peptide patterned after a phosphorylation site in ribosomal protein S6, but this IL did not alter the activity of cAMP-dependent protein kinase.
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PMID:IL-3-induced activation of protein kinases in the mast cell/megakaryocyte R6-XE.4 line. 230 40

Histone H1t has been purified from rat testes and antibodies were elicited in rabbits. Immunoblotting studies with anti-histone H1t-IgG have shown that it reacted specifically with histone H1t but not with other histone H1 subtypes, namely H1a, -b, -c, -d, -e and H10. The anti-histone H1t-IgG also did not react with chicken erythrocyte histone H5. Immunoblotting studies have also revealed that the polyclonal anti-histone H1t-IgG reacted with (a) two polypeptide fragments, NBS-N and NBS-C, derived from N-bromosuccinimide cleavage of histone H1t, (b) two polypeptide fragments, CT-N and CT-C, derived from alpha-chymotrypsin cleavage of histone H1t, and (c) GH1t, globular domain of histone H1t obtained after trypsin cleavage. The indirect immunofluorescence studies on nuclei isolated from adult rat testes with anti-histone H1t-IgG showed that the fluorescence, particularly, of the pachytene nucleus was the brightest. On the other hand, anti-histone H1t-IgG did not stain nuclei from either liver or nuclei isolated from the testes of 10-day-old rats.
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PMID:Testis-specific histone H1t is antigenically distinct among H1 subtypes. 241 27

Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid-phase radioimmunoassay (RIA). One of these (HBC-7) was specific for H2B by both techniques whereas the other (2F8) cross-reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin-digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N-terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin-sensitive, N-terminal region comprising residues 1-20. Binding of antibody HBC-7 was inhibited by in vitro ADP-ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC-7 is located at the N-terminus of H2B, probably between residues 1 and 8. We have used solid-phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 ('stripping') from high-molecular-mass chromatin had no effect on HBC-7 binding, nor was any difference observed between binding to stripped chromatin and to 146-base-pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N-terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC-7 to 146-bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non-histone proteins during nuclease digestion. Pre-treatment of high-molecular-mass chromatin or 146-bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC-7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N-terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.
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PMID:Characterization of monoclonal antibodies to histone 2B. Localization of epitopes and analysis of binding to chromatin. 242 56

Monoclonal antibodies (mAb) to ox liver histone H1 degree were produced and characterized. Two sets of mice were immunized either with pure H1(0) or with an H1(0)-yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained kappa light chains. Immunoblotting experiments using peptides derived from H1(0) or H5 treated by various proteolytic agents (trypsin, N-bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1(0). More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24-27 and were specific for H1(0). Six mAb bound to residues 27-30 and were specific for H1(0) except one of them which strongly cross-reacted with H5 and GH5. Two mAb reacted with the entire histone H1(0) but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1(0) globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1(0) and the various IgG1 mAb by increasing amounts of peptide-bovine serum albumin conjugates were then performed.
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PMID:Histone H1(0) mapping using monoclonal antibodies. 247 9

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.
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PMID:Effect of human mast cell tryptase on human plasma proenzymes. 253 Jan 78

We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.
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PMID:Purification and properties of protein kinase C from rabbit reticulocyte lysates. 282 7

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.
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PMID:Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation. 299 55

The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher Mr value than peak 1 (35,500 vs. 35,000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to Mr and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent Mr of 35,500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca2+-dependent proteinase decreased the apparent Mr by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the 'nicked' catalytic subunit was similar to that obtained with the holoenzyme.
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PMID:Catalytic subunit of the polycation-stimulated protein phosphatase. Effect of proteolysis on polycation stimulation. 302 26

C-reactive protein (CRP) is an acute phase serum protein in man which binds to phosphocholine (PC) in a calcium-dependent manner. CRP has been shown to bind to chromatin and nucleosome core particles. However, CRP does not bind to DNA and there is conflicting evidence regarding the binding of CRP to histones. In the present study, binding of CRP to chromatin was confirmed by ELISA using chromatin bound to microtiter wells. When chromatin depleted of histone H1 was used in the same assay, no CRP binding was detected. Similar results were observed using a competitive inhibition ELISA. These results indicate an important role for H1 in the binding of CRP to chromatin. Further studies were done to characterize the binding of CRP to purified individual histones. CRP binding to histones was demonstrated first by blotting. Calf thymus histones were separated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with 125I-CRP. CRP bound to H1 and H2A and to a lesser extent to H2B. Non-specific binding to H3 was seen and no binding to H4 was observed. CRP binding to purified individual histones was tested by ELISA. Essentially identical results were seen to those obtained by blotting. CRP binding to the H2A-H2B complex was observed as well as reactivity with trypsin-resistant fragments of H2A, H2B, and H3. By blotting and by ELISA all CRP reactions were blocked by PC and EDTA indicating binding through the calcium-dependent PC-binding site on CRP. These studies further characterize the nature of the binding of CRP to chromatin and histones and show that the presence of H1 on chromatin is required for CRP binding.
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PMID:Analysis of the binding of C-reactive protein to histones and chromatin. 319 19

Using limited chymotrypsin and trypsin digestion of isolated Physarum histone H1 labeled in vivo in postsynthetically added N epsilon-methyl groups of lysine we show that: --there is no postsynthetic methylation in the central globular domain of H1, --a moderate number of methylated sites occurs in the N-terminal fragment and the part of the C-terminal fragment directly adjacent to the globular domain (the main site of interphase phosphorylation), --the most intensively methylated region occurs within the sequence located in an extended part of the C-terminal fragment, distant to the globular domain and the main site of interphase phosphorylation.
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PMID:Distribution of postsynthetic methylation sites in Physarum histone H1. 322 47


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