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)

The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyanoborohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, epsilon-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of epsilon-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of epsilon-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-epsilon-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the epsilon-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of epsilon-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of epsilon-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.
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PMID:Application of reductive dihydroxypropylation of amino groups of proteins in primary structural studies: identification of phenylthiohydantoin derivative of epsilon-dihydroxypropyl-lysine residues by high-performance liquid chromatography. 643 82

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.
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PMID:Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius. 645 98

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.
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PMID:Purification and some properties of bovine liver cytosol thioltransferase. 646 49

We have investigated the effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. The sensitivity of nonglycosylated RNase A to trypsin and chymotrypsin was compared with three glycosylated species of RNase B which differed with respect to the size of the carbohydrate chain. Two forms of glycosylated RNase B were isolated by concanavalin A-Sepharose affinity chromatography, and each was shown to contain a single carbohydrate chain composed of GlcNAc2Man1 (RNase B") or GlcNAc2Man5-8 (RNase B). A third form (RNase B'), with oligosaccharide composed of GlcNAc2Man4, was prepared by partial digestion of RNase B with alpha-mannosidase. Fully glycosylated RNase B was found to be 6-10 times more resistant to trypsin digestion than nonglycosylated RNase A. RNase B' and B", with intermediate chain sizes, were 3.0- and 1.3-fold more resistant to trypsin digestion than RNase A, respectively. With chymotrypsin, however, differences in rates of digestion were much less marked, with a maximum difference of 3-fold between RNase A and B. In addition, we found that the specificity of the primary trypsin (Arg 33-Asp 34 bond) or chymotrypsin (Tyr 25-Cys 26 bond) cleavage site was not affected by the presence or size of the oligosaccharide chain. These results are consistent with the view that the size of the oligosaccharide chain and its proximity to the primary or rate-limiting cleavage site are important for expression of the carbohydrate protection against proteolytic degradation, which thus appears to be mediated by steric hindrance.
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PMID:Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. 663 Jan 85

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.
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PMID:The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP. 674 37

51Chromium-labeled rat pulmonary artery endothelial cells (EC) cultivated in MEM medium were killed, in a synergistic manner, by mixtures of subtoxic amounts of glucose oxidase-generated H2O2 and subtoxic amounts of the following agents: the cationic substances, nuclear histone, defensins, lysozyme, poly-L-arginine, spermine, pancreatic ribonuclease, polymyxin B, chlorhexidine, cetyltrimethyl ammonium bromide, as well as by the membrane-damaging agents phospholipases A2 (PLA2) and C (PLC), lysolecithin (LL), and by streptolysin S (SLS) of group A streptococci. Cytotoxicity induced by such mixtures was further enhanced by subtoxic amounts either of trypsin or of elastase. Glucose-oxidase cationized by complexing to poly-L-histidine proved an excellent deliverer of membrane-directed H2O2 capable of enhancing EC killing by other agonists. EC treated with rabbit anti-streptococcal IgG were also killed, in a synergistic manner, by H2O2, suggesting the presence in the IgG preparation of cross-reactive antibodies. Killing of EC by the various mixtures of agonists was strongly inhibited by scavengers of hydrogen peroxide (catalase, dimethylthiourea, MnCl2), by soybean trypsin inhibitor, by polyanions, as well as by putative inhibitors of phospholipases. Strong inhibition of cell killing was also observed with tannic acid and by extracts of tea, but less so by serum. On the other hand, neither deferoxamine, HClO, TNF, nor GTP gamma S had any modulating effects on the synergistic cell killing. EC exposed either to 6-deoxyglucose, puromycin, or triflupromazin became highly susceptible to killing by mixtures of hydrogen peroxide with several of the membrane-damaging agents. While maximal synergistic EC killing was achieved by mixtures of H2O2 with either PLA2, PLC, LL, or with SLS, a very substantial release of [3H]arachidonic acid (AA), PGE2, and 6-keto-PGF occurred only if a proteinase was also added to the mixture of agonists. The release of AA from EC was markedly inhibited either by scavengers of H2O2, by proteinase inhibitors, by cationic agents, by HClO, by tannic acid, and by quinacrin. We suggest that cellular injury induced in inflammatory and infectious sites might be the result of synergistic effects among leukocyte-derived oxidants, lysosomal hydrolases, cytotoxic cationic polypeptides, proteinases, and microbial toxins, which might be present in exudates. These "cocktails" not only kill cells, but also solubilize AA and several of its metabolites. However, AA release by the various agonists can be also achieved following attack by leukocyte-derived agonists on dead cells. It is proposed that treatment by "cocktails" of adequate antagonists might be beneficial to protect against cellular injury in vivo.
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PMID:Killing of endothelial cells and release of arachidonic acid. Synergistic effects among hydrogen peroxide, membrane-damaging agents, cationic substances, and proteinases and their modulation by inhibitors. 833 Sep 29

The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high-performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one-disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A.
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PMID:Nonrandom distribution of the one-disulfide intermediates in the regeneration of ribonuclease A. 863 87

With the aim to localize the structural region that becomes first accessible to proteolytic attack during thermal unfolding, the proteolysis of ribonuclease A was studied in the temperature range of 20-65 degrees C. Subtilisin, proteinase K, and elastase proved to be not appropriate as indicators of thermal unfolding, because even the native protein molecule was cleaved by these proteases. In contrast, chymotrypsin, trypsin, and thermolysin attacked ribonuclease A only after its thermal treatment. For thermolysin and trypsin, the first primary cleavage sites of ribonuclease A could be identified by blotting of the electrophoretic bands, partial N-terminal sequencing of the fragments and assignment according to their molecular masses. The results were confirmed by the separation of the proteolytic fragments by HPLC and subsequent matrix-assisted laser desorption ionization mass spectrometry. The first cleavage sites were determined to be Lys31-Ser32 and Arg33-Asn34 for trypsin and Asn34-Leu35 and Thr45-Phe46 for thermolysin. Hence the structural region from Lys31 to Leu35, together with the adjacent beta-structure containing Thr45-Phe46, is suggested to represent a labile region of the ribonuclease A molecule, which becomes exposed at thermal denaturation.
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PMID:Thermal unfolding and proteolytic susceptibility of ribonuclease A. 864 35

The thermal stabilities of ribonuclease A (RNase A) and ribonuclease B (RNase B), which possess identical protein structures but differ by the presence of a carbohydrate chain attached to Asn34 in RNase B, were studied by proteolysis and UV spectroscopy at pH 8.0. Proteolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Increasing protease concentrations led to a hyperbolic increase of the rate constants of proteolysis. With thermolysin, which attacks the unfolded molecules only, the thermal unfolding constants were determined by extrapolating the rate constants of proteolysis to infinite concentration of protease. With trypsin, the unfolding constants of RNase A could be confirmed. Subtilisin attacked even the native RNases, where RNase B was more stable toward proteolytic degradation. Kinetic stabilities (deltaG++) calculated from the unfolding constants for temperatures between 52.5 and 65 degrees C revealed a higher kinetic stability of RNase B, which results from enthalpic effects only, whereas entropic effects counteract stabilization. delta deltaG++ at the transition temperature of RNase A (60.4 degrees C) was 2.2 +/- 0.3 kJ mol(-1). Thermodynamic stabilities (deltaG) were estimated from the thermal transition curves at 287 nm for the temperature range from 55 to 70 degrees C. For 17.5-25 degrees C, deltaG values were determined from transition curves of unfolding induced by guanidine hydrochloride and extrapolation of the free energy values to those in the absence of denaturant. At all temperatures, RNase B proved to be more stable than RNase A with essentially the same enthalpy and entropy of unfolding. delta deltaG was 2.5 +/- 0.2 kJ mol(-1) at 60.4 degrees C and 2.3 kJ mol(-1) at 25 degrees C.
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PMID:Kinetic and thermodynamic thermal stabilities of ribonuclease A and ribonuclease B. 904 16

Water oxygen-17 and deuteron spin relaxation rates, measured as a function of resonance frequency, have been used to study the dynamics of protein hydration in aqueous solutions of ribonuclease A, lysozyme, myoglobin, trypsin and serum albumin. The relaxation data conform to the picture of protein hydration dynamics, proposed on the basis of previous studies of smaller proteins, where the long-lived water molecules responsible for the relaxation dispersion are identified with a small number of integrat water molecules seen in the crystal structures. These integral water molecules, with residence times in the range 10(-9)-10(-3) s, are either buried in internal cavities, trapped in narrow clefts or coordinated to metal ions. For the water molecules in the traditional hydration layer at the protein surface, the relaxation data suggest an average residence time in the range 10-50 ps, consistent with high-resolution 1H spectroscopy and computer simulations. The relaxation data also reveal some more specific features of protein hydration, relating to hydration of cavities that appear empty by crystallography, entrapment of water between structural domains of large proteins and subnanosecond 180 degrees flips in buried water clusters.
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PMID:Protein hydration dynamics in aqueous solution. 913 39


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