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)

Size-exclusion high-performance liquid chromatography was used to characterize the hydrodynamic molecular properties of estrogen receptors complexed with estradiol and the antiestrogen 4-hydroxytamoxifen. Cytoplasmic estrogen receptors complexed with [3H]-4-hydroxytamoxifen did not undergo reductions in hydrodynamic size after exposure to KCl or urea. Nuclear receptors complexed with 4-hydroxytamoxifen eluted as hydrodynamically larger molecules than nuclear receptors complexed with estradiol. Because identical hydrodynamic characterizations were obtained with the covalent ligand [3H]tamoxifen aziridine, these differences in chromatographic behavior are due to differences in ligand-mediated receptor properties and are not the result of ligand dissociation. When estrogen receptors, complexed with either [3H]estradiol or [3H]-4-hydroxytamoxifen, were exposed to trypsin, the receptors complexed with 4-hydroxytamoxifen eluted as larger hydrodynamic forms than receptors complexed with estradiol. These observations are interpreted to indicate that estradiol and 4-hydroxytamoxifen mediate contrasting transitions in the molecular orientation of estrogen receptors. The consequences of the transitions mediated by 4-hydroxytamoxifen appear to be that intermolecular associations become difficult to disrupt with KCl or urea and that the accessibility of trypsin-sensitive proteolytic sites becomes altered. Chromatin fractionation using DNase I and hypotonic Mg2+ solubilization identified a chromatin region that was less readily penetrated by receptors complexed with 4-hydroxytamoxifen than receptors complexed with estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrodynamic characterizations of estrogen receptors complexed with [3H]-4-hydroxytamoxifen: evidence in support of contrasting receptor transitions mediated by different ligands. 409 59

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.
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PMID:Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide. 608 83

Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.
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PMID:Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells. 614 92

Nucleosomes composed of 195 base pairs of DNA associated with histones H2A, H2B, H3, and H4 purified from chicken erythrocyte nuclei were used to elicit antibodies in rabbits. Specific serological reaction between the antisera and the nucleosomes is demonstrated by immunodiffusion, immunofluorescence, microcomplement fixation, solid-phase radioimmunoassay, immunosedimentation, and polyacrylamide gel electrophoresis of 5'-32P end-labeled nucleosomes. The antisera did not react with DNA extracted from these nucleosomes, core histones, or the cross-linked histone octamer from chicken erythocytes, calf thymus total histones, or chromosomal proteins HMG-1 or HMG-17. Nucleosome antigenicity was not affected by redigestion with micrococcal nuclease. Digestion with DNase I brought about 50% loss of reactivity while digestion with trypsin or proteinase K resulted in total loss of activity. The antisera reacted strongly with trimer, dimer, and monomer nucleosomes as well as with the core particle (145 base pairs of DNA) and subnucleosome (greater than 145 base pairs) obtained from chicken. It reacted less well with nucleosomes obtained from HeLa cells and was almost totally devoid of activity against chromatin particles obtained from rat liver or wheat germ. Experiments employing the technique of transferring proteins from a polyacrylamide gel to diazobenzyloxymethyl paper and visualization of antigens by autoradiography excluded the possibility that the serum contains antibodies against tissue-specific antigens which are found in small amounts but are very immunogenic. It is concluded that most of the anitbodies in the sera are directed against nucleoprotein antigenic determinants composed of the N-terminal portion of the histones and segments of DNA. Antibody binding is dependent on contact between the histone and DNA segments and is independent of the integrity of the entire nucleosome. Thus, certain histone DNA contacts remain intact even though the structure of the nucleosome has been disrupted.
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PMID:Chromatin subunits elicit species-specific antibodies against nucleoprotein antigenic determinants. 615 7

DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.
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PMID:Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs. 622 96

1. DNase I from porcine pancreas, if Mg2+ was present, hydrolyzed both sDNA and dDNA, whether free or bound to Sepharose. The hydrolysis rates were maximum at pH 7.5 with the bound DNAs and at pH 7.0 with the free DNAs negligible at pH 4.0 and pH 10.5 with the free and bound DNAs. The hydrolysis was completely inhibited by 50 mM sodium citrate. 2. With 50 mM citrate buffer (Ph 4.0), DNase I was effectively adsorbed on the DNA-Sepharoses in the absence of 5 mM Mg2+. The adsorbed enzyme was effectively eluated by the buffer containing 1 M KCl (eluate). The amounts of the eluated enzyme were approximately 1.5 X 10(5) units/mg DNA with sDNA-Sepharose and approximately 3.0 X 10(5) units/mg DNA with dDNA-Sepharose. This simple adsorption-elution of the pancreas extract resulted in approximately 300-fold purification of DNase I with a yield of 95%. In the elute, the ratios in activity of trypsin, chymotrypsin and RNase to DNase I were 1/(4.0 X 10(5)), 1/(5.3 X 10(3)), and 1/(4.1 X 10(2)) as low as in the extract, respectively. In addition, the eluate was not contaminated by kallikrein or carboxypeptidases A and B. 3. Upon repeating the adsorption-elution described above, the adsorbing capacities of DNA-Sepharoses gradually deteriorated with the whole pancreas extract, but not with the precipitate of the extract formed on 60% ammonium sulfate saturation, which contained 90% of the DNase I. With the precipitate, one dDNA-Sepharose solumn was repeatedly usable at least 20-times without deterioration. The DNase I preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis. 4. Conceivably, the above-mentioned adsorption of DNase I on DNA-Sepharoses was mainly due to the steric and electrostatic affinity of a relatively large moiety of the DNA molecule to the substrate-binding site, but not to the catalytic site, of the enzyme.
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PMID:Affinity chromatography of porcine pancreas deoxyribonuclease I on DNA-binding sepharose under non-digestive conditions, using its substrate-binding site. 625 6

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.
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PMID:Protection of actin against proteolysis by complex formation with deoxyribonuclease I. 626 44

A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.
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PMID:Stimulation of DNA polymerase alpha by a nuclear DNA/protein complex. 627 80

Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.
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PMID:Perturbation of lac operator DNA modification by tryptic core protein from lac repressor. 635 13

In this study we report the identification of an antibody in the sera of some patients with autoimmune disease that reacted with a cytoplasmic antigen localized within the Golgi apparatus. The antibody reacted with all tissues investigated, which included pancreas, kidney, testis, liver, thymus, and spleen. In addition, it reacted with some human peripheral circulating lymphocytes, murine peritoneal macrophages, and a variety of tissue culture cell lines, which included HEp-2 cells (human epithelial carcinoma), baby hamster kidney cells, a canine thymus cell line, a primary kidney cell line, Ehrlich ascites cells, Wil-2 cells, and Raji cells. The antigen is located in the same region stained by the histochemical reaction for thiamine pyrophosphatase, thus indicating that the antigen is located within the Golgi apparatus. The antigen was not demonstrated by immunodiffusion of saline extracts of rabbit thymus, pancreas, or liver. The antigen in HEp-2 cells was resistant to RNase A, DNase I, micrococcal nuclease, and to extraction with 0.1 N HC1, but was sensitive to trypsin and Proteinase K. Eight patients with anti-Golgi antibodies have been identified. Six of the eight had systemic lupus erythematosus. Autoantibodies to a Golgi apparatus antigen might serve as a useful biologic marker to study the functional relationship of the Golgi apparatus to lymphocytes and macrophages.
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PMID:Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus. 637 21


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