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)

When nucleosomal core histones were isolated from rat liver nuclei incubated with [14C]NAD+ and fractionated into the individual components (H2A, H2B, H3, and H4), [14C]adenosine diphosphate ribose (ADP-Rib) was found to be associated with all of them. However, while about 15% of the H2B molecules were modified, less than 2% of the other fractions contained radioactive ADP-Rib. The nucleotide attached to H2B was identified as a single monomer of ADP-Rib. On subjectint H2B to electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid, one single band of H2B with 5% less mobility than the unomdified control was obtained. The linkage between H2B and ADP-Rib was rapidly hydrolyzed with 0.1 N NaOH or with 1 M neutral hydroxylamine. Hydrolysis of ADP-ribosylated H2B with trypsin generated a single peptide linked to ADP-Rib, which corresponded to the sequence Pro-Glu-Pro-Ala-Lys. We were able to dansylate the NH2-terminal proline, which proved that the imino group of this amino acid was not substituted. These findings, together with the chemical properties of the linkage, which were typical of those of an ester-like bond, strongly suggest that the ADP-Rib residue was linked to the gamma-COOH group of the glutamic acid in position 2 of H2B.
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PMID:ADP ribosylation of rat liver nucleosomal core histones. 21 26

The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and H1, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments I to IV, by limited chymotryptic digestion at pH 5.0 and 15 degrees C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1--40, 41--42, 43--121 and 122--125 of the total sequence. The only new findings were microheterogeneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30% alanine). The sequence of the most basic cluster at residues 27--24, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.
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PMID:Human spleen histone H2B. Isolation and amino acid sequence. 42 50

During purification of E2R using oligo(dT)-cellulose chromatography, a receptor accessory factor (RAF) was identified in the cytosol of mouse kidney. This factor stimulates the binding of purified E2R to oligo(dT)-, oligo(dC)-, and oligo(dA)-cellulose as well as to DNA cellulose. It is a heat-stable, trypsin-resistant protein with an apparent molecular weight of between 10 and 30,000 daltons. Although structurally unrelated, similar stimulation of oligonucleotide binding was seen with calf thymus histones and, to a lesser extent, egg white lysozyme. Individual histones, especially H2a, H2B, and H3, also facilitate rebinding of purified E2R to oligo(dT)-cellulose, while H1 is less effective. Furthermore, histones stabilize the holoreceptor during sedimentation at 4 degrees and 12 degrees C. The N- and C-terminal half molecules of H2b were generated by cyanogen bromide-mediated cleavage and the N-terminal half was found to duplicate the effects of the parent molecule, both in binding and holoreceptor stabilization. These data suggest that the in vivo binding of E2R to DNA can be modulated by accessory proteins of cytosol and nuclear origin.
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PMID:Stimulation of oligonucleotide binding of estradiol receptor complexes by accessory proteins. 49 27

In this report, we introduce the use of DNA-cellulose chromatography for evaluating the strength of binding of histones to DNA under a variety of conditions. We have found that histones added directly to DNA-cellulose at physiological salt concentrations bind relatively weakly, with all histones eluting together at about 0.5 M NaCl when a salt gradient is applied. However, much tighter binding of the four nucleosomal histones to DNA-cellulose is obtained if gradual histone-DNA reconstitution conditions are used. In this case, the binding of histones H2A, H2B, H3, and H4 to DNA-cellulose closely resembles their binding to native chromatin. The nativeness of the binding is indicated both by the distinctive sodium chloride elution profile of these histones from DNA-cellulose and by their relative resistance to trypsin digestion when DNA-bound. The binding to DNA-cellulose of histones H2A, H2B, H3, and H4, which have had the first 20 to 30 amino acid residues removed from their NH2 termini, is indistinguishable from the binding to DNA-cellulose of the same intact histones, as judged by their salt elution profile. Thus, even though the NH2 termini contain 40 to 50% of the positively charged amino acid residues (thought to interact with the DNA backbone), a major contribution to the DNA binding comes from the remainder of the histone molecule. Finally, we have discovered that histones can form a "nucleosome-like" complex on single-stranded DNA. The same complex does not appear to form on RNA. Histones H3 and H4 play a predominant role in organizing this histone complex on single-stranded DNA, as they do on double-stranded DNA in normal nucleosomes. We suggest that, in the cell nucleus, nucleosomal structures may form transiently on single strands of DNA, as DNA and RNA polymerases traverse DNA packaged by histones.
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PMID:The use of DNA-cellulose for analyzing histone-DNA interactions. Discovery of nucleosome-like histone binding to single-stranded DNA. 50 Jun 33

The arrangement of 8 histones in the nucleosome core has been investigated by identifying the sites of 4 histone sequences cross-linked with a bifunctional amino-group reagent, dimethyl suberimidate, selected from among 4 diimidoesters of various linker lengths examined. H1-depleted calf thymus chromatin was allowed to react with 14C-labeled suberimidate at pH 8.5 and 0 degrees C. The cross-linked chromatin was then digested exhaustively with trypsin. Almost all the histone fragments were released from the chromatin with 0.25 M HCl and chromatographed on several columns and on paper. Cross-linked peptides were detected by analyzing the content of radioactive suberimidoylbislysine after acid hydrolysis. The chromatographic procedure developed here showed that the whole histone fragments contained 29 mol% of the total linked reagent as suberimidoylbisylsine. The 5 finally purified cross-linked peptides were identified from the total and N-terminal amino acids of each pair of peptides separated by two-dimensional cellulose thin layer chromatography after cutting the linker by ammonolysis. Thus, intramolecular cross-linking was found between Lys-5 and Lys-9 of H2A, and Lys-34 and Lys-85 of H2B, while intermolecular cross-linking was found between Lys-24 (or 27) of H2B and Lys-74 of H2A, Lys-85 of H2B and Lys-91 of H4, and Lys-120 of H2B and Lys-115 of H3 and/or Lys-77 of H4. Most of these lysine residues are located in the DNA-binding segments of the 4 histone sequences identified previously [Kato, Y. & Iwai, K, (1977) J. Biochem. 81, 621--630]. All the 5 or 6 cross-links can be located in a heterotypic tetramer consisting of one molecule each of H2A, H2B, H3, and H4, and a model of the histone arrangement in the tetramer is proposed. Two such tetramers may compose to the histone octamer in the nucleosome core.
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PMID:Identification of suberimidate cross-linking sites of four histone sequences in H1-depleted chromatin. Histone arrangement in nucleosome core. 52 33

Soluble chromatin of Trypanosoma brucei brucei procyclic culture forms was submitted to digestion with free or immobilized trypsin. Digestion with trypsin in salt solutions of low and high ionic strengths generated characteristic sets of limit histone peptides. After incubation of chromatin with immobilized trypsin in a solution of low ionic strength, histones were not degraded, whereas a selective proteolysis occurred at 50 mM NaCl. Histones a and d, which correspond to H3 and H4 of higher eukaryotes, were rapidly attacked. Histones b and c, the counterparts of H2A and H2B, were more resistant. The results indicated that probably the basic N-terminal tails of the proteins a and d are located on the surface of the core particle. The location of d on the surface differs from the internal one proposed for histone H4. The salt-induced increase of susceptibility of histones to proteolysis reflects structural changes of T.b. brucei chromatin, which may result in partial chromatin compaction.
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PMID:Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized trypsin. 135 62

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.5 M NaCl. Treatment of yeast nucleosomal particles with the amino group reagent dimethylmaleic anhydride is accompanied by selective release of histones H2A and H2B. The results indicate not large but significant structural differences between yeast and chicken nucleosomal cores. However, the in vitro transcription properties of complete and H2A.H2B-deficient nucleosomal cores are similar in the two kinds of particles: the histone octamer blocks RNA synthesis, this block being eliminated in part by the partial loss of histones H2A and H2B.
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PMID:Yeast nucleosomal particles: structural and transcriptional properties. 204 19

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.
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PMID:Accessibility and structural role of histone domains in chromatin. biophysical and immunochemical studies of progressive digestion with immobilized proteases. 210 May 22

We have identified regions within core histones that are antigenic for autoantibodies in systemic lupus erythematosus (SLE) and drug-induced lupus. An immunoblotting technique was used to determine the reactivity of lupus antibodies for intact histones and for trypsin-resistant histone fragments that lack the amino- and carboxyl-terminal amino acids that are normally exposed in native nucleosomes. In SLE, the predominant anti-histone response was restricted to epitopes in the trypsin-sensitive regions. Of 20 SLE sera that had strong antibody activity for multiple intact histones, 17 showed minimal activity with any of the corresponding trypsin-resistant fragments. A markedly different pattern of reactivity was present in sera of patients with procainamide (Pr)-induced lupus in which antibodies to H2A, H2B, and the H2A-H2B complex had strong fragment activity. Interestingly, recognition of trypsin-resistant fragments was also noted in a small number of SLE sera that contained antibodies to the H2A-H2B complex. In contrast to both SLE and Pr-induced lupus, antibodies induced by hydralazine (Hy) reacted primarily with H3 and H4. Furthermore, these antibodies bound equally well to the corresponding trypsin-resistant regions that are thought to be relatively unexposed in native nucleosomes. Thus, the specificities of anti-histone antibodies in SLE, Pr-induced lupus, and Hy-induced lupus are markedly different, but in each disease reactivity appears to be restricted to a limited number of histone determinants. The data raise the possibility that autoantigen in the form of native nucleosomes may be recognized in SLE and possibly in Pr-induced lupus. In contrast, the propensity of Hy to induce autoantibodies to determinants usually not recognized in SLE or Pr-induced lupus may suggest a different immunogenic stimulus in this disease.
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PMID:Anti-histone antibodies in idiopathic and drug-induced lupus recognize distinct intrahistone regions. 243 24

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


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