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)

Pretreatment of the purified jack bean inhibitor with enterokinase activated human pancreatic preparation for 1 hr decreased its inhibitory capacity against crystalline bovine alpha-chymotrypsin by 30% but did not affect its trypsin inhibitory activity. Preincubation of the inhibitor with bovine chymotrypsin for 60 min resulted in partial loss of the inhibitory potency. Complex formation studies by gel chromatography on Sephadex G-100 indicated that the trypsin-inhibitor and chymotrypsin-inhibitor complexes dissociated to release inactivated inhibitor and active proteinases. Gel chromatography of the inhibitor in presence of 1.5 M ammonium sulphate indicated that the inhibitor showed a tendency to aggregate without loss of biological activity. However, in 4.2 M salt medium after 3 hr, antichymotryptic activity was lost completely without any effect on antitryptic activity. Treatment with methylamine, a nucleophile, caused a greater loss of antichymotryptic activity. Trinitrobenzene sulphonate and ethylacetamidate, the amino group modifiers, affected only the antichymotryptic activity. Treatment with ninhydrin, a specific arginine modifier, at pH 9.0 abolished the antitryptic activity whereas only 50% of the antichymotryptic activity was lost. Diethylpyrocarbonate, a histidine reagent, also decreased only the antitryptic activity. Modification of tryptophan and cysteine residues of the inhibitor had no effect on its inhibitory potency. Treatment with mercaptoethanol and sodium borohydride caused nearly 50% loss of antitryptic and antichymotryptic activities. Chloramine-T, a reagent that modifies methionine residues, inactivated the inhibitor.
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PMID:Chemical modification and complex formation studies with jack bean proteinase inhibitor. 181 77

The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied. Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles. Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan. 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase. Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor. The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance. These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids.
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PMID:Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study. 185 Feb 92

Three fenugreek inhibitors (TFI-A8, TFI-N2, and TFI-B2) were isolated from an inhibitor preparation by anion exchange chromatography and subsequent preparative isoelectric focusing using immobilized pH gradients and the canal technique. The purified inhibitors inhibited the enzymes tested differently: TFI-A8 exhibited a high inhibition of trypsin (8.2 mg human trypsin/mg and 8.1 mg bovine trypsin/mg) and a very low inhibition of chymotrypsin (0.8 mg human chymotrypsin/mg and 1.0 mg bovine chymotrypsin/mg). TFI-N2 inhibited the four enzymes to about the same extent (5.0 mg/mg human and 4.1 mg/mg bovine trypsin; 4.9 mg/mg human and 3.7 mg/mg bovine chymotrypsin). TFI-B2 displayed a high inhibition of trypsin (7.5 mg/mg human and 5.1 mg/mg bovine) and a low inhibition of chymotrypsin (1.8 mg/mg human and 1.9 mg/mg bovine). On average, the human enzymes were inhibited better than the bovine ones by the purified inhibitors. The inhibitors contained high amounts of cystine (five or six disulfide bridges per molecule), aspartic acid, threonine, serine and proline, no valine and methionine and two of them also no tryptophan. Their molecular masses were about 6 kDa. Their inclusion into the Bowman-Birk soybean proteinase inhibitor family is discussed.
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PMID:Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Isolation and characterization. 187 34

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.
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PMID:Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity. 189 34

The soluble beta-galactoside-specific bovine lectin of subunit 14 kDa has been expressed in vitro by transcription and then translation in a rabbit reticulocyte lysate. The protein thus expressed shows the predicted binding to lactose coupled to Sepharose. Several mutants of the 134 amino acid protein have been expressed and insight gained into (a) the polypeptide length required to form the carbohydrate recognition domain and (b) the functional importance of some of the highly conserved amino acids. The following amino acids have been deleted: 1-9, 1-23, 88-122, 88-134, 107-134, or 124-134. In addition, a frame-shift mutant has been made in which the 23 amino acids at the C-terminal end were completely changed. Among these seven mutants only mutant 1-9 shows carbohydrate binding but with congruent to 30% of the activity of the wild-type protein (as assessed by the percentage of the protein bound to lactose-Sepharose). On the other hand, carbohydrate binding is relatively well preserved (75-90%) in mutant proteins where the C-terminal octapeptide sequence of the bovine lectin has been changed to sequences that resemble those in the chick 14-kDa lectin. When the single tryptophan at position 68 is changed by point mutagenesis to phenylalanine or to a leucine residue, a weak binding activity (congruent to 20%) is retained only with the former. When either of the cysteines 2 or 60 is changed to serine, binding activity is reduced to congruent to 60%, and when both are changed, to congruent to 20% of that for the wild-type protein. The susceptibility of the lectin to oxidative inactivation is unaffected when these 2 cysteines and cysteine 130 are changed to serine individually or in tandem (cysteines 2 and 60). In a second approach we show that the natural protein isolated from bovine heart is protected from proteolysis by trypsin and V8-protease in the presence of saccharide ligand. Although further work is required to identify residues which come into contact with the carbohydrate ligand, these results indicate that almost the complete polypeptide chain is necessary for the integrity of the carbohydrate recognition domain.
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PMID:Soluble 14-kDa beta-galactoside-specific bovine lectin. Evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain. 190 Aug 35

This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.
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PMID:The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties. 190 55

Alpha-1-proteinase inhibitor (alpha-1-PI) was isolated from goat plasma by salt fractionation, and chromatography on a DEAE-cellulose column. The inhibitor was found to be homogeneous by gel chromatography, SDS-PAGE and PAGE.Mr values by gel filtration (57 kDa), and by SDS-PAGE (52 kDa), under reducing conditions were nearly the same suggesting that the inhibitor consists of a single polypeptide chain. It contained 13.8% neutral hexose but no sialic acid residue. The values of isoionic pH, and extinction coefficient at 278 nm were 4.84, and 4.6, respectively. Fluorescence spectral properties showed tryptophan residues in the inhibitor. Solvent perturbation difference spectra suggested 74% exposure of the tryptophan residues in the native molecule. Gel filtration behaviour of the inhibitor was consistent with a Stokes radius of 3.16 nm, diffusion coefficient of 7.02 X 10(-7) cm2-sec-1 and a frictional ratio of 1.24 suggesting asymmetry and/or excessive hydration of the inhibitor molecule. Goat alpha-1-PI, unlike human alpha-1-PI was found to be potent inhibitor of bovine trypsin but a poor inhibitor of porcine pancreatic elastase. It was virtually devoid of antichymotryptic activity.
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PMID:Isolation and characterization of alpha-1-proteinase inhibitor from goat plasma. 193 Feb 50

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.
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PMID:A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus. 198 74

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.
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PMID:The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin. 208 Dec 70

Four fragments of ovomucoid representing its individual domains and their different combinations were prepared by peptic and cyanogen bromide cleavages of the protein. The fragments corresponding to domains I + II, II + III, I and III of the parent ovomucoid molecule, were found to be homogeneous by gel filtration and polyacrylamide gel electrophoresis in presence and absence of SDS. Various physico-chemical properties of these proteins, such as molecular weight, NH2- and COOH-terminal amino acid residues, sugar content, isoionic pH, specific extinction coefficient, fluorescence emission spectra, intrinsic viscosity, frictional coefficient, Stokes radius, diffusion coefficient and geometrical mean radius were determined. Analysis of the results on trypsin inhibitory activity of ovomucoid and its different fragments suggested that only domain II is involved in the antitryptic activity of the inhibitor. Optical characteristics of these fragments indicate that they are devoid of tryptophan residues. The hydrodynamic properties suggest that intact ovomucoid and two of its fragments (domain I + II and domain II + III) are significantly different from those of typical globular proteins and are asymmetric in nature. However, the shape of the two remaining fragments representing domains I and III of the intact protein appeared to be compact and globular. Furthermore, domain II of ovomucoid has been suggested to primarily contribute towards the apparent asymmetry in the intact protein.
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PMID:Ovomucoid domains: preparation and physico-chemical characterization. 209 Jan 11


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