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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glucocorticoid receptor from rat liver displays a differential sensitivity toward digestion by chymotrypsin and RNAase A that is dependent on its activation state. Unactivated (9-10 S) receptor is not digested by these enzymes, while activated 7-8 S receptor is. Chymotrypsin treatment yields an approx. 3 S form, while RNAase treatment yields a 4.9 S form that is distinct from the high-salt 4 S form. To firmly establish that the results are due to specific hydrolytic activities of the particular enzymes, we show that the chymotrypsin effect is inhibited by diisopropylfluorophosphate and not RNAasin, while the reverse is true for RNAase A. We further show that the differential sensitivity toward chymotrypsin is due to the association of a proteinase-resistant, heat-stable low molecular weight factor with the unactivated glucocorticoid receptor. When this factor is removed by warming, dialysis or molecular sieving of the receptor complex, the complex becomes sensitive to chymotrypsin. We also show that moderate chymotrypsin treatment yields a 6-7 S form of the receptor which is composed of, at least, RNA and the 4 S receptor. On the basis of these results, we propose that the 9-10 S receptor is composed of a low molecular weight stabilizing factor whose presence apparently alters the conformation of the complex such that the RNA and the RNA-binding site of the receptor are protected, a chymotrypsin-sensitive factor, RNA and the 4 S receptor itself.
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PMID:Studies with chymotrypsin and RNAase showing a heterooligomeric structure of the glucocorticoid receptor complex from rat liver which is stabilized by a low molecular weight factor. 242 6

Less than a stoichiometric amount of bovine chymotrypsin was progressively inactivated by a bovine AT III preparation. The rate of inactivation was not influenced by a synthetic substrate. Since chymotrypsin has low affinity for heparin, the polysaccharide only doubled the inactivation of the enzyme by the AT III preparation. At a low salt concentration, about two-thirds of the chymotrypsin was instantaneously inactivated by the AT III preparation in the presence of heparin. These observations indicate that inactivation of chymotrypsin is due to AT III itself, not to another contaminating inhibitor(s).
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PMID:Inactivation of bovine chymotrypsin by bovine antithrombin III. 243 74

The DEAE-cellulose-purified 4 S form of the rat liver glucocorticoid receptor can associate with cytosolic factors, as evidenced by an alteration of the sedimentation value of the 7-8 S form. On the basis of sedimentation profile, this form is indistinguishable from the activated, low-salt 7-8 S form isolated from rat liver cytosol. In addition, both the endogenous and reconstituted 7-8 S receptor can bind DNA as the 7-8 S form. In keeping with our reports that the endogenous form of the 7-8 S receptor is sensitive to RNAase digestion, treatment of the cytosol with RNAase prior to mixing with the 4 S receptor prevents the formation of the 7-8 S material. Moreover, warming the cytosol to 50 degrees C prior to mixing with the 4 S receptor also eliminates the ability to form the heavier material. Since RNA is heat-stable, this suggests that other factors may be involved. Treatment of the cytosol with N-ethylmaleimide, a reagent reported to be specific for sulfhydryl groups, also eliminates 7-8 S generating ability. These observations suggest that a protein may be a component of the 7-8 S generating material. This is substantiated by the observation that trypsin or chymotrypsin treatment of the cytosol mitigates the ability of the cytosol to form the 7-8 S material and results in the appearance of a form of the receptor that sediments at approximately 6 S. Protease treatment of partially purified material eliminates the 7-8 S generating activity entirely. We conclude that the 7-8 S form of the receptor can be reconstituted from the 4 S receptor via association with at least two other cytosolic factors, a protein and an RNA.
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PMID:The ability to convert the 4 S glucocorticoid receptor to the 7-8 S form is dependent on both RNA and protein factors. 244 56

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3'-triiodo-L-thyronine (T3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 +/- 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T3 at 22 degrees for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 +/- 1.47 x 10(5) M-1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 +/- 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T3-receptor complex using trypsin alpha-chymotrypsin, elastase, and papain produced distinct T3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.
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PMID:Differences in nuclear thyroid hormone receptors among species. 250 Mar 75

Conformational changes of alpha-chymotrypsin, induced by pH and pressure, have been studied with Raman spectroscopy. The secondary structure of alpha-chymotrypsin, chymotrypsinogen and DFP-chymotrypsin has been calculated by a singular value analysis of the Raman amide-I band. The changes in secondary structure, with pH and pressure titration of alpha-chymotrypsin, indicate a conformational transition. The salt bridge between Asp-194 and Ile-16 is disrupted, and the enzyme becomes inactive. No changes are observed for chymotrypsinogen. It is concluded that the proenzyme exhibits the same conformation at different pH values as alpha-chymotrypsin at alkaline pH. The results for DFP-chymotrypsin indicate that the active conformation is stabilized by the presence of the DFP inhibitor in the binding site.
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PMID:Raman spectroscopic study of the changes in secondary structure of chymotrypsin: effect of pH and pressure on the salt bridge. 259 7

The electric dichroism of alpha-chymotrypsin has been measured in buffers of various pH values and ion compositions. The stationary dichroism obtained as a function of the electric field strength is not compatible with an induced dipole mechanism and clearly shows that alpha-chymotrypsin is associated with a substantial permanent dipole moment. After correction for the internal directing electric field according to a sphere model, the dipole moment is 1.6 X 10(-27) C m at pH 8.3 (corresponding to 480 D). This value decreases with decreasing pH (to 1.2 X 10(-27) C m at pH 4.2), but is almost independent of the monovalent salt concentration in the range from 2 to 12 mM and of Mg2+ addition up to 1 mM. The assignment of the permanent dipole moment is confirmed by analysis of the dichroism rise curves. The dichroism decay time constants of (31 +/- 1) ns at 2 degrees C can be represented by a spherical model with a radius of 25-26 A, which is consistent with the known X-ray structure. The limiting linear dichroism is slightly dependent on the buffer composition and demonstrates subtle variations of the protein structure. As a complement to the experimental results, electric and hydrodynamic parameters of alpha-chymotrypsin have been calculated according to the known X-ray structure. Bead model simulations provide the center of diffusion, which is used to calculate dipole moments according to the equilibrium charge distribution evaluated from standard pK values.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The nature of protein dipole moments: experimental and calculated permanent dipole of alpha-chymotrypsin. 262 62

Bovine granulosa cells were disrupted by nitrogen cavitation and the resulting membrane vesicles were isolated by centrifugation using a self-generating Percoll gradient. Transmission electron microscopy and marker enzyme assays revealed a highly enriched preparation of plasma membrane vesicles with little contamination from intracellular organelles. The membranes were examined for their ability to bind [3H]heparin under a variety of physical conditions. Binding was dependent largely on electrostatic interactions which were sensitive to alterations in the ionic strength and pH of the medium. Optimal binding was obtained in the absence of added salt and at pH 6.5 but reduced by 50% at 150 mM-NaCl or at pH values above 7.5. Heparin binding to the membranes was abolished by a 1-h pretreatment with chymotrypsin, plasmin, pronase or trypsin. Detergent treatment of the membranes had various effects, depending on the ionic characteristics of the detergents used. Sodium dodecyl sulphate-polyacrylamide gels of plasma membrane proteins revealed a complex pattern of polypeptides with Mr of 10,000-120,000. Autoradiographic analysis of plasma membrane proteins on Western blots labelled with 125I-labelled heparin revealed 3 major heparin-binding proteins with molecular weights of 14,000-16,000. These studies report a new method of rapidly obtaining purified membranes from a limited population of granulosa cells. The characterization of the binding domains as membrane-associated proteins provides opportunities for numerous additional studies. Detergent solubilization of the membranes without appreciable loss in binding activity should simplify attempts to purify the binding proteins. Further analysis of the interactions of these molecules with native follicular fluid GAGs at various stages of granulosa cell development should provide useful insights into the role of complex carbohydrates in follicular maturation.
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PMID:Properties of heparin binding to purified plasma membranes from bovine granulosa cells. 262 5

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C. The synthesized protein constituted about 1% of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.
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PMID:Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells. 268 Jul 70

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.
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PMID:Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage. 271 76

The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of Mr 36,000, 34,000, and 33,000 and two chymotryptic fragments of Mr 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.
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PMID:Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation. 275 25


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