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)

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.
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PMID:Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity. 300 Jul 82

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.
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PMID:Accessibility of tryptophan residues in Na,K-ATPase. 303 Oct 29

Trypsin and chymotrypsin were used as probes of structure-divalent cation relationships in G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. The tryptic product of G-actin, 33 kDa is a protease-resistant fragment in the presence of divalent cations. However, once divalent cations are eliminated from the solution during the digestion, the 33 kDa fragment starts to degrade into smaller peptides via a 30 kDa fragment. On the other hand the chymotryptic product of G-actin, 35 kDa (precursor of 33 kDa) is rather stable even in the absence of divalent cations. In addition it is observed that the presence of divalent cation is necessary for the degradation of G-actin to the 33 kDa fragment by trypsin. The ultra violet and intrinsic tryptophan fluorescence spectra of G-actin are changed after the elimination of divalent cations. These results suggest that the structure of G-actin molecule depends on the presence or absence of divalent cations, and that the divalent cation-dependency of G-actin structure is still conserved even after the tryptic digestion.
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PMID:Effect of divalent cation on the structure of skeletal muscle G-actin molecule. 335 76

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and activity of trypsin in reverse micelles. 336 18

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.
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PMID:DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1. 351 Oct 54

We examined whether the generation of reactive oxygen metabolites (as quantified by measuring luminol-amplified chemiluminescence) by isolated rat glomeruli could be triggered enzymatically. No response was observed with thrombin (1 or 10 U/ml), collagenase (100, 200, or 400 U/ml), or plasmin (0.1 or 1 U/ml). In contrast, chymotrypsin and trypsin caused a dose-dependent (10-200 micrograms/ml) increase in chemiluminescence from glomeruli. The peak response with chymotrypsin (100 micrograms/ml) and trypsin (50 micrograms/ml) was as follows: resting, 16 +/- 2 X 10(3) cpm/mg protein, n = 17; chymotrypsin, 233 +/- 58 X 10(3) cpm/mg protein, n = 17; and trypsin, 221 +/- 38 X 10(3) cpm/mg protein, n = 10. Tubules had only a minor response. Soybean trypsin inhibitor and aprotinin caused marked inhibition, indicating the dependency of the chemiluminescence response on the protease enzyme activity. The chemiluminescence response was by glomeruli rather than by "contaminating" leukocytes, since a similar marked response (n = 6) was observed in glomeruli isolated from cyclophosphamide-treated leukopenic (leukocyte less than 1,000/mm3) rats. Superoxide dismutase, a scavenger of superoxide, and free-radical scavengers benzoate and tryptophan inhibited the glomerular chemiluminescence response to trypsin and chymotrypsin. Neutral proteases from infiltrating leukocytes and/or renal tissue have been shown to be released in glomerular diseases; our results, which show the generation of chemiluminescence in response to neutral proteases, suggest a potential mechanism for the production of reactive oxygen metabolites in glomerular diseases.
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PMID:Trypsin- and chymotrypsin-induced chemiluminescence by isolated rat glomeruli. 359 31

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.
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PMID:Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers. 359 1

The photoreactive arylsufenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (STI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-STI indicated that only one of the two tryptophans, 93 or 117, present in STI was modified. Amino acid analysis of the two separated CNBr-cleavage products of 2,4-NAPS-STI showed that only tryptophan 93 underwent modification. 2,4-NAPS-STI fully retained its inhibitory activity against trypsin. The covalent attachment of 2,4-NAPS-STI to tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride. Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas were prepared by selective modification of the epsilon-amino groups of 2,4(5)-NAPS-Cl. The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.
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PMID:Photoreactive, active derivatives of trypsin and chymotrypsin inhibitors from soybeans and chickpeas. 379 89

The tryptophan environments in crystalline alpha-chymotrypsin were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was resolved by time-correlated fluorescence spectroscopy. The fluorescence decays at 296-nm laser excitation and various emission wavelengths could be characterized by a triple-exponential function with decay times tau 1 = 150 +/- 50 ps, tau 2 = 1.45 +/- 0.25 ns, and tau 3 = 4.2 +/- 0.4 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 325, 332, and 343 nm. The three decay components in this enzyme can be correlated with X-ray crystallographic data [Birktoft, J.J., & Blow, D.M. (1972) J. Mol. Biol. 68, 187-240]. Inter- and intramolecular tryptophan-tryptophan energy-transfer efficiencies in crystalline alpha-chymotrypsin were computed from the accurately known positions and orientations of all tryptophan residues. These calculations indicate that the three fluorescence decay components in crystalline alpha-chymotrypsin can be assigned to three distinct classes of tryptophyl residues. Because of the different proximity of tryptophan residues to neighboring internal quenching groups, the decay times of the three classes are different. Decay tau 1 can be assigned to Trp-172 and Trp-215 and tau 2 to Trp-51 and Trp-237, while the tryptophyl residues 27, 29, 141, and 207 all have decay time tau 3.
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PMID:Study of the time-resolved tryptophan fluorescence of crystalline alpha-chymotrypsin. 381 86

The amino acid sequence of rabbit skeletal muscle heat-stable inhibitor of the cAMP-dependent protein kinase has been determined by microsequencing techniques. Proof of the structure involved a series of nonoverlapping tryptic fragments for primary identification of 86% of the amino acids. Complementary fragments generated by cleavage with chymotrypsin, Staphylococcus aureus V8 proteinase, and mast cell proteinase II contributed to proof of the structure. The inhibitor is a single polypeptide chain of 75 residues and has a molecular weight of 7829. It lacks tryptophan, proline, and sulfur-containing amino acids. The amino terminus of the inhibitor is blocked by an unidentified group. The amino-terminal region of the molecule contains the kinase inhibitory domain, and synthetic peptides based on the sequence of residues 11-30 are potent competitive inhibitors of the cAMP-dependent protein kinase [Scott, J. D., Fischer, E. H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383]. Residues 14-22 show considerable homology to the "hinge-regions" of the regulatory subunits of the cAMP-dependent protein kinase. The remainder of the molecule shows no similarity to the known amino acid sequence of any protein.
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PMID:Amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase from rabbit skeletal muscle. 389 70


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