Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, alpha-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.
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PMID:Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis. 329 May 76

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.
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PMID:The steroid binding domain of porcine estrogen receptor. 330 Jul 75

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.
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PMID:Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity. 362 Apr 50

We have previously shown, in the estrogen-unresponsive C3H mouse mammary tumor that the affinity of the estrogen receptor (ER) for calf thymus DNA in vitro is four-times higher than that of uterine ER [Baskevitch, P. P., Vignon, F., Bousquet, C. and Rochefort, H. (1983) Cancer Res. 43, 2290]. By mixing cytosols from this tumor and uterus, we describe a tumor factor capable of increasing ER affinity for DNA, as assayed by DNA-cellulose chromatography and saturation studies. The activity of this factor was inhibited by alpha-chymotrypsin-inhibitors such as N-tosylphenylalanylchloromethane and chymostatin. Using the fluorogenic substrate glutarylglycylglycylphenylalanyl-N-naphthylamide, we assayed such a protease in the C3H mammary tumor cytosol. This protease and the factor altering ER-DNA binding were eluted together from chromatography on DEAE-cellulose, AcA 44, and carboline-agarose and were sensitive to the same inhibitors. The partially purified factor decreases the molecular mass of the estrogen receptor as seen by denaturing electrophoresis after covalent labelling of the ER with [3H]tamoxifen aziridine. We suggest that the increase of ER affinity for DNA and the decrease of ER molecular size are due to the same protease with an alpha-chymotrypsin-like specificity.
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PMID:A cytosol protease from the estrogen-resistant C3H mammary carcinoma increases the affinity of the estrogen receptor for DNA in vitro. 388 20

Ultrafiltration was applied to the fractionation of neutral vs. charged peptides of similar size. The peptides, produced from gliadins, a major fraction of wheat storage proteins, were obtained by limited hydrolysis with alpha-chymotrypsin in water-ethanol 80/20 (v/v). Peptides, according to their elution by RP-HPLC, were quasineutral (repetitive peptides) irrespective of pH, or positively charged (nonrepetitive peptides) at pH below 5. The transmission through the membranes of the nonrepetitive peptides was less (until sevenfold) than that of the repetitive ones, because of the role of electrostatic repulsion involved in the retention of charged solutes. The difference of transmission was more efficient at acidic pH (3) and low ionic strength with inorganic membranes and in a wider range of pH and ionic strength with membranes modified by coating of positively charged polymers (polyvinylimidazole PVI, polyethyleneimine PEI). A continuous diafiltration process using an inorganic membrane of low molecular cut-off permitted the selective enrichment of the retentate in nonrepetitive peptides (up to 80%) and of the permeate in repetitive peptides (up to 80%) from hydrolysate feed containing about 60/40% of repetitive and nonrepetitive peptides, respectively, with a diafiltration volume of 4.
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PMID:Fractionation of gliadin hydrolysates in water-ethanol by ultrafiltration with modified or unmodified membranes. 995 23