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)

Mononucleosomes prepared from goose erythrocyte nuclei exhibited limited heterogeneity with respect to number of electrophoretic components, histones and DNA composition. The components differ slightly in ionic strength induced self-association. Thermal denaturation of each component gave only two dominant, highly cooperative, melting transitions, T" and T"'. Urea and trypsin were used to establish the differential lability of these two transitions. Comparison of the morphologies of the mononucleosomes at various stages throughout the melting profile indicated that the 13.3 +/- 1.5 nm diameter mononucleosomes start to disrupt only in the latter half of transition T" and do not unfold until after reaching T"'. The resultant, open ended (17.4 +/- 2.2 nm diameter) toroids are still largely negatively staining and much more uniform in shape if fixed simultaneously with gluteraldehyde.
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PMID:Alteration in nucleosome structure induced by thermal denaturation. 67 53

1) S. mutans strains of serotypes a, d and g were strongly agglutinated with soluble glucans and dextran T2000. Homologous glucan did not in all cases produce agglutination. 2) The quantity of low molecular weight dextrans bound (T20 and T70) does not correspond to the agglutination induced by glucan or T2000. 3) The agglutination and binding of high molecular weight glucan by B13 cells was sensitive to heat, trypsin, dextranase, EDTA, SDS and urea, whereas no inhibition of binding of T20 and T70 was seen. 4) Pretreatment of B13 cells with anti-d, or anti-glucan sera, or Con A, RCA I, or RCA II completely inhibited agglutination by T2000 and caused a significant reduction of the binding of glucan. No reduction in the binding of T20 and T70 occurred. 5) An agglutination-negative mutant was agglutinated by sucrose but not by T2000 or high molecular weight glucan. It bound normal levels of T20 and T70. 6) The results indicate that B13 cells possess multiple glucan binding sites and that the site responsible for agglutination consists of both polysaccharide and protein. 7) Inhibition studies on agglutination and adherence using B13 cells indicate that the two processes involve different mechanisms.
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PMID:Dextran/glucan binding by Streptococcus mutans: the role of molecular size and binding site in agglutination. 74 9

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5 - 12. Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II. Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.
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PMID:Conformational studies on murein-lipoprotein from the outer membrane of Escherichia coli. 79 57

O-Acetylserine-O-acetylhomoserine sulfhydrylase [EC class 4.2.99], catalyzing the sulfhydrylation of both O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) (O-acetyl-L-serine(O-acetyl-L-homoserine) + H2S leads to L-cysteine (L-homocysteine) + acetate), was extracted and purified from bakers' yeast by an improved method. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecylsulfate and by ultracentrifugal analysis. The apo-enzyme was protected by pyridoxal phosphate (PALP) from inactivation by heat, urea, and trypsin [EC 3.4.21.4], suggesting that the binding of PALP to the apo-enzyme rendered the conformation of the protein more stable. The holo-enzyme showed absorption peaks at 420 and 330 nm due to bound PALP, in addition to a peak at 280 nm. Upon reduction with borohydride, the 420-nm peak disappeared and an increase in the 330-nm peak occurred concomitant with loss of the catalytic activity. Lysine appeared to be the pyridoxal binding site, based on identification of pyridoxyl-lysine in the hydrolyzate of the holo-enzyme. It was shown by both spectral and chemical determinations that 4 moles of PALP could bind to 200,000 g of apo-protein. The apo-enzyme showed a lower association constant with PALP than some other enzymes. Pyridoxal inhibited the activity competitively with respect to PALP. Based on these findings, it appears that the reaction mechanism of this enzyme is similar to those of other pyridoxal enzymes.
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PMID:O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Further purification and characterization as a pyridoxal enzyme. 79 6

When Rana cancrivora were collected from fresh water and dehydrated (weight loss 4-10%) by exposure to saline, the plasma titre of hydro-osmotic activity, measured by amphibian bladder assay, was increased three-to fourfold. This activity, which was abolished by thioglycollate and by incubation with tyrosinase or trypsin, was ascribed to vasotocin. The plasma vasotocin activities (hydrated and dehydrated frogs respectively) were estimated to be 0-03-0-5 and 0-15-0-25 mug/1; if referred to oxytocin as a standard the equivalent values were 10 and 30-60 mu./ml. Assuming that the increase represented released pituitary hormone, the amount of vasotocin released by osmotic dehydration was calculated to be of the order of 1 ng. Pituitary glands of hydrated and dehydrated frogs were estimated to have 0.15 and 0-18 mug vasotocin/gland respectively. The possible physiological function of released vasotocin in promoting reabsorption of urea from the urinary bladder is discussed in relation to the euryhaline ability of R. cancrivora.
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PMID:Vasotocin-like activity in the plasms of the euryhaline frog (Rana cancrivora) after transfer from fresh water to saline. 81 47

Ribonuclease T1 [EC 3.1.4.8] was selectively hydrolyzed by digestion with trypsin at the peptide bond of the single arginine residue at position 77. Selective hydrolysis was achieved by blocking the xi-amino group of Lys-41 with 2-methoxy-5-nitrotropone and subsequent digestion with trypsin in the presence of 2M urea. The trypsin-digested ribonuclease T1 was composed of two polypeptide chains containing 77 and 27 residues, though the two chains were covalently linked by a disulfide bond between Cys-6 and Cys-103. The modified enzyme lost enzymatic activity toward RNA and the ability to bind to 3'-GMP. The circular dichroic spectrum of the modified protein suggested that its conformation was extensively destroyed. It is concluded from the present results that the continuity of the peptide chain at the arginine residue is extremely important for maintaining the active conformation of the enzyme protein and for the enzymatic function of ribonuclease T1.
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PMID:Preparation and properties of trypsin-digested ribonuclease T1 split at the single arginyl peptide bond. 82 Jun 95

Proteinase inhibitor II, an inhibitor of chymotrypsin and trypsin, is a heat-stable protein with a dimeric molecular weight of 21 000 that is a component of Russet Burbank potato tubers. Four monomeric isoinhibitor species of molecular weight 10 500 comprise inhibitor II and were isolated by chromatography on phosphocellulose in 8 M urea. Upon removal of the urea, each monomeric species dimerized to yield homogeneous dimers. The three major protomer species, called B, C, and D, and their homogeneous dimers were further characterized. They have similar molecular weights and amino acid compositions, and each has an N-terminal alanine residue. Dimers of purified protomers B, C, and D exhibited full cross-reactivities with each other in immunological double-diffusion assays. Reconstituted dimers possess two binding sites for bovine alpha-chymotrypsin, indicating that each monomer possesses one binding site for this enzyme. Significant differences were noted among the reconstituted dimers in their isoelectric points, immunoelectrophoretic mobilities, ion-exchange properties, and their inhibitory reactivities against trypsin. The properties of the inhibitor II dimeric species are similar but not identical to inhibitors IIa and IIb reported from Japanese potatoes (variety "Danshaku-Imo"), indicating the existence of intervarietal, as well as intravarietal, differences among potato tuber inhibitor II isoinhibitors.
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PMID:Proteinase inhibitor II from potatoes: isolation and characterization of its protomer components. 82 19

The ability of C1 to bind to the Fc5mu-fragment of a monoclonal IgM was determined by means of a haemolytic C1-inhibition assay. Fc5mu fragments were produced by trypsin digestion at five different temperatures ranging from 54-62 degrees (2 degrees increments) and were purified by immunoadsorption through a column of monospecific anti-Fabmu and by molecular exclusion chromatography. Analytical ultracentrifugation showed the final preparations to be free of aggregates. A plot of mug Fc5mu required to inhibit 50% of available C1 versus temperatures of production of the fragment yielded a curve with a minimum at 58-60 degrees. Upon mild reduction and alkylation of these Fc5mu fragments their C1-fixing capacity became approximately the same irrespective of temperature of production. Fc5mu was also prepared at 25 degrees in the presence of 5 M urea, purified by immunoadsorption as before and aliquots then exposed to temperatures ranging from 40-70 degrees (5 degrees increments) for 15 min. After aggregates had been removed by chromatography a similar minimum in C1-fixation was again observed at 60 degrees. Reduction and alkylation once more abolished these differences. Fc5mu and its reduced and alkylated subunits, produced at 60 degrees and then exposed to various concentrations of urea (0-7 M) for 24 hd did not yield a minimum in C1 fixation. Reduced and alkylated Fcmu incubated at various temperatures (40-70 degrees) also did not fix C1 differentially. Examination in the near and far u.v. region of the circular dichroism spectra of different Fc5mu preparations showed a gradual loss of structure associated with restricted aromatic chromophores and secondary (beta) structure with increased temperature. Urea denaturation had a more pronounced and irreversible effect on Fc5 mu conformation. These changes could not be correlated with the CU-fixation patterns observed. It would therefore appear that elevated temperatures induce a static change in the pentameric FC-part of IgM which in turn directly influences or modulates the availability of the C1-binding site. The importance of disulphide bonds in maintaining these temperature-induced changes in Fc5mu was also indicated.
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PMID:The importance of quaternary structure in the expression of the C1-binding site of IgM. 82 51

Two forms of proacrosin have been purified from ejacualted boar spermatozoa. The isolation method utilized benzamidine to inhibit the premeture activation of the zymogen and included pH precipitation, ammonium sulfate fractionation, and sodium chloride precipitation. Further purification was achieved by Sephadex G-200 FILTRATION OF THE PREPARATION AFTER IT WAS TREATED WITH 8 M urea. The overall proacrosin yield was 58% with a specific acitivity of 253 units/mg of protein. The molecular weights of the proacrosins determined by sodium dodecyl sulfate disc gel electrophoresis were 55,000 and 53,000. Proacrosin autoactivation followed the classical S-shaped activation curve and calcium was not required to obtain full activation. Time course activation studies in 0.1 M Tris/HCl, pH 8.4, at 0 degrees were monitored with sodium dodecyl sulfate-disc gel eletrophoresis and anlytical gel electrophoresis with staining techniques for protein and enzymatic activity. Under the conditions used, the zymogens were sequentially degraded to three different active specise of acrosin (alpha, beta, and gamma). The approximate molecualr weights of the acrosins were 49,000, 39,000, and 25,000 for the alpha, beta, and gamma forms, respectively. The autoactivation is concentration-dependent and can be proteolytically stimulated with either alpha- or beta-acrosin and trypsin, indicating the activation of proacrosin can via a bimolecular process.
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PMID:Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. 84 51

Procedures were developed for isolating highly purified cytoplasmic granules of basophilic leukocytes from guinea pig peripheral blood. The methods involved disruption of cells in 0.34 M sucrose followed by a series of membrane filtrations and fractionation on sucrose density gradients. These preparations, up to 95% pure basophil granules by electron microscopy, contained a mixture of neutral esterases-proteases including caseinolytic activity; both trypsin- and chymotrypsin-like serine hydrolases were identified by means of appropriate inhibitors. Localization of at least one such activity to the basophil granule was confirmed by a cytochemical method; this activity was absent in contaminating lymphocytes and eosinophils. By contrast, several lysosomal enzymes, lactic dehydrogenase, and plasminogen activator activity, present in cell homogenates, were absent from purified granules. The granule matrix of guinea pig basophils, unlike the cytoplasmic granules of other granulocytes or mast cells, was little altered by high or low salt concentration but was disrupted into insoluble fragments by 0.01 N HCl and by Triton X-100. Granules were solubilized by papain and by urea-SDS but enzyme activity was destroyed. Triton X-100 incubation with freeze-thawing proved to be the optimal method for extracting esterase activities. Esterase activities were not released from basophils under conditions of anaphylactic degranulation that liberated the great majority of basophil granule histamine.
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PMID:Isolation of the cytoplasmic granules of guinea pig basophilic leukocytes: identification of esterase and protease activities. 87 25


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